Culture medium

Graefes Arch Clin Exp Ophthalmol, 1998 Apr, 236(4), 312 - 9
A sensitive method for testing the quality of organ culture media and of individual medium components in a cornea bank; Engelmann K et al.; BACKGROUND: It has been suggested that variations in the quality of organ culture preservation media are responsible for variations in early postoperative graft morphology . Spates of such variations have been observed repeatedly for short periods . This paper reports the results of a series of grafts with low postoperative clearing observed during a period of 6 weeks . Simultaneously, preoperative phase-contrast microscopy evaluation of the corneal endothelium revealed that an unusually large proportion of donor corneae were unsuitable for transplantation . METHODS: The corneal storage media were therefore rigorously screened, paying particular attention to specific components and properties of the medium, including L-glutamine, amphotericin B, water quality, pH, and the glassware used . Possible toxic effects were identified by means of a sensitive growth assay performed using isolated human corneal endothelial cells . RESULTS: The evaluation demonstrated that both the water quality and the L-glutamine which had been used for preparation of the medium were substandard during the period in which poor clinical results were obtained . CONCLUSION: It is recommended that cornea banks undertaking long-term organ culture use standardized protocols and carefully monitored equipment . The quality of the basal media and supplements should be routinely checked.

Lipids, 1998 Mar, 33(3), 285 - 93
Effects of polyunsaturated fatty acids and their n-6 hydroperoxides on growth of five malignant cell lines and the significance of culture media; Noding R et al.; We examined effects of polyunsaturated fatty acids (PUFA), their corresponding hydroperoxy fatty acids (hp-PUFA), as well as various pro- and antioxidants on the growth of tumor cells in culture . When cultured in RPMI 1640 medium, A-427 and WEHI clone 13 cells were both highly sensitive to hydroperoxy docosahexaenoic acid (hp-DHA), but they were far less sensitive in minimum essential medium (MEM) . In contrast, A-427 cells were also sensitive to DHA in both culture media, while WEHI clone 13 cells, as well as other cell lines, tested in their respective media, were resistant . The lower sensitivity of the cell lines to hp-DHA in MEM-medium was apparently due to a more rapid reduction of hp-DHA to the corresponding hydroxy-DHA in MEM-medium . Addition of glutathione (GSH) to the culture medium abolished the effects of hp-DHA, but not the effects of DHA, while depletion of intracellular GSH levels by L-buthionine-S,R-sulfoximine strongly enhanced the cytotoxic effect of hp-DHA, but not the cytotoxic effect of DHA . alpha-Tocopherol protected A-427 cells against the toxic effect of DHA and abolished the induced lipid peroxidation, while it did not protect against the toxic effects of hp-DHA in A-427 or WEHI clone 13 cells . Ascorbic acid reduced the cytotoxic effect of DHA, but potentiated the toxic effect of hp-DHA while selenite essentially abolished the toxicity of both DHA and hp-DHA . These results indicate that sensitivity of tumor cell lines to PUFA and their oxidation products depends on their antioxidant defense mechanisms, as well as culture conditions, and establishes hp-DHA as a major, but probably not the sole, metabolite responsible for cytotoxicity of DHA.

FEBS Lett, 1998 Mar 27, 425(2), 371 - 5
A possible involvement of endogenous polyamines in the TNF-alpha cellular sensitivity; George P et al.; A critical step in the cytotoxic action mechanism of tumor necrosis factor-alpha (TNF-alpha) involves, among mitochondrial dysfunctions, an early change of the inner membrane permeability displaying the characteristics of permeability transition . Cytosolic polyamines, especially spermine, are known to inhibit it . Our results show that spermine is only detectable in the TNF-alpha resistant C6 cells while N1-acetylspermidine is present in the TNF-alpha sensitive WEHI-164 cells, and putrescine and spermidine are found in both . TNF-alpha treatment does not change this distribution but only induces a quantitative alteration in TNF-alpha sensitive cells . Omission of glutamine (energetic substrate) from the culture media alters neither the TNF-alpha responsiveness of both cell lines nor their polyamine distributions, only their quantitative polyamine contents.

Am J Hum Genet, 1985 Jul, 37(4), 798 - 808
Type IX Ehlers-Danlos syndrome and Menkes syndrome: the decrease in lysyl oxidase activity is associated with a corresponding deficiency in the enzyme protein; Kuivaniemi H et al.; Type IX of the Ehlers-Danlos syndrome (E-D IX) and the Menkes syndrome are X-linked recessively inherited disorders characterized by abnormalities in copper metabolism . These abnormalities are associated with a severe reduction in the activity of lysyl oxidase, the extracellular copper enzyme that initiates crosslinking of collagens and elastin . No increase in this deficient enzyme activity was obtained when culture media from fibroblasts of patients with E-D IX or the Menkes syndrome were incubated with copper under various conditions in vitro . A distinct, although small, increase in lysyl oxidase activity was obtained, however, when copper-supplemented media were used during culturing of the fibroblasts, although even under these conditions, the enzyme activity in the media from the affected cells remained markedly below that of the controls . Immunoprecipitation, dot-blotting, and immunoperoxidase staining experiments with antisera to human lysyl oxidase indicated that fibroblasts from patients with E-D IX or the Menkes syndrome do not secrete into their medium, or contain inside the cell, any significant amounts of a copper-deficient, catalytically inactive lysyl oxidase protein . These findings appear to be consistent with the hypothesis that synthesis of the lysyl oxidase protein itself is impaired . The possibility is not excluded, however, that a copper-deficient enzyme protein may be synthesized in normal amounts but become degraded very rapidly inside the cell . The failure to obtain any large increase in the deficient lysyl oxidase activity upon various forms of copper administration suggests that it may not be possible to obtain any significant improvement in the connective tissue manifestations of these disorders by copper therapy.

Calcif Tissue Int, 1998 Apr, 62(4), 341 - 9
Effect of metal ions on calcifying growth plate cartilage chondrocytes; Litchfield TM et al.; The effects of the trace metals zinc (Zn), manganese (Mn), and cadmium (Cd) on the metabolism of growth plate chondrocytes was examined using a mineralizing culture system . Supplementation of serum-free primary cultures of growth plate chondrocytes with 10-100 mu m Zn resulted in an increase in cell protein and greatly increased alkaline phosphatase (AP) activity; however, above 25 mu m Zn mineralization of the cultures was reduced . The effects of Zn on cellular protein and AP activity were enhanced by the addition of the albumin to the culture media . Removal of Zn from basal culture media resulted in recoverable reductions in cellular protein and AP activities . Cadmium was acutely toxic to chondrocyte cell cultures at concentrations above 5 mu m . Even at very low concentrations (0.25 mu m) Cd caused significant reductions in DNA, cellular protein, and matrix protein synthesis . In contrast, Cd had negligible effects on AP activity or culture mineralization . Manganese treatment (50 mu m) resulted in reduced levels of proteoglycan, cell protein, DNA synthesis, and collagen synthesis, although AP specific activity did not change . At 10 mu m, Mn significantly reduced mineralization but had only minor influence on other culture parameters . Both Zn (200 mu m) and Cd (0.1 mu m), but not Mn, induced the synthesis of metallothionein . The physiological and biochemical effects of specific metal ions is largely dependent on their physicochemical properties, especially their ligand affinities . Knowledge of these properties allows predictions to be made regarding whether the organic or the mineral phase are most likely to be affected in a mineralized tissue.

Biol Reprod, 1998 Apr, 58(4), 988 - 94
Endometriotic lesions synthesize and secrete a haptoglobin-like protein; Sharpe-Timms KL et al.; To explore the identity and possible function of endometriosis protein-I (ENDO-I), which is an acidic glycoprotein synthesized and secreted by endometriotic lesions, partial amino acid sequence and cDNA sequence were determined . Partially purified, de novo-synthesized rat endometriosis glycoproteins were separated by two-dimensional SDS-PAGE, transferred to polyvinyl difluoride membranes, and stained with Coomassie blue . Protein corresponding to the size and pI of ENDO-I was cut from the membranes and analyzed by automated Edman degradation . ENDO-I amino acid sequence analysis identified 15 residues that shared significant homology with the beta-chain of rat, mouse, and human haptoglobin (Hp) and human Hp-related protein . Western blot analyses using anti-Hp antibody demonstrated cross-reactivity with de novo-synthesized ENDO-I protein in endometriosis culture media . For nucleotide sequence analysis, poly A-enriched mRNA was isolated from rat endometriotic tissues . A gene-specific oligonucleotide primer was designed and used for 3' rapid amplification of cDNA ends (RACE) . Automated sequencing of RACE cDNA fragments identified 859 base pairs, of which 858 were identical to rat Hp . Reverse transcription-polymerase chain reaction was used to demonstrate that ENDO-I transcripts are differentially expressed by endometriosis but not by uterine tissues . In the human, distinct subtypes of Hp as well as proteins sharing epitopes with Hp have been used to diagnose a variety of diseases; therefore, Hp-like ENDO-I may prove to be a nonsurgical diagnostic tool to assess endometriosis . Hepatic Hp, induced by acute-phase stimuli, modulates macrophage function and angiogenic activity . If ENDO-I possesses similar activities, it may be involved with anomalies of the immune system or the etiology and pathophysiology of endometriosis.

Biochim Biophys Acta, 1998 Apr 1, 1397(1), 27 - 30
Sequencing and high level expression in Escherichia coli of the tropomyosin allergen (Der p 10) from Dermatophagoides pteronyssinus; Asturias JA et al.; The cDNA encoding an allergen from the dust mite Dermatophagoides pteronyssinus has been cloned and sequenced . The allergen (Der p 10) is a tropomyosin that shared more than 65% identical residues with other invertebrate tropomyosins . The final recovery of recombinant Der p 10 from the culture media after a single purification step was as much as 26 mg/l . The recombinant allergen is reactive to shrimp antitropomyosin IgG antibodies and has a 5.6% frequency of IgE reactivity in sera from mite-allergic patients.

Biochem J, 1998 Mar 1, 330 ( Pt 2), 1051 - 8
Soluble FasR ligand-binding domain: high-yield production of active fusion and non-fusion recombinant proteins using the baculovirus/insect cell system; Mahiou J et al.; We used the recombinant baculovirus/insect cell system to express two soluble forms of the mouse Fas receptor (mFasR) extracellular domain (ECD): a monomer comprising the entire ligand-binding portion of mFasR followed by a carboxy-terminal hexa-histidine extension aiding purification by immobilized metal affinity chromatography and an immunoadhesin in which the same 148 residues were fused to the Fc portion of a truncated human IgG1 immunoglobulin heavy chain . Both constructs harboured a 24 base pairs insertion placed upstream of the initiating ATG {Peakman, Charles, Sydenham, Gewert, Page, and Makoff (1992) Nucleic Acids Res . 20, 6111-6112} . Despite its hexa-histidine extension, the monovalent recombinant protein from crude culture media failed to bind immobilized Ni2+ unless proteins were first precipitated twice by ammonium sulphate . The overall procedure then yielded approximately 10mg/l of protein which could be purified to near homogeneity using two additional chromatographic steps . The glycosylated polypeptide migrated as a band of Mr=(21-31) x 10(3) in SDS/PAGE and was monomeric in physiological buffers . Under non-reducing conditions, denaturation in 6 M guanidinium chloride was reversible after slow removal of the denaturing agent . The mFasR immunoadhesin was secreted (approximately 5-10 mg/l) as a disulphide-linked homodimer, and endowed with ligand-binding activity since it could bind FasL on the surface of D11S, FasL-expressing cells . When tested for their ability to inhibit FasR-dependent cell lysis, the soluble dimeric immunoadhesin markedly inhibited FasL-mediated cytotoxicity (IC50 approximately 30 nM), and was approximately 6 times as effective as its monomeric counterpart.

Pharmacotherapy, 1998 Mar-Apr, 18(2), 341 - 4
Cyclosporine-induced beta-adrenergic receptor down-regulation in bovine pulmonary artery smooth muscle cells: a pilot study; Dickens GR et al.; We attempted to determine the effects of cyclosporine on beta-adrenergic receptors in bovine pulmonary artery smooth muscle cells . Bovine pulmonary artery smooth muscle cells were exposed to cyclosporine at a concentration of 100 ng/ml in culture media for 5 days, and control bovine pulmonary artery smooth muscle cells were exposed to only culture media for the same 5-day period . Beta-adrenergic receptors were measured as total binding capacity (Bmax) by nonlinear least squares fit of the specific binding curve . In a separate experiment beta1- versus beta2-adrenergic receptor subtypes were identified by computer modeling (LIGAND) of 17-19 point CGP20712A-125ICYP competition curves . Cyclosporine significantly (p=0.02) decreased bovine pulmonary artery smooth muscle beta-adrenergic receptor density by 54%+/-7% . The Bmax for control versus treated cells was 38.9+/-18 versus 17.7+/-12 fmol/mg protein, respectively . Subtype determination of beta-receptors revealed 70% or more beta2- and 30% or less beta1-adrenergic receptors . Cyclosporine caused a 54% reduction in overall beta-adrenergic receptor density in bovine pulmonary artery smooth muscle cells . The reduction in Bmax is suspected not to be a result of selective down-regulation of beta1-adrenergic receptors alone . We believe that cyclosporine may also contribute to a decrease in beta2-adrenergic receptors.

Curr Eye Res, 1998 Mar, 17(3), 276 - 85
Growth factor and cytokine modulation of trabecular meshwork matrix metalloproteinase and TIMP expression; Alexander JP et al.; PURPOSE: We hypothesize that regulated trabecular extracellular matrix (ECM) turnover, initiated by the matrix metalloproteinases, is critical for the maintenance of normal aqueous humor outflow rates . However, very little is known about the regulation of trabecular ECM turnover . To identify candidate trabecular regulators, we evaluated the effects of several growth factors and cytokines on trabecular matrix metalloproteinase and TIMP expression . METHODS: Porcine trabecular meshwork cells were treated with several doses of a variety of growth factors and cytokines and culture media was analyzed after 24, 48, and 72 h . Zymograms were used to evaluate stromelysin, gelatinase A and B activity levels, while immunoblots of Western transfers were used to evaluate stromelysin, collagenase, TIMP-1 and TIMP-2 protein levels . RESULTS: A phorbol mitogen (TPA), and TNF alpha and beta, interleukin-1 alpha and PDGF BB stimulate gelatinase B, stromelysin, interstitial collagenase and TIMP-1 expression, while having negligible effects on gelatinase A expression; TIMP-2 levels are reduced by TNF but not affected by the other treatments . Acidic and basic FGF, IL-1 beta, TGF beta and PDGF AB produce similar but smaller effects, while HGF, VEGF, EGF, KGF, and LIF produce small to moderate elevations in stromelysin with minimal other responses . PDGF AA, gamma INF, oncostatin-M and endothelin-1 produce negligible changes in these proteinases and inhibitors . CONCLUSIONS: In addition to providing potential ways to modulate trabecular metalloproteinase and TIMP levels, the responsiveness of these cells to some of these growth factors and cytokines suggests possible roles in normal or pathogenic trabecular cell regulation and some may affect aqueous humor outflow.

Biochim Biophys Acta, 1997 Dec 31, 1362(2-3), 208 - 20
Proteoglycan breakdown from bovine nasal cartilage is increased, and from articular cartilage is decreased, by extracellular ATP; Brown CJ et al.; The addition of ATP, but not ADP or AMP, to the culture media of bovine nasal cartilage explants caused an acceleration in the rate of proteoglycan loss from the tissue . The ATP-stimulated loss of proteoglycan was not inhibited by the IL1-receptor antagonist protein, but was partially inhibited by the presence of ADP or AMP . The proteolytic events resulting from the presence of ATP were found to be similar to those following treatment with IL1, in that inhibitors of the cysteine-peptidase cathepsin B, serine-proteinases with trypsin-like specificity, and of some of the matrixins, could all prevent proteoglycan loss, which was mediated, at least in part, by the action of 'aggrecanase' . In contrast to its effects on nasal cartilage, ATP inhibited basal and stimulated proteoglycan release from articular cartilage . Both ADP and AMP had no effect on proteoglycan release in articular cartilage but enhanced the response to ATP when added concurrently . We conclude that extracellular ATP, probably acting via P2-purinoceptors, stimulates proteoglycan breakdown from bovine nasal cartilage and thus, may have a role in diseases which primarily involve destruction of non-articular cartilage . Extracellular ATP has, in contrast, a chondroprotective effect on bovine articular cartilage.

J Reprod Fertil, 1998 Jan, 112(1), 149 - 56
Characterization of conceptus-produced goat interferon tau and analysis of its temporal and cellular distribution during early pregnancy; Guillomot M et al.; Two proteins (17 and 22-24 kDa) produced by day 17 goat conceptuses were purified from in vitro culture media . Analysis of their N-terminal amino acid sequences and of their antiviral activity confirmed that both proteins belonged to the interferon tau family characteristic of ruminant conceptuses . The two molecules were glycosylated (22-24 kDa) or nonglycosylated (17 kDa) isoforms of the same protein . The time course of secretion was plotted and immunoblotting of the protein contents of uterine flushings from day 13 to day 21 of pregnancy was performed . The nonglycosylated isoform (17 kDa) was first detected on day 16; both isoforms were present at day 17 and, thereafter during pregnancy, the two proteins were not present in uterine flushings . Immunohistochemistry was used to show that the goat interferon tau was present in the trophoblastic cells as early as day 14 and until day 17 . However, immunostaining was not uniform along the conceptus; labelling was greater at the abembryonic pole than at the embryonic pole . By day 18, as implantation proceeded, goat interferon tau was no longer detected . These results confirmed that the goat conceptus secretes interferon tau during the period of maternal recognition of pregnancy but its rapid decrease suggests that other factors need to be present by day 18 to take over its role in the maintenance of luteal function.

J Androl, 1998 Jan-Feb, 19(1), 92 - 9
Oxidative stress differentially regulates the expression of gamma-glutamyl transpeptidase mRNAs in the initial segment of the rat epididymis; Markey CM et al.; Reactive oxygen species (ROS) have a powerful cytotoxic effect on spermatozoa and have been implicated in spermatozoal dysfunction and male infertility . gamma-Glutamyl transpeptidase (GGT) is essential to the metabolism of the antioxidant glutathione and, as such, is believed to be important in protecting spermatozoa against oxidative stress . The aims of this study were 1) to establish in vitro conditions in which ROS were generated and 2) to determine whether oxidative stress regulated the expression of GGT mRNAs I-IV in the initial segment of the epididymis . Initial segments were collected from adult male rats and incubated in culture media to which ROS-generating compounds, hypoxanthine and xanthine oxidase, were added . By 6.5 hours, incubation of tissue in high-oxidative stress conditions caused a 56% decrease in reduced glutathione concentration, a concomitant 240% increase in oxidized glutathione concentration, and a 25% decrease in adenosine triphosphate concentration . RNase protection analyses demonstrated an approximate 70% up-regulation of GGT mRNAs II-IV in a differential manner, depending on the concentration of oxidizing agents and the type of ROS generated . gamma-Glutamyl transpeptidase mRNA I was not expressed . These results support the hypothesis that expression of GGT mRNAs is regulated by oxidative stress in the initial segment of the rat epididymis.

Biochem J, 1998 Feb 15, 330 ( Pt 1), 35 - 40
Mutagenesis of the aspartic acid ligands in human serum transferrin: lobe-lobe interaction and conformation as revealed by antibody, receptor-binding and iron-release studies; Mason A et al.; Recombinant non-glycosylated human serum transferrin and mutants in which the liganding aspartic acid (D) in one or both lobes was changed to a serine residue (S) were produced in a mammalian cell system and purified from the tissue culture media . Significant downfield shifts of 20, 30, and 45 nm in the absorption maxima were found for the D63S-hTF, D392S-hTF and the double mutant, D63S/D392S-hTF when compared to wild-type hTF . A monoclonal antibody to a sequential epitope in the C-lobe of hTF reported affinity differences between the apo- and iron-forms of each mutant and the control . Cell-binding studies performed under the same buffer conditions used for the antibody work clearly showed that the mutated lobe(s) had an open cleft . It is not clear whether the receptor itself may play a role in promoting the open conformation or whether the iron remains in the cleft.

Biol Trace Elem Res, 1998 Mar, 61(3), 237 - 52
Different selenium-containing proteins in the extracellular and intracellular media of leucocytes cultivated in vitro; Liu Q et al.; The purpose of this communication is to elucidate if selenium plays a role in the function of granulocytes and lymphocytes . Thus, the incorporation of selenium in proteins from granulocytes and lymphocytes cultured with 1 microCi/mL radioactive Na2(75)SeO3 was studied . The protein peaks containing 75Se from two columns of Heparin Sepharose CL-6B and Sephacryl S-200 HR were separated further by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis . The results showed that the incorporation of 75Se into granulocytes was about six times higher than that of lymphocytes during a 96-h cultivation, however, the GSH-Px activity in granulocytes did not change significantly . On the other hand, the GSH-Px activity of lymphocytes rose significantly after three days cultivation . These data indicated that the main chemical form of selenium in granulocytes was not GSH-Px . Results from SDS-PAGE revealed a strongly 75Se-labeled protein band with subunit molecular weight of 15 kDa in the supernatant of granulocyte homogenate . However, the main chemical forms of selenium in the culture media of granulocytes and lymphocytes were found to be selenoprotein P . The different forms of selenium-containing proteins in the intracellular and extracellular media of granulocytes indicated the different functions of these proteins.

Biosci Biotechnol Biochem, 1998 Feb, 62(2), 309 - 15
Overproduction of 1,2-alpha-mannosidase, a glycochain processing enzyme, by Aspergillus oryzae; Yoshida T et al.; A recombinant strain of Aspergillus oryzae has been constructed in which 1,2-alpha-mannosidase, an intracellular glycochain processing enzyme with specificity toward 1,2-alpha-mannosidic linkages, has been overexpressed . For the construction, the N-terminal signal-encoding sequence of the 1,2-alpha-mannosidase gene (msdC) from Penicillium citrinum was replaced with that of the aspergillopepsin I signal, and the fused gene was inserted between amyB promoter-terminator elements in the expression plasmid pTAPM1 . A transformant of A . oryzae (the strain PM-1) secreted a great deal of heterogeneous 1,2-alpha-mannosidase into the culture media, which was purified by CM ion-exchange chromatography . Approximately 21 mg of the purified enzyme was obtained per liter of culture . N-terminal amino acid analysis indicated that the signal peptide was removed from the secreted enzyme . The Penicillium 1,2-alpha-mannosidase expressed in A . oryzae did not show any notable difference from the enzyme from P . citrinum in such properties as M(r), specific activity, CD spectra, or kinetic parameters . Man7GlcNAc2 accumulated temporarily during the degradation of Man9GlcNAc2 to Man5GlcNAc2 by fungal 1,2-alpha-mannosidase.

Thromb Haemost, 1998 Mar, 79(3), 631 - 4
Atrial natriuretic peptide inhibits the expression of tissue factor and plasminogen activator inhibitor 1 induced by angiotensin II in cultured rat aortic endothelial cells; Yoshizumi M et al.; The pharmacological characteristics of atrial natriuretic peptide (ANP), such as natriuresis, vasodilation, or suppression of smooth muscle cell proliferation, are well investigated . However, this is the first study to report its role on blood coagulation and fibrinolysis mediated by vascular endothelial cells . In this study, the effects of ANP on the enhanced expression of tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1) by angiotensin II (Ang II) in cultured rat aortic endothelial cells (RAECs) were examined . The expressions of TF and PAI-1 mRNA were detected by northern blotting methods . The activities of TF on the surface of RAECs and PAI-1 in the culture media were measured by chromogenic assay . ANP suppressed mRNA expressions of TF and PAI-1 induced by Ang II in a concentration-dependent manner . This suppression was accompanied by the decreased activities of TF and PAI-1.

Biochem Mol Biol Int, 1998 Feb, 44(2), 347 - 62
Transfer of cholesterol from macrophages to lymphocytes in culture; de Bittencourt Junior PI et al.; A major feature of macrophage metabolism is its capacity to produce and export cholesterol . Several reports have shown that the manipulation of lymphocyte cholesterol content elicits important changes in lymphocyte proliferation . These findings lead to an inquiry as to whether macrophage-derived cholesterol released into the lymphocyte surroundings may be transferred to the latter thus affecting lymphocyte function . In this study, cholesterol transfer from macrophages to lymphocytes was examined in vitro using rat cells in culture . The findings indicate that there may be a significant transfer of cholesterol from {4-14C}cholesterol labeled resident peritoneal macrophages to mesenteric lymph node resting lymphocytes (up to 173.9 +/- 2.7 pmol/10(7) lymphocytes/10(7) macrophages when co-cultivated for 48 h), in a lipoprotein-dependent manner . This represents the mass transfer of ca . 17 nmoles of cholesterol molecules per 10(7) lymphocytes from 10(7) macrophages (calculated on the basis of specific radioactivity incorporated into macrophages after the pre-labelling period), which suggests that macrophages are capable of replacing the whole lymphocyte cholesterol pool every 21 h . Moreover, an 111%-increase in the total cholesterol content of lymphocytes was found after co-cultivation with macrophages for 48 h . When compared to peritoneal cells, monocytes/macrophages obtained from circulating blood leukocytes presented a much higher cholesterol transfer capacity to lymphocytes (3.06 +/- 0.10 nmol/10(7) lymphocytes/10(7) macrophages co-cultivated for 24 h) . Interestingly, inflammatory macrophages dramatically reduced their cholesterol transfer ability (by up to 91%, as compared to resident macrophages) . Cholesterol transfer may involve a humoral influence, since it is not only observed when cells are co-cultivated in a single-well chamber system (cells in direct contact), but also in a two-compartment system (where cells can communicate but not by direct contact) . Co-cultivation with macrophages decreased the basal incorporation of {2-14C}thymidine into lymphocyte DNA and the addition of cholesterol to lymphocyte culture media also impaired the lymphocyte proliferative response to the mitogens concanavalin A (Con A) and bacterial lipopolysaccharide (LPS) . The above results suggest that macrophages may transfer cholesterol to lymphocytes (from both lymph nodes and blood), thus regulating lymphocyte function by raising the intracellular cholesterol content and suppressing lymphocyte proliferative activity . If this is so, a modulatory role for the transfer of cholesterol in both physiological (e.g . immune response) and pathological conditions (e.g . atherosclerosis) may be postulated . This hypothesis is currently under investigation in our laboratory.

Am J Physiol, 1998 Mar, 274(3 Pt 2), F463 - 72
Potential autocrine and paracrine mechanisms of recovery from mechanical injury of renal tubular epithelial cells; Anderson RJ et al.; The present studies were done to clarify potential pathways of the nephrogenic repair process . Media removed from mechanically injured vascular smooth muscle cells and LLC-PK1 renal tubular epithelial cells significantly stimulated {3H}thymidine uptake and cell number in quiescent LLC-PK1 cells, demonstrating the existence of potential autocrine and paracrine pathways of nephrogenic repair . The effect of mechanical injury resulting in release of one or more growth factors into culture media was also found in the opossum kidney OK renal tubular cell line . The nonspecific peptide growth factor antagonist suramin inhibited the effect of media from injured LLC-PK1 cells to stimulate {3H}thymidine uptake in quiescent LLC-PK1 cells . Exposure of quiescent LLC-PK1 cells to six growth factors, including acidic and basic fibroblastic growth factors (aFGF and bFGF), platelet-derived growth factors AA and BB (PDGF-AA and PDGF-BB), endothelin-2, and hepatocyte growth factor, reproduced the biological responses seen when quiescent LLC-PK1 cells were exposed to media from injured cells . Immunoblotting and enzyme-linked immunosorbent assay experiments demonstrated the presence of aFGF, bFGF, and PDGF-BB but not other candidate growth factors in the media from injured LLC-PK1 cells . A neutralizing antibody directed against bFGF attenuated the effect of media from injured cells to stimulate {3H}thymidine uptake in serum-starved LLC-PK1 cells . These results demonstrate that mechanical injury to renal tubular epithelial cells results in release of aFGF, bFGF, and PDGF-BB into the media and suggests that bFGF may be involved in an autocrine fashion to promote recovery from injury.

J Chromatogr A, 1998 Feb 6, 795(2), 277 - 87
Bovine whey fractionation based on cation-exchange chromatography; Hahn R et al.; Bovine whey proteins have potential applications in veterinary medicine, food industry and as supplements for cell culture media . A fractionation scheme for the economically interesting proteins, such as IgG, lactoferrin and lactoperoxidase, based on cation exchangers was the goal of our investigations . A chromatographic process was developed where alpha-lactalbumin passes through the column and separation of the desired proteins is achieved . Four different cation-exchange media (S-HyperD-F, S-Sepharose FF, Fractogel EMD SO3- 650 (S) and Macro-Prep High S Support) were compared in regard to their dynamic binding capacity for IgG and their different elution behaviours when sequential step gradients with NaCl buffers were applied . Peak fractions were analyzed by size-exclusion chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis . Lactoperoxidase activity was monitored by the oxidation of o-phenylenediamine . In order to explain the different resolution behaviours, isocratic runs with pure standards of whey proteins were performed . The k' values were calculated and plotted against salt concentration . Fractogel EMD had the highest binding capacity for IgG, 3.7 mg/ml gel at a linear flow-rate of 100 cm/h, but the resolution was low compared to that with the other three media . S-Hyper D and S-Sepharose FF showed lower capacities, 3.3 and 3.2 mg/ml gel, respectively, but exhibited better protein resolution . These effects could be partially explained by the k' versus salt concentration plots . The binding capacity of Macro-Prep S was considerably lower compared to that of the other resins investigated because its selectivity for whey proteins was completely different . S-Sepharose FF and S-Hyper D combine relatively high dynamic capacity for IgG and good resolution . Compared to studies with standard proteins, such as 100 mg/ml bovine serum albumin for S-Hyper D, their binding capacities were very low . Even after removal of low-molecular-mass compounds, the capacity could not be improved significantly . The running conditions (low pH) were responsible for the low protein binding capacity, since low-molecular-mass compounds in the feed do not compete with the adsorption of whey protein . The dynamic capacity did not decrease to a large extent within the range of flow-rates (100-600 cm/h) investigated . The dynamic capacity of HyperD and Fractogel was at least five times higher when pure bovine IgG was used for determination . In conclusion, S-Sepharose FF, S-Hyper D-F and Fractogel EMD SO3- 650 (S) are considered as successful candidates for the large-scale purification of bovine whey proteins.

J Allergy Clin Immunol, 1998 Mar, 101(3), 363 - 70
Characterization of recombinant Mercurialis annua major allergen Mer a 1 (profilin); Vallverdu A et al.; BACKGROUND: Two major allergens (Mer a 1A and Mer a 1B), tentatively identified as profilin, have been described in the euphorbiacea, Mercurialis annua . OBJECTIVES: We sought to clone and characterize these major allergens from M . annua pollen and to obtain the immunologically active and soluble recombinant allergen, which could then be used for diagnostic procedures and therapy . METHODS: Isolation of cDNA clones was performed by polymerase chain reaction amplification with degenerate primers . Expression in Escherichia coli BL21 (DE3) was carried out with a vector based in the T7 expression system, and the recombinant allergen was isolated by affinity chromatography on poly-(L-proline)-Sepharose . Electrophoretic (sodium dodecylsulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and 2-dimensional polyacrylamide gel electrophoresis) and immunochemical methods (Western blot and ELISA) were used for the characterization of the recombinant allergen . RESULTS: Two cDNA inserts coding for M . annua pollen profilin (Mer a 1) were cloned and sequenced . Full-length Mer a 1 cDNA was expressed in E . coli as nonfusion protein . The final yield of recombinant Mer a 1 from the culture media after a single purification step on poly-(L-proline)-Sepharose was as much as 5 mg per liter . The reactivity of recombinant Mer a 1 with IgE antibodies present in sera from patients allergic to M . annua, Olea europaea, and Ricinus communis pollens was comparable to that of the natural counterparts, but latex profilin had no cross-reactivity with M . annua profilin . Recombinant Mer a 1 was shown to share B-epitopes with sunflower profilin . CONCLUSION: This approach is suitable for the production of defined and purified recombinant allergens, which could allow more detailed immunologic characterization of these proteins and the development of much more accurate diagnostic measures and specific anti-allergic treatments.

Biol Chem, 1998 Feb, 379(2), 219 - 24
Sorting of non-glycosylated human procathepsin S in mammalian cells; Nissler K et al.; Cathepsin S, a lysosomal cysteine protease, is synthesized as inactive precursor . It is activated in the lysosomes by a proteolytic cleavage of the propeptide . HEK 293-cells which do not express cathepsin S were transfected with cDNA of either wild type human procathepsin S or a mutant procathepsin S in which Asn of the only glycosylation site in the proregion was replaced by Gln . The cells expressed glycosylated and non-glycosylated procathepsin S, respectively . Large amounts of the precursors were secreted into the culture media by both transfectants . Secreted wild type procathepsin S contained Man-6-phosphate in the oligosaccharide chain . Wild type procathepsin S was activated in the cells but no maturation occurred in the culture media . In vitro processing of glycosylated as well as of non-glycosylated procathepsin S gave fully active enzymes thus indicating that the oligosaccharide chain was not necessary for proper folding . A reuptake of the glycosylated and non-glycosylated procathepsin S by HEK 293-cells could be observed . Small amounts of mature cathepsin S were detected in the lysosomes of the mutant transfectants . Subcellular fractionation showed non-glycosylated procathepsin S in the membrane fraction . Non-glycosylated procathepsin S was bound to the plasma membrane at 2 degrees C, suggesting an additional sorting motif in the cathepsin S molecule besides the Man-6-phosphate residue.

Neuroscience, 1998 May, 84(1), 7 - 10
Nuclear estrogen receptor-independent neuroprotection by estratrienes: a novel interaction with glutathione; Green PS et al.; Post-menopausal estrogen replacement therapy is associated with a reduction in the risk of Alzheimer's disease and has been reported to improve cognitive functioning in several small clinical trials . The present study evaluates the dependence of estrogenic neuroprotection on the presence of estrogen receptors using the murine neuronal cell line, HT-22, exposed to the neurotoxic beta-amyloid peptide . These cells lack functional estrogen receptors . The amyloid peptide killed 50-60% of these cells and concurrent treatment with either of three estratrienes, beta-estradiol, alpha-estradiol, or estratrien-3-ol, resulted in a dose-dependent protection . The potency of this estrogen neuroprotection was dependent on the presence of glutathione in the culture media . The presence of reduced glutathione in the media increases the neuroprotective potency of estrogens by an average of 400-fold . These results demonstrate that a nuclear estrogen receptor is not necessary for the neuroprotective actions of estrogens; however, the presence of an appropriate antioxidant in the extracellular milieu is needed for estratriene neuroprotection at physiologically and pharmacologically relevant doses . These data suggest the possibility of combined estrogen-antioxidant therapy for neurodegenerative diseases such as Alzheimer's disease.

Biochem Pharmacol, 1998 Mar 1, 55(5), 697 - 701
In vitro enzymatic processing of radiolabelled big ET-1 in human kidney; Russell FD et al.; We have investigated enzymatic processing of big ET-1 in sections of human renal cortex by examining selected binding characteristics of the radiolabelled precursor and cleaved peptide . Sections of histologically normal human kidney obtained from patients undergoing nephrectomy for hypernephroma (50-74 years, N = 10, male or female) were incubated with 0.1 nM {125I}-ET-1, {125I}-Tyr13 big ET-1 or {125I}-Tyr31 big ET-1 in culture media at 37 degrees to facilitate enzymatic activity . Specific binding measured from sections incubated with {125I}-Try13 big ET-1 (which would yield {125I}-ET-1 on enzymatic cleavage) was 39.7 +/- 2.5% . This was significantly reduced to 19.0 +/- 2.0% following co-incubation with 10 microM thiorphan, an inhibitor of neutral endopeptidase (NEP) but not the putative endothelin converting enzymes (ECE) . No further reduction in specific binding was obtained with 100 microM thiorphan, indicating that this is a maximal effect . However phosphoramidon (100 microM), an inhibitor of ECE and NEP, almost abolished specific binding, indicating that both NEP and ECE cleave big ET-1 in the kidney . No specific binding was detected when sections were labelled with {125I}-Tyr31 big ET-1 (which would be expected to yield {125I} labelled C-terminal fragment) . Binding of the product of processed {125I}-Tyr13 big ET-1 was inhibited mainly by the ET(B) selective antagonist (BQ788 = 75.1 +/- 2.1% inhibition; FR139317 = 9.7 +/- 7.3% inhibition), consistent with the predominance of this subtype in human kidney . We conclude that big ET-1 is processed by NEP and ECE in human kidney and that the cleaved product binds predominantly to the ET(B) receptor subtype . ECE may be a therapeutic target in the attenuation of renal diseases in which ET-1 has been implicated.

Clin Exp Metastasis, 1998 Feb, 16(2), 193 - 203
Effect of MRC-5 fibroblast conditioned medium on breast cancer cell motility and invasion in vitro; Heylen N et al.; We used Transwell chambers to study separately cellular motility and invasion . In order to assess the cellular motility, polycarbonate microporous filters were coated with extracellular matrix proteins which adsorbed on the filters without clogging the pores . To investigate the invasive behavior of tumor cells, filters were covered with a layer of Matrigel which clogged the pores . The motility and the invasion of breast cancer cell lines (MDA-MB-231, MCF-7/6 and MCF-7/AZ cells) were assessed quantitatively in different culture media: defined (serum-free), serum-containing and normal human fibroblast MRC-5 conditioned media . In serum-containing medium, tumor cells migrated and invaded through the coated and covered filters . Their motility and invasion potentials were considerably lower in defined medium, whereas medium conditioned by MRC-5 fibroblasts stimulated both motility and invasion but not growth . The MRC-5 conditioned medium induced also the spreading of clusters of MCF-7/6 cells grown on Matrigel-coated plates.

Glycoconj J, 1997 Nov, 14(7), 809 - 19
Secretion of alpha1,3-galactosyltransferase by cultured cells and presence of enzyme in animal sera; Cho SK et al.; Glycosyltransferases are normally synthesized as membrane-anchored proteins . However, we recently found that the murine enzyme UDP-Gal:Gal beta1 -->4GLcNAc (Gal to Gal) alpha1,3 galactosyltransferase (alpha1,3GT) is secreted in a soluble form into media by mouse teratocarcinoma F9 cells (Cho SK, Yeh J-C, Cho M, Cummings RD (1996) J Biol Chem 271: 3238-46) . To study the biosynthesis of this enzyme and whether secretion of the soluble enzyme is a general phenomenon, a solid-phase assay was developed for the alpha1,3GT activity . A recombinant and soluble form of the murine alpha1,3GT was produced in H293 cells (H293-alpha1,3GT) to aid in optimizing the assay . Desialylated orosomucoid was used as an immobilized acceptor in coated microtiter plates . The formation of product was detected by a biotinylated human-derived anti-alpha-Gal IgG and streptavidin conjugated to either alkaline phosphatase or the recombinant bioluminescent protein aequorin . Enzyme activity was dependent on the concentrations of asialoorosomucoid, UDP-Gal, alpha1,3GT and the time of incubation . The assay was also useful in monitoring alpha1,3GT activity during enzyme enrichment procedures . Using this assay, we found that alpha1,3GT activity was present in both cell extracts and culture media of several mammalian cell lines . Enzyme activity was also present in the sera from several mammals, but activity was absent in the sera from either humans or baboons . Our results demonstrate the development of a novel assay for the alpha1,3GT and provide evidence that secretion of the enzyme is a common biological phenomenon.

Anal Chem, 1998 Mar 1, 70(5), 923 - 30
Investigation of protein patterns in mammalian cells and culture supernatants by matrix-assisted laser desorption/ionization mass spectrometry; van Adrichem JH et al.; The direct protein profiling of mammalian cells and bacteria has a growing influence in biotechnology as a high information bearing method for characterization of cells and cell states . Monitoring of proteins excreted in culture media not only serves to produce data on product yield and quality but provides important information on cell viability and nutrient supply that forms the basis for future process and expression optimization . Fast and simple MALDI mass spectrometry approaches were developed to efficiently characterize such complex biological systems . Several mammalian cell lines including CHO DXB11, CHOSSF3, and hybridomas were investigated; the lysis process, the sample pretreatment, and the matrix preparation were optimized for MALDI conditions . Initial experiments to observe the success of protein translation in gene expression experiments were performed . Using MALDI-compatible detergents, it was possible to extend the mass range detectable by MALDI mass spectrometry from the current range of 16,000 to 75,000 Da . In this mass range, the data are complementary (offering a better mass accuracy) to those obtained by SDS-PAGE electrophoresis experiments . These new methods were used to monitor a large-scale cultivation of hybridoma cells expressing an antibody of the IgG type . The increase in whole antibody and antibody light-chain protein, 8650 Da, and the decrease of insulin were followed during the monitoring period . Quantitative measurements of the IgG level during the cultivation compared favorably with those obtained by affinity HPLC.

Gac Med Mex, 1997, 133 Suppl 1, 105 - 10
{Importance of molecular methodology in diagnosis}; Escobar-Gutierrez A et al.; The National Institute for Epidemiological Diagnosis and Reference (INDRE) partially supports epidemiological surveillance programs through the identification of most infectious agents prevalent in the country . The success of a program for the control or eradication of a particular infectious disease mainly depends on the opportune and accurate identification of the corresponding etiologic agent . For laboratory diagnosis at INDRE, both conventional methodology using direct or microscopic examinations of specimens or growth in culture media followed by physiological or immunological characterization of the isolate, as well as new techniques based in biochemical, immunochemical and molecular biology procedures are carried out . Antigens can be detected in clinical samples by ELISAs with polyclonal or monoclonal antibodies . Specific nucleic acids can be extracted, identified and typed with techniques like electrophoresis, hybridization with genomic probes, polymerase chain reaction or fragment restriction length polymorphism . Recombinant molecules or highly purified antigens are being obtained and used for the determination of antibodies, mainly with indirect ELISA, IgM capture-ELISA and Western Blot . The better performance, specificity and sensitivity of these laboratory procedures, provide faster results, with equal or greater accuracy than traditional ones, at lower cost.

Transpl Int, 1998, 11(1), 58 - 62
The effect of organ preservation solutions on kidney tubular and endothelial cells; Moutabarrik A et al.; Organ preservation solutions have primarily been tested in whole organ animal models . In the current study, we have examined the effect of commonly used organ preservation solutions on both kidney tubular and endothelial cells . Primary human endothelial and kidney tubular cells were incubated at 4 degrees C in the following solutions: 0.9% saline (NS), EuroCollins (EC) . University of Wisconsin (UW), or Hank's balanced salts with 5% polyethylene glycol (PEG) . Cell viability was assessed by colorometric measurement of mitochondrial reduction of 3 (4,5-dimethylthiazol-2-yl)-2-,5-diphenyltetrazolium bromide (MTT) to purple 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan . After hypothermic storage, cells were incubated at 37 degrees C in media with MTT, and the amount of reduced formazan present was quantified . Endothelial cells preserved in PEG displayed the best viability (P < 0.05) . UW provided better cellular viability than EC or NS (P < 0.05) . Control endothelial cells preserved in culture media at 37 degrees C displayed the highest absorbance values (P < 0.01) . For kidney tubular cells, UW and PEG provided the best cellular protection (P < 0.05) . Control kidney tubular cells cultured in complete media at 37 degrees C displayed the highest absorbance values (P < 0.01) . Although the model presented here was not part of a truly morphological study, it may be more reliable for the rapid assessment of preservation-induced cell injury than models presented in previous morphological studies and may help in the development of improved preservation techniques.

Eur J Clin Invest, 1998 Jan, 28(1), 48 - 58
Immunoreactive osteocalcin forms in conditioned media from human osteoblast culture and in sera from healthy adult control subjects and patients with bone pathologies; Diaz Diego EM et al.; BACKGROUND: The aim of this work was to study the immunoreactive forms of bone Gla protein (BGP) present in conditioned media of human osteoblast cultures (BGP released from osteoblast) and in the sera of healthy adult control subjects and patients with bone pathologies (chronic renal failure on haemodialysis, Paget's disease of bone and post-menopausal osteoporosis) . METHODS: The technical procedure used was a combination of high-performance liquid chromatography (HPLC) and different BGP assays with several specificities to analyse BGP levels in the different HPLC fractions . Aliquots of conditioned media or sera were purified through a Sephadex G-50m column and by HPLC (C4 reverse-phase column) in a 25-40% acetonitrile gradient . Two-minute fractions were collected and divided into three aliquots in order to determine osteocalcin content using three different assays: (a) ELSA-OST-NAT IRMA, which only detects intact osteocalcin; (b) ELSA-OSTEO IRMA, which detects intact osteocalcin and N-terminal fragments; and (c) OSCA Test RIA, which detects intact osteocalcin, C-terminal and other fragments . RESULTS: We found different immunoreactive forms of osteocalcin in the culture medium of human osteoblasts and in sera from control subjects and patients for the bone pathologies studied . We did not find great qualitative differences between the immunoreactive osteocalcin profile found in the culture medium from human osteoblasts and the sera from healthy control subjects . However, the different bone pathologies show different characteristic patterns of immunoreactive forms of osteocalcin . CONCLUSIONS: An interesting finding has been the detection, both in sera and in osteoblast culture media, of several immunoreactive forms of intact osteocalcin that eluted from HPLC at different acetonitrile percentages, and therefore correspond to different molecular forms.

J Adolesc Health, 1998 Mar, 22(3), 205 - 8
Diagnosis of Trichomonas vaginalis in adolescent females: InPouch TV culture versus wet-mount microscopy; Ohlemeyer CL et al.; PURPOSE: This study compared the InPouch TV culture to wet-mount, Diamond's culture medium, and Papanicolaou (Pap) smear for the diagnosis of trichomonas infection in sexually active adolescents . METHODS: A total of 467 subjects were recruited among 12-18-year-old girls who received pelvic examinations at two urban adolescent clinics . All girls were tested by wet-mount and InPouch TV . In addition 339 of 467 had cultures in Diamond's medium and 366 of 467 had Pap smears . Specimens were collected for InPouch TV and Diamond's cultures and read at 24-48 h and 5 days, and in the case of Diamond's cultures, also at 7 days . In a subset of subjects (268 of 467) who had all four tests done, sensitivities and specificities were calculated using Diamond's culture as the "gold standard." RESULTS: In the 467 subjects, 73 (15.6%) tested positive for trichomonas by at least one method . In the subset with all four tests done, sensitivities of the wet-mount and InPouch TV were 36% and 81%, respectively; while that of the Pap smear was 56% . The culture media were equally efficient in identifying Trichomonas vaginalis . There were no differences found between subjects with or without trichomonas infections in gynecological symptoms, previous history of sexually transmitted diseases, or use of a condom at last intercourse . CONCLUSIONS: InPouch TV culture is a good diagnostic method for T . vaginalis because of its long shelf-life, relatively low expense, and high sensitivity (over twice as sensitive as wet-mount).

Hepatology, 1998 Mar, 27(3), 720 - 6
Fibrogenic effect of oxidative stress on rat hepatic stellate cells; Svegliati Baroni G et al.; Oxidative stress is associated with liver fibrosis and with hepatic stellate cell (HSC) activation in vivo . However, it remains controversial whether oxidative stress contributes to HSC activation either directly or through a paracrine stimulation by damaged hepatocytes . A medium containing products released from cells undergoing oxidative stress was obtained after incubation of hepatocytes with (HCM/Fe) or without (HCM) 0.1 mmol/L ferric nitrilotriacetate complex (FeNTA) . Exposure of HSC to HCM/Fe for 24 hours significantly increased the number of proliferating HSC compared with HCM and to controls at all dilutions tested . The simultaneous coincubation of HSC with HCM/Fe and desferrioxamine (50 micromol/L) did not reduce the observed increase in cell proliferation, thus excluding a role for eventually contaminating iron in HCM/Fe . HCM/Fe induced also a significant increase in collagen type I accumulation in HSC culture media . To study the cellular mechanism underlying HCM/Fe effects, we evaluated the activity of the Na+/H+ exchanger, which plays a role in regulating HSC proliferation . The incubation of HSC for 24 hours with HCM/Fe significantly increased baseline intracellular pH (pHi) and Na+/H+ exchanger activity, indicating a plausible role of this antiport in mediating cell response . In conclusion, hepatocytes undergoing oxidative stress release factors which are fibrogenic for HSC, thereby, confirming what has been only hypothesized in vivo . In addition, HSC proliferation is associated with changes in the Na+/H+ exchanger activity, thus providing a useful target for the evaluation of inhibitors of this pathway for the treatment of hepatic fibrosis.

Am J Obstet Gynecol, 1998 Feb, 178(2), 255 - 8
The induction of cyclooxygenase-2 by interleukin-4 in human umbilical vein endothelial cells: a possible role in regulation of fetal vascular tone; Lantz ME et al.; OBJECTIVE: We sought to determine a possible role for interleukin-4 in the control of umbilical cord blood flow by evaluating its effect on cyclooxygenase-2 production of a vasoactive prostaglandin . STUDY DESIGN: Human umbilical vein endothelial cells in culture were incubated for 16 hours in media containing interleukin-4 in concentrations from 5 to 100 ng/ml . Prostaglandin E2 concentrations in the culture media were measured using a monoclonal enzyme-immunoassay . Concentrations of cyclooxygenase-1 and cyclooxygenase-2 were determined by Western blot analysis on cell homogenates . Statistical comparisons between prostaglandin E2, cyclooxygenase-1, and cyclooxygenase-2 concentrations for each interleukin-4 concentration were performed using a one way analysis of variance . RESULTS: Incubation of human umbilical vein endothelial cells in media containing interleukin-4 resulted in a significant increase in both prostaglandin E2 and cyclooxygenase-2 for interleukin-4 concentrations greater than 50 ng/ml (p < 0.05) . Cyclooxygenase-1 levels were not affected . CONCLUSIONS: We suggest that interleukin-4 may have a role in the regulation of umbilical blood flow mediated through the induction of cyclooxygenase-2.

Microvasc Res, 1998 Jan, 55(1), 29 - 42
Vascular endothelial growth factor increases release of gelatinase A and decreases release of tissue inhibitor of metalloproteinases by microvascular endothelial cells in vitro; Lamoreaux WJ et al.; The present study was designed to determine the influences of vascular endothelial growth factor (VEGF) on cell proliferation and the release of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) from human dermal microvascular endothelial cells . Treatment of cultures with 10 ng/ml or more of VEGF significantly increased cell proliferation . The effect of VEGF treatment on the levels of specific MMPs and TIMPs in the media was subsequently examined in cultures that were treated with 10 ng/ml VEGF . Zymography and Western blot analyses demonstrated that gelatinase A levels in the media were increased by VEGF treatment . Collagenase was detected by Western blots in both VEGF-treated and untreated culture media, but the levels were not significantly increased by the VEGF treatment . An ELISA assay confirmed that VEGF treatment significantly increased gelatinase A levels but did not significantly increase collagenase levels . Western blot and ELISA data showed that VEGF treatment significantly decreased TIMP-1 and TIMP-2 levels compared to untreated cultures . The data suggest that VEGF may modulate endothelial cell-derived MMP activity by: (1) increasing the abundance of gelatinase A; (2) disinhibiting gelatinase A by decreasing the abundance of TIMP-2; and (3) disinhibiting preexisting collagenase by reducing levels of TIMP-1 . These actions could contribute to the ability of VEGF to promote endothelial cell invasion of new territory .

Br J Ophthalmol, 1997 Dec, 81(12), 1093 - 8
Endotoxins modulate the autocrine function of organ cultured donor corneas and increase the incidence of endothelial cell death; Sobottka Ventura AC et al.; BACKGROUND/AIMS: Bacterial endotoxin is a potent inflammatory stimulator, the local and systemic responses thereby elicited being mediated via the release of cytokines from diverse cell types . Under physiological conditions, the corneal endothelium is protected from these toxins by the epithelial and vascular barriers, but in organ culture these safeguards are no longer operative, and such substances will therefore have ready access to this cell layer . The consequences of such exposure may take the form of overt damage to the endothelium and/or a more discreet influence on the cornea's immunological status, the effects of which may be realised only after transplantation, by its poor performance . The media bathing organ cultured donor corneas were monitored for the presence of various cytokine mediators of the inflammatory response before and after incubation with endotoxin, and these data compared with those pertaining to endothelial cell morphology and numerical density . METHODS: Six pairs of fellow donor corneas were cultured for an initial equilibration period of 10 days and then transferred to fresh medium; thereafter, one of each pair was incubated in the absence, and the other in the presence, of endotoxin (50 micrograms/ml = 25,000 units/ml), and culturing continued for a further 10 days . Samples of medium were withdrawn at regular intervals throughout the 20 days and screened for the cytokines IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, GM-CSF, AND TNF by ELISA; endothelial cell morphology and area density were assessed on days 0, 10, and 20 . RESULTS: Spiking of organ culture media with endotoxin led to a substantial increase in the level of IL-8, and a smaller one in that of IL-6, but none of the other cytokines were detected . In five of the six stimulated corneas, these changes coincided with an increased incidence of endothelial cell loss, compared with that incurred by the fellow control, and the surviving population also evinced signs of degeneration not seen in the latter . CONCLUSION: Endotoxin induced increases in the levels of IL-6 and IL-8 appear to be correlated with endothelial cell loss . Since no adverse effects of this toxin on long term cultured monolayers of human corneal endothelial cells have been previously observed, the damage incurred in corneal organ culture may well be attributable to the influence of cytokines produced by other corneal cells or a non-intrinsic (passenger) cell population, such as macrophages, Langerhans cells or lymphocytes present under these latter conditions.

Fertil Steril, 1998 Feb, 69(2), 329 - 34
Improved clinical outcomes for in vitro fertilization with delay of embryo transfer from 48 to 72 hours after oocyte retrieval: use of glucose- and phosphate-free media; Carrillo AJ et al.; OBJECTIVE: To evaluate clinical outcomes of day 2 versus day 3 ET using a culture media with no glucose or phosphate . DESIGN: Retrospective clinical study . SETTING: Hospital-based fertility clinic . PATIENT(S): One hundred seventy-six IVF-ET patients undergoing controlled ovarian supraovulation . INTERVENTION(S): IVF and delaying the ET by 1 day . MAIN OUTCOME MEASURE(S): Number of blastomeres per embryo, implantation and pregnancy rates . RESULT(S): Delaying the ET from day 2 to day 3 after oocyte retrieval significantly increased implantation rates (13% versus 24%) and ongoing/delivered pregnancy rates per retrieval (26% versus 44%) . Day 3 embryos with > or = 8 blastomeres resulted in a significantly higher pregnancy rate (53%) than day 3 embryos with < 8 cells (23%) and day 2 embryos with > or = 4 cells (31%) or < 4 cells (11%) . CONCLUSION(S): Day 3 ET was associated with a significant increase in implantation and pregnancy rates . Delaying the ET until day 3 may permit the selection of more viable embryos than on day 2 . The absence of glucose and phosphate from the culture media is compatible with good IVF outcomes.

J Assist Reprod Genet, 1998 Jan, 15(1), 50 - 3
Effectiveness of Menuzo's B2 medium with buffalo rat liver cells for development of in vitro matured/in vitro fertilized bovine oocytes; Krisher RL et al.; PURPOSE: The effectiveness of two culture media on the development of bovine embryos in a buffalo rat liver (BRL) coculture system was investigated . METHODS: Bovine oocytes were matured and fertilized in vitro, then cocultured, 25 per well, for 7 days in 500 microliters of modified M199 or modified Menuzo's B2 medium over a BRL cell monolayer at 39 degrees C in an atmosphere of 5% CO2 in air . Medium 199 was modified by the addition of 10% of (v/v) fetal bovine serum (FBS), 9 mg/ml bovine serum albumin, 2 mM glycine, 1 mM alanine, and 0.1 mM nonessential amino acids (NEAA) . Menuzo's B2 medium was modified by the addition of 10% (v/v) FBS, 1 mM alanine, and 0.1 mM NEAA . RESULTS: Modified Menuzo's B2 medium improved embryo development to the morula or blastocyst stage compared to modified M199 (121/353, 34.3%, versus 99/362, 27.3%, P < 0.05) . CONCLUSIONS: These results suggest that Menuzo's B2 medium with modifications in a BRL coculture system can provide a significant benefit for culture of early bovine embryos over the traditional use of medium 199.

Mol Reprod Dev, 1998 Mar, 49(3), 333 - 41
Serotonin inhibition of steroid-induced meiotic maturation in the teleost Fundulus heteroclitus: role of cyclic AMP and protein kinases; Cerda J et al.; The transduction of the serotonin (5-HT) signal in Fundulus heteroclitus ovarian follicles leading to the inhibition of oocyte meiosis reinitiation (oocyte maturation) in vitro induced by the naturally occurring maturation-inducing steroid 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17,20 beta P) was investigated . Steroid-induced oocyte maturation was inhibited by 5-HT in a dose-dependent manner; maximum inhibition (90%) was observed with 10(-4) M 5-HT . Groups of follicle-enclosed oocytes were cultured in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and treated with increasing doses of 5-HT . Serotonin was found to slightly increase the levels of follicular 3',5'-cyclic adenosine monophosphate (cAMP) in a dose-dependent manner; 10(-4) M 5-HT induced approximately a 3-fold increase in cAMP with respect to the controls . The changes in cAMP were then evaluated in follicles treated with 17,20 beta P in IBMX-free culture media in the presence or absence of 10(-4) M 5-HT . The exposure of follicles to 17,20 beta P alone produced a small and transient reduction in cAMP (40%) within 1-3 hr of steroid stimulation, and these early changes in cAMP appeared associated with a high incidence of germinal vesicle breakdown (80% GVBD) by 24 hr of incubation . Under these conditions, treatment of follicles with 5-HT also increased significantly the production of cAMP, and when 5-HT was combined with 17,20 beta P, the steroid-mediated reduction in cAMP was prevented and the levels of GVBD inhibited by 95% . Meiosis also was reinitiated with either the protein kinase A (PKA) inhibitor H8 or the protein kinase C (PKC) activator PMA, and the 5-HT inhibitory action on GVBD was found to be 100-fold reduced or completely ineffective, respectively . Preincubation of follicles with the PKC inhibitor GF109203x abolished PMA-induced GVBD in a dose-dependent manner, whereas this inhibitor had no effect on 17,20 beta P-triggered meiotic maturation, indicating that activation of PKC is apparently sufficient but not necessary to reinitiate meiosis . Taken together, these findings suggest that 5-HT may inhibit 17,20 beta P-induced meiotic reinitiation through the activation of a cAMP-PKA transduction pathway and that PKC possibly induces oocyte maturation by a different pathway than the steroid and thus is not affected by 5-HT.

Endocrinology, 1998 Mar, 139(3), 1249 - 57
Characterization and hormonal regulation of a rat ovarian insulin-like growth factor binding protein-5 endopeptidase: an FSH-inducible granulosa cell-derived metalloprotease; Resnick CE et al.; Previous studies established the existence of an FSH-inducible rat granulosa cell-derived insulin-like growth factor binding protein (IGFBP)-5 endopeptidase . It was the objective of this communication to characterize this activity in some detail . Exposure of {125I}rhIGFBP-5 substrate to media conditioned by FSH-treated granulosa cells (a cell-free assay) produced two rhIGFBP-5 cleavage products (estimated size 19.5 and 17.5 kDa) . The acquisition of IGFBP-5 endopeptidase activity in culture proved FSH (or PMSG) to be dose and time dependent . The addition of oFSH or rhFSH to the cell-free assay in turn, proved without effect on IGFBP-5 endopeptidase activity, thereby arguing against the possibility of an FSH receptor-independent phenomenon or of contaminating pituitary-derived contribution . The ability of FSH to induce IGFBP-5 endopeptidase activity proved relatively specific in that other granulosa cell agonists such as activin-A, IGF-I, GnRH, interleukin-1beta, TNF alpha, TGF beta1, EGF, or endothelin-1 failed to do so . However, the concurrent provision of GnRH, TNF alpha, EGF, or endothelin-1 proved inhibitory to the IGFBP-5 endopeptidase-inducing property of FSH . Activin-A and TGF beta1 in turn further stimulated the FSH effect . Sensitivity to EDTA, 1,10 phenanthroline, and high concentrations (> or = 0.1 mM) of Zn2+ suggested a Zn2+ metalloprotease . Insensitivity to TIMP-1 and TIMP-2 argued against a matrix metalloprotease (MMP) . Relative insensitivity to PMSF, AMPSF, aprotinin, TPCK, and benzamidine argued against the possibility of a serine protease . Insensitivity to pepstatin A and E64 argued against aspartic and cysteine proteases, respectively . Insensitivity to plasminogen activator inhibitor-1 (PAI-1) and the presumed lack of free plasminogen in serum-free culture media argued against plasmin . Proteolysis was completely inhibited over the acid pH range but proceeded unencumbered at neutral and basic pH . Competition studies using unlabeled IGFBPs (1-6) as well as cell-free proteolysis assays of {125I}-labeled IGFBP-1, 2, 3, and 6 suggested a significant level of specificity for the FSH-induced/IGFBP-5-directed endopeptidase . Centricon-mediated fractionation of FSH-conditioned media revealed the IGFBP-5 endopeptidase activity in the fraction representing proteins of molecular weight >100K . Taken together, these observations document a secreted, granulosa cell-derived, high molecular weight, FSH-inducible, IGFBP-5-selective, neutral/basic pH-favoring, non-MMP Zn2+ metalloprotease.

J Investig Allergol Clin Immunol, 1997 Nov-Dec, 7(6), 611 - 8
Weekly variation of fungal colonies in the atmosphere of Palencia (Spain) throughout the year 1992; Herrero B; We have carried out a qualitative and quantitative study of the fungal colonies developed in two different culture media: Czapecdox and Sabouraud, throughout the year 1992 in Palencia city . A volumetric trap was used . We collected daily samples of aerovagans spore from the atmosphere through a cellulose esther filter, half of which was cultivated on Petri dishes . The following genera were identified: 26 Deuteromycetes (54%), four Zygomycetes (28%), and three bacteria, which along with Actinomycetes, reached 18% of all the registered colonies . Fifty-two percent of the colonies were developed in Czapecdox culture medium and 48% in Sabouraud medium . Most of the bacteria were grown in Sabouraud medium . The highest number of colonies recorded belonged to the following three genera: Mucor (25%), Aspergillus (23%) and Penicillium (16%) . Most colonies were grown in autumn (32%), while spring was the second most frequent season when 28% of the colonies were registered.

Blood, 1998 Feb 15, 91(4), 1304 - 17
Studies of multimerin in human endothelial cells; Hayward CP et al.; Multimerin is a novel, massive, soluble protein that resembles von Willebrand factor in its repeating, homomultimeric structure . Both proteins are expressed by megakaryocytes and endothelial cells and are stored in the region of platelet alpha-granules resembling Weibel-Palade bodies . These findings led us to study the distribution of multimerin within human endothelial cells . Multimerin was identified in vascular endothelium in situ . In cultured endothelial cells, multimerin was identified within round to rod-shaped, dense-core granules, some of which contained intragranular, longitudinally arranged tubules and resembled Weibel-Palade bodies . However, multimerin was found primarily in different structures than the Weibel-Palade body proteins von Willebrand factor and P-selectin . After stimulation with secretagogues, multimerin was observed to redistribute from intracellular structures to the external cellular membrane, without detectable accompanied secretion of multimerin into the culture media . In early passage endothelial cell cultures, multimerin was associated with extensive, fibrillary, extracellular matrix structures, in a different distribution than fibronectin . Although multimerin and von Willebrand factor are stored together in platelets, they are mainly found within different structures in endothelial cells, indicating that there are tissue-specific differences in the sorting of these soluble, multimeric proteins.

Clin Exp Immunol, 1998 Feb, 111(2), 278 - 85
Human CD4+ T lymphocytes recognize a highly conserved epitope of human T lymphotropic virus type 1 (HTLV-1) env gp21 restricted by HLA DRB1*0101; Kitze B et al.; HTLV-1 causes two distinct human diseases, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T cell leukaemia/lymphoma (ATL) . Persistently infected individuals carry a risk of <1% of developing either disease . These basic epidemiological data imply that virus-host interactions, especially immunogenetic factors, influence the outcome of infection . Several studies showed that the HLA class II DR1 DQ5 haplotype is over-represented in HAM/TSP, but rare in ATL . Therefore, we selected four patients with HAM/TSP and one seronegative control who all carried the HLA DR1 DQ5 haplotype . We analysed the CD4+ T lymphocyte response against eight synthetic peptides of HTLV-1 envelope (env) glycoprotein gp21, a crucial target antigen in HAM/TSP . The first of two immunodominant epitopes corresponded to a domain of the HTLV-1 envelope protein which had previously been shown to be essential for HTLV-1 envelope function . The second immunodominant epitope overlapped a highly conserved sequence of the retroviral transmembrane envelope protein . DR1 (DRB1*0101)-restricted T lymphocytes were activated by the conserved peptide sequence in nanomolar concentrations . In contrast, this conserved sequence can also induce non-specific, cAMP-mediated immunosuppressive effects on T cells when added in micromolar concentrations to culture media, as shown by Haraguchi S, Good RA, James-Yarish M, Cianciolo GJ, Day NK, Proc Natl Acad Sci USA 1995; 92:5568-71 . Hence, HTLV-1 env gp21 might exert either stimulating immunological or immunosuppressive effects in HTLV-1-infected individuals, depending on the level of its expression and the presence of HLA DRB1*0101.

Trop Med Int Health, 1998 Jan, 3(1), 46 - 51
Secretory acetylcholinesterase of Setaria cervi microfilariae and its antigenic cross-reactivity with Wuchereria bancrofti; Sharma S et al.; Setaria cervi, a bovine filarial parasite, secretes acetylcholinesterase during in vitro cultivation . A significant amount of enzyme activity was detected both in culture media and somatic extracts of different developmental stages of the parasite . The microfilarial stage showed a higher level of AChE activity than adult worms, with females being considerably more active than males . The secretory enzyme from microfilariae preferentially utilized acetylthiocholine iodide as substrate and showed two electrophoretically distinct isoforms in native PAGE . Secretory enzyme was purified from the excretory/secretory products of microfilariae using edrophonium chloride linked to epoxy-activated sepharose . Analysis of purified acetylcholinesterase by SDS-PAGE revealed the existence of two proteins of 75kD and 45kD under nonreducing conditions . These secretory enzymes are antigenic and cross-reactive with Wuchereria bancrofti-infected asymptomatic microfilaraemic human sera when tested by enzyme linked immunosorbent assay and immunoblotting . The secretory AChE(s) from S . cervi microfilariae may be utilized for diagnosis of early filarial infections.

Glia, 1998 Mar, 22(3), 237 - 48
Astrocytes protect neurons from neurotoxic injury by serum glutamate; Ye ZC et al.; Serum is used widely for culturing neurons and glial cells, and is thought to provide essential, albeit undefined, factors such as hormones, growth factors, and trace elements that promote the growth of cells in vitro . Moreover, serum can have profound effects on cell proliferation, differentiation, and cell morphology, and may even influence cell fate decisions . Despite the overall growth-promoting influence of serum on cell culture, frequent media changes have been shown to be detrimental to neuronal cultures, significantly reducing the yield of viable neurons . The reason for this loss of neurons by frequent media changes has been puzzling . We demonstrate that bovine and horse sera, the most popular serum complements for CNS cell culture, are a significant source for glutamate, supplying glutamate at concentrations sufficient to kill primary cultured hippocampal neurons . By using the bioluminescence detection method, we determined the glutamate concentration {Glu} in several batches of fetal bovine (calf) sera (FBS) to be close to 1 mM, and that of horse sera to be approximately 0.3 mM . Thus 10% serum supplement to culture media results in {Glu} of 30-100 microM due to serum alone . We subsequently produced glutamate depleted media (GDM) by using primary cultures of hippocampal astrocytes to absorb glutamate from media containing 10% FBS . Within 3 h, astrocytes reduced the {Glu} in the medium from approximately 90 microM to less than 1 microM . Sister cultures of hippocampal neuron that underwent frequent media changes with GDM or GDM + partial untreated media demonstrated that GDM significantly increase neuronal survival (10-fold at 21 DIV) . Subsequent exposure to glutamate provided by either untreated serum or by equivalent doses of exogenous glutamate added to GDM led to dose-dependent neuronal cell death . The relative sensitivity of hippocampal neurons to glutamate increased with increasing culture age from initial ED50 values of > 100 microM (< 6 DIV) to approximately 6 microM in cultures maintained for 3 weeks or longer . The relative sensitivity to exogenous glutamate was at least 2-fold higher in neurons cultured in GDM than in sister cultures maintained in media containing untreated serum . The death of neurons exposed to untreated media was blocked by the NMDA receptor antagonist MK-801 . These experiments suggest that the vulnerability of neurons to media changes can be solely explained by excitotoxicity resulting from serum-borne glutamate . Moreover, we propose that use of GDM may be advantageous for culturing hippocampal neurons and may eliminate the possible selection for glutamate resistant neurons . The use of GDM could be particularly important for studies of excitotoxicity; our study predicts that the ED50 for neuronal culture with regular serum will be artificially high and may not adequately reflect the in vivo state.

J Biol Chem, 1998 Jan 30, 273(5), 3027 - 32
Identification of the major oxidatively damaged proteins in Escherichia coli cells exposed to oxidative stress; Tamarit J et al.; In the present study we have analyzed protein oxidation on Escherichia coli when these cells were submitted to different stress conditions such as hydrogen peroxide, superoxide-generating compounds, and iron overloading . Carbonyl groups on oxidized cell proteins were examined by Western blot immunoassay . When anaerobically grown E . coli cells were exposed to hydrogen peroxide stress, alcohol dehydrogenase E, elongation factor G, the heat shock protein DNA K, oligopeptide-binding protein A, enolase, and the outer membrane protein A were identified as the major protein targets . A similar immunostained band pattern was found when cells were shifted from anaerobic to aerobic conditions in the presence of different concentrations of iron; it is relevant to note that oxidation of outer membrane protein C, not observed in peroxide stress conditions, was clearly detected as the concentration of iron was increased in the culture media . The hydrogen peroxide stress performed under aerobic conditions affected the beta-subunit of F0F1-ATPase; the rest of the oxidized protein pattern was very similar to that found for anaerobic conditions, with the exception of alcohol dehydrogenase E, a protein not synthesized aerobically . Cells submitted to superoxide stress using menadione showed a more specific pattern in which elongation factor G and the beta-subunit of F0F1-ATPase were affected significantly . When paraquat was used, although the degree of oxidative damage was lower, the same two modified proteins were detected, and DNA K was also clearly damaged . Cell viability was affected to different extents depending on the type of stress exerted . The results described in this paper provide data about the in vivo effects of oxidative stress on protein oxidation and give insights into understanding how such modifications can affect cellular functions.

Am J Respir Cell Mol Biol, 1998 Feb, 18(2), 255 - 64
Expression of RANTES by normal airway epithelial cells after influenza virus A infection; Matsukura S et al.; The chemokine regulated on activation, normal T cells expressed and secreted (RANTES), is a C-C chemokine and a potent chemoattractant for monocytes, T lymphocytes, basophils, and eosinophils . Its expression by human airway epithelium has been demonstrated both in vitro and in vivo . We investigated whether RANTES is expressed by normal human airway epithelial cells after influenza viral infection and examined its bioactivity . Epithelial cells were obtained from bronchial tissue or nasal polyps of patients who had undergone lobectomy for lung cancer or polypectomy for nasal polyps . These cells were cultured by the outgrowth method . Cultured cells were infected with influenza virus A (subtype H3N2) after which the supernatants and the cells were collected 8 to 72 h after infection . RANTES mRNA (messenger RNA) was analyzed by the reverse transcriptase-polymerase chain reaction and Southern blot analysis of its product . Concentrations of RANTES in the supernatants were analyzed by enzyme-linked immunosorbent assay . RANTES protein and mRNA were not detected in the media of uninfected cells . PCR products for RANTES were clearly detected in nasal and bronchial epithelial cells 24 h after infection . Southern blot analysis confirmed that the PCR products were indeed specific for RANTES mRNA . Twenty-four to 72 h after infection, significant levels of RANTES protein were detected in culture media . We also investigated the chemotactic activity of the supernatant of cultured cells . The supernatant of the cells 48 h after infection had potent chemotactic activity for eosinophils, which was attenuated by the addition of anti-RANTES antibodies . These findings suggest that influenza virus infection may induce expression of bioactive RANTES by normal human bronchial and nasal epithelial cells.

Heart Vessels, 1997, Suppl 12, 194 - 7
Tyrosine kinases mediation of c-fos expression by cell swelling in cardiac myocytes; Sadoshima J et al.; We investigated the signal transduction mechanism initiated by hypotonic cell swelling in cardiac myocytes using c-fos expression as a nuclear marker . Treatment of myocytes with hypotonic culture media rapidly induced c-fos expression, whereas hypertonic stress had no effect on it . Extensive pharmacological studies indicated that only tyrosine kinase inhibitors suppressed the hypotonic swelling-induced c-fos expression; down-regulation of protein kinase C or a Ca2+ chelator (EGTA) had no effect . Hypotonic stress immediately (within 5s) increased tyrosine kinase activity . Thus tyrosine kinase is one of the earliest signaling molecules activated by hypotonic stress and plays an essential role in hypotonic swelling-induced c-fos expression . Rapid activation of tyrosine kinase may be a common early signaling mechanism in response to mechanical stresses that increase cell membrane tension.

FASEB J, 1998 Feb, 12(2), 189 - 97
Interleukin-10 attenuates experimental fetal growth restriction and demise; Rivera DL et al.; Premature labor, fetal demise, and fetal growth restriction are accompanied by indices of inflammation or infection of the uteroplacental unit . To understand whether these events are causally related, we established an animal model of fetal demise and growth restriction and evaluated the potential utility of the anti-inflammatory cytokine interleukin-10 (IL-10) . We administered low-dose endotoxin (lipopolysaccharide, or LPS, 100 microg/kg, i.p.) to third trimester rats (gestational days 14-20) . Control rats received normal saline . A third group received IL-10 (100 microg/kg; s.c.) concomitantly with LPS for 7 prenatal days . Cytokine gene expression (IL-10 and TNF-alpha) was evaluated by RT-PCR and tissue levels (TNF-alpha) were determined by ELISA . Apoptosis was evaluated by TdT-mediated dUTP nick end labeling immunohistochemistry, and nitric oxide (NO) levels were quantified by microelectrode electrochemical detection in explants in culture media . LPS exposure resulted in 43% fetal demise and reduced the size of the surviving fetuses . Placental weight was not altered by LPS . IL-10 attenuated the LPS-induced fetal death rate (to 22%) and growth restriction (P<0.05) . In normal rats, IL-10 did not affect fetus size or the incidence of resorptions, although placental size was marginally smaller . Increased uterine TNF-alpha content and NO release and apoptosis of uterine epithelia and muscularis were hallmarks of the LPS model . All were normalized by IL-10 . IL-10 may represent a new therapeutic option for the treatment of a variety of perinatal complications . Benefit may result from the suppression of TNF-alpha- and NO-mediated cell death.

Metabolism, 1998 Feb, 47(2), 185 - 9
The effect of glucose and calcium on Ca2+-adenosine triphosphatase in pancreatic islets isolated from a normal and a non-insulin-dependent diabetes mellitus rat model; Levy J et al.; Regulation of calcium balance is important in the secretory function of pancreatic islets . Ca2+-adenosine triphosphatase (ATPase) is altered in tissues of non-insulin-dependent diabetes mellitus (NIDDM) rats, and they have an impaired response to glucose, "glucose blindness." We propose that the glucose blindness of the diabetic islet is the result of defective cellular calcium metabolism . Since Ca2+-ATPase activity is important in the regulation of calcium balance, we investigated the effect of glucose and/or calcium on Ca2+-ATPase activity in pancreatic islets in vitro and compared it with the effect in freshly isolated islets from controls and from rats with NIDDM induced by streptozotocin neonatally . Islets were isolated using collagenase and were stored fresh or cultured up to 2 days in RPMI 1640 in the presence of different concentrations of glucose and calcium . Membrane Ca2+-ATPase activity, insulin secretion, and insulin content were determined . Ca2+-ATPase activity was 1.30 +/- 0.20 micromol/L Pi/microg membrane protein in normal noncultured islets and 1.02 +/- 0.15 in islets cultured in 5.6 mmol/L glucose . Ca2+-ATPase activity progressively decreased to 0.56 +/- 0.10 and 0.34 +/- 0.14 micromol/L Pi/microg membrane protein when glucose was increased in the culture media to 16.6 and 27.7 mmol/L, respectively . Decreasing glucose to 2.8 mmol/L did not alter Ca2+-ATPase activity . Increasing or decreasing the Ca2+ content of the media did not significantly change Ca2+-ATPase activity . Islets isolated from NIDDM rats had lower basal Ca2+-ATPase activity and insulin content compared with normal controls . Incubation of islets from diabetic rats in high glucose further decreased the Ca2+-ATPase content, but incubation in low glucose did not reverse it . Insulin secretion was responsive to glucose and calcium in normal islets, but was suppressed in islets from diabetic animals . From these studies, we conclude that high glucose, but not calcium, decreases Ca2+-ATPase activity in islets from normal rats . Islets from NIDDM rats with glucose blindness have decreased Ca2+-ATPase activity, likely due to the glucose status . We suggest that this decreased Ca2+-ATPase activity may contribute to the pancreatic islets' glucose blindness.

Biol Reprod, 1998 Jan, 58(1), 88 - 93
Gonadotropins and reproductive function in the anuran amphibian, Rana esculenta; Polzonetti-Magni AM et al.; In this study, the measurement both of peripheral gonadotropins (FSH and LH) and of sex steroids in male and female wild anuran, Rana esculenta, was performed during the annual reproductive cycle; moreover, the role of gonadotropins in the vitellogenic process and in ovarian steroidogenesis was investigated through in vitro experiments . LH plasma changes in males showed high values during autumn-winter months and during the mating period, when high androgen levels were found . Conversely, for the first time in male vertebrates, a clear correspondence between plasma FSH and estradiol-17beta (E2) was shown . In females, FSH peak values were found at the beginning of the mating period in parallel with those of plasma vitellogenin (VTG) and E2; in contrast, high LH levels went together with ovarian weight (gonadosomatic index), which is considered a good marker for the plasma sequestration of VTG by growing oocytes . The in vivo results are corroborated by in vitro studies showing the direct effects of both FSH and LH in inducing hepatic VTG synthesis and release in the culture media . Lastly, although it is not yet known whether or not FSH and LH have separate functions in amphibians, it was clearly shown that they induce ovarian steroid production . These results are discussed in terms of the high seasonality previously demonstrated in this wild frog.

Microbiology, 1998 Jan, 144 ( Pt 1), 103 - 7
Haemolysin production by strains of Verocytotoxin-producing Escherichia coli; Chart H et al.; Twenty-one strains of Verocytotoxin-producing Escherichia coli (VTEC) that hybridized with DNA probe CVD419 were examined for the ability to produce haemolysin . With solid media, all strains produced most haemolysin when grown in blood agar tubes and least when grown on blood agar plates incubated in air . Haemolysin production was increased considerably by incubating blood agar plates in an atmosphere comprising 8% carbon dioxide, 40% hydrogen and 52% nitrogen at 37 degrees C for 16 h, followed by 6 h at 21 degrees C in air . Haemolysin production was also increased when strains were grown on L-agar containing the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid) prior to subculture on blood agar . Intracellular haemolysin was detected in five out of the 21 strains of E . coli grown on L-agar in the atmosphere described above, but haemolysin was not detected in L-broth culture supernatants . The haemolysins lysed guinea pig, mouse and ferret erythrocytes, but not human, rabbit, rat, turkey or chicken erythrocytes . Also, the addition of calcium ions to culture media was not required for haemolytic activity . It was concluded that haemolysins produced by VTEC appear to be quite distinct from E . coli alpha-haemolysin and resemble a form of beta-haemolysin.

Dent Mater, 1997 Jan, 13(1), 62 - 8
In vitro attachment of osteoblast-like cells to osteoceramic materials; Keller JC et al.; OBJECTIVE: The objective of this work was to examine osteoblast-like cell attachment and morphology in vitro to osteoceramic materials with three different surface morphologies . METHODS: Osteoceramic composite disks were fabricated from tricalcium phosphate and magnesium-aluminate spinel (MgAl2O4) in a 50 vol% ratio . The disks were prepared with three different surface morphologies, including as-fired (irregular), etched (rough), or polished through 1 mm diamond paste (smooth) . Osteoblast-like cell cultures were plated onto the prepared disks for 2 h, and the number of attached cells was determined . ANOVA and Student Newman-Kuels tests were used to test for significant differences in cell attachment (p < 0.05) . SEM was used to visually evaluate the nature of the cellular adaptation on the osteoceramic surfaces . RESULTS: Some additional surface roughening resulted from the interaction between the osteoceramic disks and the biological culture media during the attachment assay . A statistically larger number of cells was found to be attached to the etched osteoceramic surfaces compared to the as-fired and polished osteoceramic surfaces or the tissue culture plastic control . Cellular adaptation was extensive on all three osteoceramic surfaces at 2 h . SIGNIFICANCE: These results are consistent with previous in vivo work and continue to support the hypothesis that osteoceramic materials have potential for implants and bone substitute materials.

In Vitro Cell Dev Biol Anim, 1997 Nov-Dec, 33(10), 796 - 802
Establishment and characterization of a novel in vitro angiogenesis model using a microvascular endothelial cell line, F-2C, cultured in chemically defined medium; Chen CS et al.; The behavior of vascular endothelial cells (EC) is an important factor in the processes involved in angiogenesis, but the regulatory mechanisms of angiogenesis, especially underlying the tubulogenesis by EC are not yet clear . Although a number of in vitro experimental models of tubulogenesis have been developed by use of cultured EC, most of those models are too complex to be easily handled and further, the culture media are usually supplemented with serum, creating problems in interpretation of experimental results . To generate a simple in vitro angiogenesis study model under serum-free culture conditions, we adapted a murine microvascular endothelial cell line, F-2, to a chemically defined medium, Cos Medium 001, and successfully established a subline of F-2, designated F-2C, which revealed a unique growth pattern . In Cos Medium 001, F-2C proliferates in a cobblestone pattern at an early growth stage, but, at a late growth stage, spontaneously differentiates to form three-dimensional honeycomblike tubular structures without the supplementation of any specific factors . The cell aggregation activity of F-2C in the presence of Ca2+ was much greater than that of F-2 . The amount of subendothelial matrix deposited by F-2C was significantly higher than that by F-2, and increased prominently after the F-2C cells reached the differentiating stage of tubulogenesis . These findings indicate that F-2C is a new EC line in which tubulogenesis is spontaneously induced by the marked deposition of basement membrane analog to the subendothelial matrix and by the enhancement of presumable cadherin activity . We suggest that this cell line, F-2C, represents a simple and useful in vitro angiogenesis model.

J Anim Sci, 1998 Jan, 76(1), 66 - 73
Lithium chloride does not inhibit the proliferation of L6 myoblasts by decreasing intracellular free inositol; Laurenz JC et al.; We conducted a series of experiments to determine whether lithium chloride (LiCl) inhibited the proliferation of L6 myoblasts by reducing the availability of intracellular free inositol . After the myoblasts were plated in DMEM + 10% fetal bovine serum (FBS) for 24 h, medium was replaced with DMEM + 10% FBS containing 0 (control), 5, 10, or 20 mM LiCl . Cell number, protein content, and {3H}thymidine incorporation into DNA were determined at 24-h intervals . Control cells exhibited a 3.8-fold increase in cell number by 96 h in culture . Although 5 mM LiCl did not affect the rate or extent of proliferation, 10 and 20 mM LiCl caused 36 and 86% decreases, respectively (P < .05), in cell number by 96 h in culture . The effects of LiCl could not be overcome by the addition of free inositol (up to 20 mM) to the medium . Lithium chloride caused 4.6- and 7.3-fold increases (P < .05) in lactate dehydrogenase activity in culture media after 96 h of exposure to 10 and 20 mM LiCl, respectively, indicating loss of viability after chronic treatment . However, the acute effects of LiCl after 24 h of treatment were reversible, as indicated by a rapid resumption of proliferation following removal of LiCl . Concentrations of 5, 10, and 20 mM LiCl caused 4.7-, 8.2-, and 9.1-fold increases (P < .05), respectively, in the accumulation of {3H}inositol within the inositol monophosphate pool . Treatment of cells with 10 and 20 mM LiCl also increased (P < .05) label recovered as inositol bisphosphate . Rather than depress phosphoinositide synthesis, the addition of 10 and 20 mM LiCl dose-dependently increased (P < . 05) the incorporation of {3H}inositol into phosphatidylinositol and phosphatidylinositol-4-phosphate . These results indicate that LiCl does not decrease proliferation of L6 myoblasts via a depletion in the intracellular free inositol pool . Instead, LiCl may block the hydrolysis of phosphatidyl inositides.

Exp Eye Res, 1997 Oct, 65(4), 569 - 74
Gefarnate stimulates secretion of mucin-like glycoproteins by corneal epithelium in vitro and protects corneal epithelium from desiccation in vivo; Nakamura M et al.; The effect of drugs for gastritis and gastric ulcer (ecabet sodium, gefarnate, teprenone, and troxipide) on the secretion of mucin-like glycoproteins from rat cornea were investigated in vitro and on a short-term, rabbit dry eye model in vivo . For the studies in vitro, cultured rat cornea sections (3 mm diameter) were incubated with radiolabeled sodium sulfate, rinsed, and then incubated for 30 min in the presence of one of the drugs . The culture media were reacted with Dolichos biflorus agglutinate (DBA)-lectin, and the radioactivity of DBA-bound mucin-like glycoproteins was measured . A cytotoxicity assay confirmed that mucin-like glycoproteins had not leaked from damaged cells . For studies in vivo, eye drop vehicle or drops containing gefarnate were instilled in the eyes of nine anesthetized rabbits, and then the eyes were kept open with specula for two hours . These rabbits and two control rabbits not subjected to ocular drying were killed, and their eyes were enucleated and stained with methylene blue . Corneal epithelial damage from desiccation was evaluated based on the extent of methylene blue staining . Among the four kinds of drugs for gastritis and gastric ulcers, only gefarnate significantly increased the mucin-like glycoprotein secretion from cultured rat corneas in vitro; this stimulatory effect of gefarnate was dose-dependent . In vivo, the instillation of gefarnate reduced corneal epithelial damage from desiccation in a dose-dependent fashion . These results suggest that gefarnate reduces desiccation of corneal epithelium, perhaps by stimulating secretion of mucin-like glycoproteins from corneal epithelium.

FEBS Lett, 1998 Jan 2, 421(1), 12 - 4
CD146: biosynthesis and production of a soluble form in human cultured endothelial cells; Bardin N et al.; We previously identified the S-Endo 1-associated antigen (CD146), an endothelial member of the immunoglobulin superfamily with a characteristic V-V-C2-C2-C2 Ig domain structure . In cultured human endothelial cells, we investigated its biosynthesis by immunoprecipitation and pulse-chase labeling . CD146 was synthesized as a 100 kDa precursor form, which was processed into a 120 kDa mature form . In the culture media of endothelial cells, we observed a CD146 soluble form that was about 10 kDa smaller than cell-associated CD146 . In parallel with soluble forms of other members of the immunoglobulin superfamily, soluble CD146 could modulate and control the functions of the molecule.

Lab Invest, 1998 Jan, 78(1), 89 - 100
Increased expression of interleukin-6 and tumor necrosis factor-alpha in pathologic biliary epithelial cells: in situ and culture study; Yasoshima M et al.; We examined the pathologic significance of the expression of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), both proinflammatory cytokines, on intrahepatic biliary epithelial cells, using immunohistochemical and in situ hybridization techniques as well as culture study . IL-6 and TNF-alpha were expressed in the cytoplasm of biliary epithelial cells of damaged small bile ducts and bile ductules, particularly in primary biliary cirrhosis . Their expression on the bile ducts was mild to moderate in other hepatobiliary diseases and mild or absent in normal livers . Signals of IL-6 mRNA and TNF-alpha mRNA were detected in the cytoplasm of biliary epithelial cells, especially in primary biliary cirrhosis . Immunoelectron microscopic study supported this . TNF receptor and to a lesser degree IL-6 receptor alpha-chain were detected on these damaged bile ducts, suggesting an autocrine effect . By Western blotting and enzyme-linked immunosorbent assay, IL-6 and TNF-alpha were frequently detected in gallbladder bile from primary biliary cirrhosis, and their titers were higher compared with other hepatobiliary diseases . Culture of intrahepatic biliary epithelial cells revealed that they expressed IL-6 and secreted IL-6 in the culture media . These results suggest that the intrahepatic biliary epithelial cells are able to synthesize IL-6 and probably TNF-alpha and are involved in the production of bile duct lesions by means of receptor-mediated processes, particularly biliary epithelial proliferation and destruction and autoimmune augmentation, in primary biliary cirrhosis.

Lab Invest, 1998 Jan, 78(1), 79 - 87
Expression of matrix metalloproteinase 7 (matrilysin) in human osteoarthritic cartilage; Ohta S et al.; Matrix metalloproteinases (MMP) consisting of at least 16 different molecules are thought to be involved in the degradation of extracellular matrix (ECM) macromolecules under various pathologic conditions . Among them, MMP-7 (matrilysin) is unique in that it has high specific activity against various ECM components such as cartilage proteoglycan . In the present study, we examined the expression and tissue localization of MMP-7 in articular cartilages of human osteoarthritis (OA) . Immunohistochemistry using a monoclonal antibody specific to MMP-7 demonstrated that the proteinase is localized to the OA chondrocytes mainly in the superficial and transitional zones in 92% of the OA cases examined (36 of 39 cases) . On average, approximately 30% of the total chondrocytes (29.1%+/-30.2%) were immunostained in the positive OA cartilage samples . In contrast, MMP-7 staining was found in 8% of the normal cartilage cases (1 of 12 cases), and only a few chondrocytes (0.15%+/-0.67%) in the superficial zone were immunostained . There was a linear correlation between degree (%) of the immunostained chondrocytes and Mankin scores (rho {rho} = 0.84) . Immunoblot analysis of the culture media from the cartilage explants demonstrated MMP-7 in 65% of the OA cases (15 of 23 cases) and 8% of the normal specimens (1 of 12 cases) . Reverse transcription-PCR demonstrated the specific amplicon in 68% of the OA cartilage cases (17 of 25 cases), whereas only 18% of the control (2 of 11 cases) amplified the product . In situ hybridization revealed that the chondrocytes in OA cartilage express MMP-7 mRNA . MMP-7 gene expression in cultured OA chondrocytes was enhanced by the treatment with interleukin-1alpha and/or tumor necrosis factor-alpha . These data demonstrate for the first time that MMP-7 is overexpressed in human OA cartilage and suggest that cytokine-induced MMP-7 may play an important role in the degradation of ECM macromolecules in the OA cartilage.

Kidney Int, 1998 Feb, 53(2), 287 - 95
Synergistic effect of dexamethasone and isoproterenol on the expression of angiotensinogen in immortalized rat proximal tubular cells; Wang L et al.; To investigate whether the expression of angiotensinogen (ANG) in rat kidney proximal tubules is stimulated by dexamethasone and isoproterenol, immortalized rat proximal tubular cells (IRPTC) were cultured in a monolayer . Immunoreactive rat ANG (IR-rANG) in the culture medium was measured by a specific radioimmunoassay (RIA) for rANG . This RIA was developed by employing rabbit antiserum against the purified recombinant rat ANG (rANG) . The purified rANG from plasma and the iodinated rANG were used as the hormone standard and tracer, respectively . The RIA is specific for rat ANG and it has no cross-reactivity with other pituitary hormone preparations or other rat plasma proteins . The sensitivity of detection of the RIA is approximately 2 ng of rANG . The levels of IR-rANG in the culture media of IRPTC ranged from 2 to 5 ng/ml/24 hr/10(6) cells . The addition of dexamethasone (10(-13) to 10(-5) M) stimulated the expression and secretion of rANG from IRPTC in a dose-dependent manner, whereas the addition of isoproterenol alone had no effect . However, a combination of both dexamethasone and isoproterenol synergistically stimulated the expression and secretion of rANG by IRPTC . The synergistic effect of dexamethasone and isoproterenol was blocked by the presence of RU 486 (a glucocorticoid receptor antagonist) or propranolol (beta-adrenoceptor blocker) . These studies suggest that the addition of dexamethasone and isoproterenol acts synergistically to stimulate the expression and secretion of ANG protein in rat proximal tubules in vivo.

Hum Reprod Update, 1997 Jul-Aug, 3(4), 367 - 82
Culture and selection of viable blastocysts: a feasible proposition for human IVF?
Gardner DK, Lane M.
In human in-vitro fertilization (IVF) embryos are routinely transferred to the uterus on day 2 or day 3 of development . Resultant implantation and pregnancy rates are disappointingly low, with only approximately 10% of embryos transferred leading to a live birth . The ability to culture embryos to the blastocyst stage should help to resolve this problem by synchronizing the embryo with the female reproductive tract, and by identifying those embryos with little developmental potential . Co-culture has offered a possible means of producing blastocysts capable of high implantation rates . However, recent developments in the field of embryo physiology and metabolism have led to the formulation of new sequential serum-free culture media capable of supporting the development of viable blastocysts in several mammalian species, including the human . It is therefore proposed that blastocyst transfer should be considered for routine use in human IVF . The high viability of blastocysts cultured in the appropriate sequential media means that fewer embryos are required for transfer to achieve a pregnancy, culminating in fewer multiple births . Furthermore, the development of suitable non-invasive tests of embryo viability should further increase the overall success of human IVF by the ability to select before transfer those blastocysts most able to establish a pregnancy.

Am J Reprod Immunol, 1998 Jan, 39(1), 16 - 23
IL-15, a novel cytokine produced by human fetal membranes, is elevated in preterm labor; Fortunato SJ et al.; PROBLEM: Interleukin (IL)-15 is a novel cytokine known to have functions similar to those of IL-2 in the cell-mediated immune response . The objectives of this study were to determine whether IL-15 levels change in labor or preterm labor and to identify the regulatory agents and the site of production of IL-15 . METHOD OF STUDY: Amniochorionic membranes were cultured in an organ explant system and were stimulated with lipopolysaccharides (LPSs) . Samples were subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) using specific primers for IL-15 and IL-2 . The localization of mRNA and protein was accomplished by in situ hybridization and immunocytochemistry . IL-15 was measured in culture media and amniotic fluid from term and preterm gestations by enzyme-linked immunoadsorbent assay (ELISA) . RESULTS: RT-PCR indicated the expression of IL-15 mRNA in the amniochorion . In situ hybridization and immunocytochemistry documented that mRNA and peptide for IL-15 are found in amnion, chorion, and decidual cells . ELISA results indicated no significant increase of IL-15 peptides in the culture media after LPS stimulation . Maximum levels of this cytokine were seen in the amniotic fluid (AF) of women with preterm labor compared to term labor . AF levels were not higher in preterm-labor patients with proved infection compared with those without infection . RT-PCR-based detection also showed the presence of two isoforms of IL-15 mRNA known to code for two different leader peptide sequences . IL-2 mRNA expression was not observed in the fetal membranes . CONCLUSIONS: The presence of IL-15 mRNA and peptide in the amniochorion and decidua and its increased presence in the AF during preterm labor suggests a possible role for IL-15 in preterm labor . Amniochorion is also shown to possess two IL-15 isoform leader sequences, the differential expression of which may be involved in the regulation of IL-15 secretion.

J Neurochem, 1997 Nov, 69(5), 2048 - 54
Amidation of beta-amyloid peptide strongly reduced the amyloidogenic activity without alteration of the neurotoxicity; Forloni G et al.; Beta-amyloid accumulates in cerebral deposits in Alzheimer's disease, so to test the correlation between the neurotoxic and fibrillogenic capacity of beta-amyloid, we synthesized a peptide homologous to fragment 25-35 of beta-amyloid (beta25-35) and amidated at the C-terminus (beta25-35-NH2) . As the amidation strongly reduced the amyloidogenic capacity of beta25-35, we compared its neurotoxic activity in the amidated (beta25-35-NH2) and nonamidated forms . The viability of primary cultures from fetal rat hippocampus was reduced in a dose-related manner (10-100 microM) similarly by beta25-35 and beta25-35-NH2, whereas a scrambled peptide, amidated or nonamidated, did not alter the neuronal viability . The neurotoxic activity of beta25-35-NH2 is mediated by apoptosis as demonstrated by morphological and biochemical investigations . Electron microscopy examination of culture media with beta25-35 or beta25-35-NH2 incubated with neuronal cells for 7 days confirmed the high level of fibrillogenic activity of beta25-35 and the almost total absence of fibrils in the solution with beta25-35-NH2 . Furthermore, staining with thioflavine S was used to identify amyloid fibrils, and only the cultures exposed to beta25-35 exhibited intense staining associated with neuronal membranes . These data indicate that the neurotoxic activity of the beta-amyloid fragment is independent of the aggregated state of the peptide.

J Gen Virol, 1997 Oct, 78 ( Pt 10), 2467 - 76
Establishment of persistent hepatitis C virus infection and replication in vitro; Seipp S et al.; Hepatitis C virus (HCV) is a major cause of chronic viral hepatitis . Development of anti-viral strategies has been hampered by the lack of efficient cell systems to propagate HCV in vitro . To establish a long-term culture system, we tested human hepatoma (HuH7, HepG2) and porcine non-hepatoma (PK15, STE) cell lines, as well as several culture and infection conditions . As a marker for virus replication, minus-strand HCV RNA in infected cells was detected by an enhanced detection system using nested RT-PCR followed by hybridization analysis . Short-term efficiency of HCV infection (10 days) was slightly increased by addition of polyethylene glycol (PEG) and/or dimethyl sulfoxide (DMSO) to culture media during inoculation of HuH7, PK15 and STE cells, but no augmentation in long-term culture was achieved, suggesting enhanced attachment of HCV to cells rather than more efficient infection . A stabilizing effect on HCV propagation was observed for 50 days in a serum-free medium with stimulation of the low-density lipoprotein (LDL) receptor expression by lovastatin . Using partially serum-free culture conditions, long-term persistence of HCV in cells and release of virions into supernatant was achieved for up to 130 days . Infectivity of released virions in supernatants after long-term culturing (day 30-80) was shown by successful infection of fresh cells . In conclusion, supplementation with PEG, DMSO and lovastatin during inoculation did not enhance virus replication substantially, but continued stimulation of LDL-receptor expression resulted in infections which persisted for over 4 months . These data support the hypothesis of an LDL-receptor mediated uptake of HCV into cells in vitro.

Fertil Steril, 1998 Jan, 69(1), 84 - 8
Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers; Gardner DK et al.; OBJECTIVE: To determine whether the transfer of blastocysts on day 5, developed in sequential culture media, resulted in an increase in implantation rate compared with embryos transferred on day 3 . DESIGN: Comparative study of embryo culture regimes . SETTING: Private practice assisted reproductive technology center . PATIENT(S): Twenty-three patients undergoing routine IVF cycles . INTERVENTION(S): Culture of embryos to day 3 in either standard culture conditions or a serum-free chemically defined medium . One hundred one embryos were subsequently cultured from day 3 to day 5 in a second serum-free medium specifically designed to support development of the blastocyst . MAIN OUTCOME MEASURE(S): Embryo cell number and quality on day 3 . Blastocyst development on day 5 . Implantation rate (determined by fetal heart) and ongoing pregnancy rate (PR) . RESULT(S): Implantation rates for embryos transferred at the blastocyst stage of development were twice that observed for embryos transferred on day 3, around the eight-cell stage . Significantly more embryos were required for transfer on day 3, compared with day 5, to establish similar PRs . CONCLUSION(S): Viable human blastocysts can be obtained in sequential culture media in the absence of coculture and serum . Transfer of blastocysts in IVF will facilitate high PRs while limiting the number of embryos transferred and therefore minimizes the risk of multiple gestation.

Eur Spine J, 1997, 6(6), 376 - 84
Elevated synthesis of biglycan and decorin in an ovine annular lesion model of experimental disc degeneration; Melrose J et al.; The aim of this study was to extend our earlier observations on the changes that occur in the proteoglycans (PGs) of discs subjected to experimental injury to the annulus fibrosus (AF) . We employed the alginate bead culture method to examine the metabolism of the dermatan sulphate (DS) containing PGs by cells derived from different regions of ovine discs that had been subjected to experimental annular injury . This was compared with the metabolism of the DS-PGs by cells isolated from equivalent regions of normal sham-operated discs . Six months after induction of the annular lesion, AF cells isolated from the lesion produced significantly higher levels of decorin and biglycan in alginate bead culture than did cells from equivalent zones of the controls . Decorin and biglycan were identified in culture media samples by immunoblotting, using specific antibodies (6-B-6, LF-96), and also by positive identification of their de-glycosylated core proteins . The core protein of the DS-PGs has been shown to inhibit type I/II collagen fibrillogenesis, to negatively regulate the action of transforming growth factor-beta (TGF-beta) and to diminish cellular proliferation in vitro; events which may be detrimental to tissue repair . The findings are therefore consistent with our previous observation the annular lesions in the avascular inner annulus have no capacity to heal.

Virus Res, 1997 Nov, 52(1), 25 - 41
Biologic properties of human herpesvirus 7 strain SB; Black JB et al.; The growth characteristics of human herpesvirus 7 strain SB (HHV-7 (SB)) were studied in human umbilical cord blood lymphocyte (CBL) cultures . The virus has approximately a 4-day growth cycle, as measured by immunofluorescence analysis, quantitation of the relative viral DNA concentration, and examination of infected cells by electron microscopy on consecutive days post-infection . By systematically varying the culture media components, improved culturing conditions were established . Activated lymphocytes were required for virus growth . HHV-7(SB) grew best in phytohemagglutinin-stimulated CBL cultured in media containing 0.01 mg/ml hydrocortisone . Addition of recombinant human interleukin 2 (IL-2) at concentrations exceeding 1-10 U/ml inhibited virus growth in most CBL cultures . Addition of exogenous IL-2 to the culture media had no effect on viral DNA production . However, the percentage of virus antigen-positive cells was highest when 0.1-1 U/ml was added to the media . Differences in the ability of individual CBL cultures to replicate HHV-7(SB) was not explained by differing CD4+ cell concentrations . However, individual cultures varied in the level of endogenous IL-2 production, which may contribute to the virus growth variability in CBL . HHV-7(SB) grew in the CD4-positive T-cell line SupT1, but not in a variety of other lymphocyte, fibroblast, or epithelial cell lines . Nine compounds were tested for antiviral activity against HHV-7 in vitro . Phosphonoformic acid inhibited virus growth with a 50% effective concentration of 4.8 microM . Ganciclovir (200 microM) and phosphonoacetic acid (100 microM) inhibited more than 90% of virus production . None of the compounds were cytotoxic at concentrations which inhibited the virus . A generalized increase in host cell protein synthesis was also observed in virus-infected cells similar to that seen in CBL infected with human herpesvirus 6.

Pflugers Arch, 1998 Jan, 435(2), 211 - 8
Physiological changes in extracellular sodium directly control human proximal tubule growth and transport; Johnson DW et al.; In order to examine the nature and potential mechanisms of action of extracellular sodium on human proximal tubule growth and transport, quiescent primary cultures of human proximal tubule cells (PTC) were incubated for 24 h in serum-free, growth-factor-free culture media containing low (130 mmol/l), control (140 mmol/l) or high (150 mmol/l) Na+ . Compared to control conditions, cells exposed to a high Na+ concentration demonstrated stimulated thymidine incorporation (121.8 +/- 7.6%, P < 0.05) and increased cellular protein content (139.7 +/- 9.9%, P < 0.05); the latter arising from suppressed protein degradation ({3H}valine release 72.3 +/- 2.5%, P < 0.01) and unchanged protein synthesis ({3H}valine incorporation 98.5 +/- 2.6%, P > 0.1) . Substitution of choline chloride for NaCl did not replicate these effects . Conversely, cells incubated in low-Na+ media showed reduced thymidine incorporation (77.2 +/- 4.4%, P < 0 . 05), reduced protein synthesis (60.6 +/- 4.3%, P < 0.01), reduced protein degradation (79.5 +/- 1.8%, P < 0.01) and an unaltered protein content (102.4 +/- 8.8%) . A role for apical Na+/H+ exchange (NHE) activity in mediating Na+-dependent alterations in PTC growth was suggested by the findings of increased apical, ethylisopropylamiloride- (EIPA)-sensitive 22Na+ uptake in the presence of a high Na+ concentration (159 +/- 19% of control, P < 0 . 05) and concentration-dependent inhibition of cellular growth by EIPA at levels corresponding to those producing inhibition of apical NHE . Conditioned media from low Na+, control or high Na+ PTC contained comparable amounts of platelet-derived growth factor-AB (1 . 19 +/- 0.23, 1.14 +/- 0.22 and 1.28 +/- 0.20 ng/mg protein, P > 0.1) and transforming growth factor-beta1 (1.76 +/- 0.32, 1.73 +/- 0.33 and 1.45 +/- 0.28 ng/mg protein, P > 0.1), and did not exhibit autocrine growth factor activity on separate PTC following adjustment of Na+ concentrations to 140 mmol/l by dialysis . Similarly, low-Na+, control or high-Na+ media did not modify the mitogenic responsiveness of PTC to insulin-like growth factor-I (IGF-I) or alter the affinity or number of PTC IGF-I binding sites . The results confirm that physiological increases in extracellular Na+ concentration directly stimulate human proximal tubule growth and Na+ transport . Such stimulation does not appear to be mediated by altered PTC secretion of, or responsiveness to, cytokines known to affect tubule growth and transport.

Differentiation, 1997 Dec, 62(3), 139 - 47
Intestinal-type fibroblasts selectively influence proliferation rate and peptide synthesis in the murine entero-endocrine cell line STC-1; Ratineau C et al.; The intestinal epithelium consists of enterocytes, endocrine cells, goblet cells and Paneth cells, which differentiate from pluripotent stem cells located at the crypt bases . The role of the epithelial-mesenchymal inter-actions has been well documented for the differentiation of enterocytes, but the mechanisms that control endocrine cell differentiation are poorly understood . We have cultured the intestinal endocrine cell line STC-1, which synthesizes most of the intestinal peptide hormones, in media conditioned by several subepithelial fibroblast cell lines from three distinct sites of intestine . The fibroblast Swiss 3T3 cell line was used as a non-intestinal control . Our results show that culture media from intestinal fibroblasts inhibit the proliferation rate of STC-1 cells, while those from Swiss 3T3 fibroblasts do not . As regards peptide hormone gene expression, Swiss 3T3-conditioned media have no effect, whereas media from intestinal fibroblasts variably affect cholecystokinin, glucagon, secretin and somatostatin mRNA levels . In particular, clonal subepithelial myofibroblasts do not exert the same effects as mixed subepithelial fibroblasts from homologous intestinal segment . Taken together, these results suggest that cultured fibroblasts of intestinal origin release soluble factors that inhibit STC-1 cell proliferation and modulate, in a region-specific manner, the expression of hormonal peptide genes in this nonspecialized endocrine cell line.

J Comp Pathol, 1997 Oct, 117(3), 191 - 9
Equine pulmonary mycosis due to Aspergillus niger and Rhizopus stolonifer; Carrasco L et al.; Invasive pulmonary mycosis caused by Aspergillus niger and Rhizopus stolonifer is reported in a 2-year-old horse, one of three that died after being housed in a disused, uncleaned stable . Lesions were characterized by thrombosis of the blood vessels with haemorrhage and tissue necrosis . Fungal hyphae were observed both in thrombosed vessels and in adjacent necrotic tissue . In culture media inoculated with lung samples and samples from the bedding hay, two types of colony were recorded and identified as A . niger and R . stolonifer . This study is the first description of equine pulmonary mucormycosis and of A . niger as an aetiological agent in the horse.

J Biol Chem, 1998 Jan 23, 273(4), 2222 - 31
Identification of functional domains of rat intestinal phospholipase B/lipase . Its cDNA cloning, expression, and tissue distribution; Takemori H et al.; A cDNA encoding a rat intestinal Ca(2+)-independent phospholipase B/lipase (PLB/LIP) was cloned from an ileac mucosa cDNA library using a probe amplified by polymerase chain reaction based on the purified enzyme's sequence . PLB/LIP consists of an NH2-terminal signal peptide, four tandem repeats of about 350 amino acids each, and a hydrophobic domain near the COOH terminus . The enzyme purified previously was found to be derived from the second repeat part . To examine the function of each domain, the full-length PLB/LIP, individual repeats, and a protein lacking the COOH-terminal hydrophobic stretch were expressed in COS-7 cells . The results showed that the second repeat, but not the other repeats, had all the activities (phospholipase A2, lysophospholipase, and lipase) found in the purified natural and expressed full-length enzymes, suggesting repeat 2 is a catalytic domain . The full-length enzyme was mainly present in membrane fractions and efficiently solubilized by treatment with 1% Triton X-100, but not with phosphatidylinositol-specific phospholipase C . Deletion of the COOH-terminal hydrophobic stretch caused the secretion of > 90% of synthesized PLB/LIP into culture media . These results suggest the hydrophobic domain is not replaced by a glycosylphosphatidylinositol anchor but serves as a membrane anchor directly . A message of the full-length PLB/LIP was abundantly expressed in the ileum and also, in a smaller, but significant amount, in the esophagus and testis . Immunohistochemistry showed that PLB/LIP is localized in brush border membranes of the absorptive cells, Paneth cells, and acrosomes of spermatid, suggesting its roles related and unrelated to intestinal digestion.

Dev Biol, 1997 Dec 15, 192(2), 572 - 84
The effects of angiotensin II and specific angiotensin receptor blockers on embryonic cardiac development and looping patterns; Price RL et al.; The role of angiotensin II (Ang II) in the early embryonic development of the heart has not been examined . We have used RT-PCR to identify mRNA for angiotensinogen, angiotensin-converting enzyme, and the Ang II AT1 and AT2 receptors in embryonic day 10.25 Sprague-Dawley rats, and have used confocal microscopy to localize the AT1 receptor to the greater curvature of the developing ventricle in these animals at embryonic days (ED) 9.25 and 10.25 . The antibodies used in immunolocalization studies did not distinguish between the AT1a and AT1b receptor subtypes . In whole embryo culture, Ang II added to the culture media resulted in increased ventricular growth and myocyte hypertrophy when treated embryos were compared to cultured littermate controls . Use of Losartan and PD123,319 to block the Ang II AT1 and AT2 receptors resulted in reduced ventricular development and cardiac dilation when compared to control and Ang II-treated embryos . Addition of Ang II and PD123,319 to the culture media also resulted in cardiac loop inversions which may be associated with disruption of normal myofibrillar development . These results clearly indicate an important role for Ang II in the early embryonic development of the heart.

Folia Parasitol (Praha), 1997, 44(4), 297 - 301
Some factors which influence the in vitro maintenance of Anisakis simplex (Nematoda); Iglesias L et al.; Several culture media as well as some factors that may affect the in vitro development of the nematode Anisakis simplex Rudolphi, 1809 have been studied . After testing six media and four temperatures, the conditions for the in vitro culture selected were as follows: RPMI-1640 + 20% (v/v) heat-inactived fetal bovine serum or Meyer's M3 (without agar) media, at 37 degrees C, under 5% CO2 in air atmosphere, and renewal of the medium twice a week . The average survival rates of the larvae were significantly increased when the pH of the culture medium was increased (from 4.0 to 7.2) or decreased (from 7.2 to 4.0) after L3 to L4 moulting . The length of the larvae at the onset of culture affected the survival and moulting of themselves, but these were culture medium-dependent . On the other hand, we have observed that several L3 and L4 were attached, by means of a brown unknown substance apparently secreted by themselves, to the bottom of the substratum . Frequently, when a larva was spontaneously detached, a "cap" of the brown substance blocked, apparently, its mouth . The possible absorption of nutrients through the L3 larvae cuticle of A . simplex is discussed.

Hum Reprod, 1997 Nov, 12(11), 2493 - 8
The supplementation of culture medium with protease improves the hatching rate of mouse embryos; Lee DR et al.; Mammalian embryos are known to exhibit delayed development and have lower hatching rates in vitro than in vivo because of inadequate culture condition . These discrepancies may be due to a deficiency of the paracrine factors and proteolytic enzymes which exist in the oviduct and uterus . In order to evaluate the effects of proteases on embryonic development and hatching, 2-cell mouse embryos were cultured for 72 h with or without proteases . The addition of 1.0 microg/ml pronase (PE) and/or 0.1 microg/ml proteinase K (PK) did not affect embryonic development up to the blastocyst stage (94.1% versus 88.2%; 92.2% versus 90.2%, respectively) but significantly increased the hatching rate (60.4% versus 39.2%, 71.8% versus 35.3%, respectively) . However, the addition of alpha-chymotrypsin (Chymo) was detrimental to embryonic development and hatching . Changes in the structure of the zona pellucida (ZP) structure of embryos which had been cultured in human tubal fluid (HTF) medium with PE and PK were assessed by fluorescein isothiocyanate-conjugated (FITC)-casein . Embryos cultured in HTF-PE and PK were not stained with FITC-casein . When these embryos were cultured within oviducts, their perivitelline space (PVS) became strongly stained with FITC-casein which was easily removed by phosphate-buffered saline washing . This suggests that PE and PK altered the structure of the ZP . We suggest that the addition of PE and PK to culture media may accelerate the hatching of embryo, by structurally altering the ZP and PVS . This may provide a valuable and effective assisted hatching technique for human in-vitro fertilization-embryo transfer.

C R Acad Sci III, 1997 Oct, 320(10), 805 - 10
{Production of hyaluronidase by cultured human tumor cells}; Victor R et al.; The presence of hyaluronidase was detected at pH 3.8 in eight out of twelve human cancer cell line culture media . Eight cell lines derived from primary tumours and four from metastases . In three culture media the enzymatic activity was lower than 0.035 pU/cell/h . In five others (in a hepatoma cell line and in four metastasis-derived cell lines) the activity was higher than 0.057 pU/cell/h . A tumour-derived fibroblast culture was negative . The optimal activity was observed at a pH comprised between 3.6 and 4 . Salt inhibition of hyaluronidase was reversible . The enzyme was denaturated by a 10-min heating at 70 degrees C . The enzyme was not strictly specific for hyaluronan hydrolysis but also digested chondroitin sulfates . PH20, a spermatozoid protein that has homologies with the bee venom hyaluronidase, was not expressed by cell lines tested.

Exp Cell Res, 1997 Dec 15, 237(2), 247 - 58
Phenotypic modulation of hamster acinar cells by culture in collagen matrix; Yuan S et al.; The aim of this study was to assess the effect of different culture conditions on the survival and morphological phenotype of cultured acinar cells . Acinar fragments isolated from hamster pancreas were embedded in rat-tail collagen . Four groups were established: Medium 1-5% NuSerum + basic medium (basic medium = DMEM/F12 supplemented with dexamethasone, 3-isobutyl-2-methylxanthine, and antibiotics); Medium 2-10% NuSerum + basic medium . Medium 3-Medium 2 supplemented with epidermal growth factor and cholera toxin; and Medium 4:-Medium 3 supplemented with soybean trypsin inhibitor . Freshly isolated acinar cells were retrieved morphologically intact . In Medium 1, more than 80% of cells retained a normal histological appearance at 34 days in culture . Immunostaining for amylase was observed at the apical pole of the cells . The remaining cells showed variable degrees of degeneration . In Medium 2, approximately 50% of acinar cells appeared normal at 34 days in culture, while the remainder were severely degenerated . A few cystic structures were also observed . Positive immunostaining for amylase was limited to the cells with a normal histological appearance . The cells grown in Media 3 and 4 had similar courses of morphological changes . After 8 days in culture, most acinar fragments disappeared and were replaced by cystic structures, lined by a single layer of cuboidal cells . Some amylase-positive immunoreactive cells were integral components of the cystic wall . Cellular amylase activity was a function of the different culture media, a more rapid decrease in amylase activity being observed in Media 3 and 4 . Uptake of {3H}thymidine did not show any significant differences between the media . It was also found that the ductlike cells cultured in Medium 4 had a limited capacity to redifferentiate into acinar cells . This study shows that the acinar cell phenotype can be maintained in vitro for more than 1 month . This study also suggests that ductal-like epithelial structures arise from transformation of acinar cells.

Melanoma Res, 1997 Oct, 7(5), 353 - 63
In vitro characterization of lectin-induced alterations on the proliferative activity of three human melanoma cell lines; Lorea P et al.; Lectin binding is known to be able to elicit signalling events relevant for various aspects of cell physiology . The influence of lectin binding on melanoma cells remains relatively unexplored . The aim of our study was to investigate the in vitro effects of five plant lectins, namely peanut (PNA), wheat germ (WGA), concanavalin A (Con-A), Griffonia simplicifolia (GSA-IA4) and Phaseolus vulgaris (PHA-L) agglutinins, on the cell proliferation of melanoma cell lines (SK-MEL-28, HT-144 and C32) cultured in media supplemented with either 10% or 1% fetal calf serum (FCS) . Cell proliferation was assessed by means of the tetrazolium derivative reduction (MTT) assay . Four lectin concentrations were tested, namely 0.05, 0.5, 5 and 50 micrograms/ml, in four experimental settings, namely 1, 3, 5 and 7 days after the addition of each lectin to the culture media . Determination of the cell gain compartment (percentage of cells in the S and G2 phases of the cell cycle) was done by means of digital cell image analysis assessed on Feulgen-stained nuclei . Our results demonstrated that of the five lectins under study, four had a globally significant dose-dependent toxic effect on melanoma cell proliferation . The fifth lectin, PNA, had a significant stimulatory effect on the C32 cell line . Low doses of lectins may produce a transient increase in cell proliferation . Increasing the FCS from 1% to 10% in the culture media significantly antagonized lectin-induced toxicity in the three cell lines . The cell kinetics measurements showed that the inhibition of cell growth was merely due to cell death . The present data strongly suggest that some lectins might influence the proliferation of melanoma cells . In addition, because lectins are present in our diet and are able to pass into the systemic circulation, we speculate that lectins may exert an influence on melanoma growth under clinical conditions.

Gene, 1997 Dec 12, 203(2), 141 - 8
Secretion of functional Renilla reniformis luciferase by mammalian cells; Liu J et al.; The soft coral Renilla reniformis luciferase enzyme is a monomeric soluble intracellular protein that is used increasingly as a marker of gene expression . Here the Renilla luciferase gene was engineered to encode a protein product secreted by mammalian cells . The 5' end of the Renilla luciferase gene was fused in frame with the 3' end of a short DNA sequence encoding the signal peptide from human interleukin-2 (IL-2) protein . This construct was cloned under transcriptional control of the cytomegalovirus (CMV) promoter in a mammalian expression vector . Simian COS-7 cells were transiently transfected with the construct, and light emission was measured from cell lysates and from cell culture media . The results of these experiments indicated that Renilla luciferase was secreted as a functional enzyme by mammalian cells . The advantages and disadvantages of secreted Renilla luciferase as a marker of gene expression in comparison to other secreted protein markers are discussed.

J Vasc Surg, 1997 Dec, 26(6), 994 - 9; discussion 999-1001
Reduced growth of dermal fibroblasts from chronic venous ulcers can be stimulated with growth factors; Stanley AC et al.; PURPOSE: Although the slow healing rate of venous ulcers is well known, the underlying defect in the healing process is not well understood . The purpose of this study was to examine the cellular characteristics of fibroblasts taken from venous ulcers (wound-fb) and compare them with the fibroblasts of normal tissue (normal-fb) . METHODS: Biopsy specimens were obtained from wound margins and normal tissue of the upper thigh in each patient . Dermal fibroblasts were isolated from explant cultures in Dulbecco's modified Eagle's medium supplemented with 10% calf serum . These cells were then plated at 1000 cells per plate, and total cells per plate were counted over time so that growth curves could be generated . In further experimentation, media was supplemented with additional calf serum (20%, 30%, 40%, 50%) and growth factors (epidermal growth factor, basic fibroblast growth factor, interleukin-1 beta) in an attempt to stimulate growth . RESULTS: Two major differences were noted: (1) normal-fb replicated more rapidly than wound-fb; and (2) the morphologic features of wound-fb were different . Normal-fb were compact and tapered, with well-defined nuclear morphologic features . Wound-fb were larger and polygonal in shape, with less-uniform nuclear morphologic features . Additional calf serum in tissue culture media enhanced normal-fb growth but had no effect on wound-fb . Supplementation of media with growth factors stimulated the growth of wound-fb . Statistically significant differences were noted at day 10 and 14 with basic fibroblast growth factor supplementation (p = 0.02 and 0.0001, respectively) and at day 14 with epidermal growth factor (p = 0.008) . Although interleukin-1 beta stimulated cell growth in five of six patients, the differences observed were not statistically significant . CONCLUSIONS: Our data demonstrate that wound-fb proliferate at a slower rate and are morphologically distinct from normal-fb . These characteristics are typical of aged or senescent cells . This decreased growth can be stimulated by growth factors basic fibroblast growth factor, epidermal growth factor, and interleukin-1 beta . Slowed growth may be partially responsible for the defect in healing of venous stasis ulcers . Furthermore, we believe that in some patients ulcer healing may be improved by exogenous provision of specific growth factors.

Acta Anat (Basel), 1997, 158(4), 247 - 54
11.5-day rat embryos cultured in vitro after introduction of immunoglobulin G into the vitelline circulation; Mensah-Brown EP et al.; The fate of rat immunoglobulin G (IgG) in the 11.5-day-rat conceptus cultured in vitro has been studied utilizing the intravitelline cannulation technique . When IgG bound to colloidal gold was introduced into the vitelline circulation, gold particles were detected on the luminal surface of embryonic endothelial cells, in both coated pits and vesicles and in various portions of the vacuolar system of the embryonic endothelial cell . By means of the radiolabeled macromolecule, it has been demonstrated that the internalized IgG was not degraded . In comparison, digested products of radiolabeled bovine serum albumin (BSA) were detected in culture media after the macromolecule was introduced into the conceptus . It was therefore concluded that the 11.5-day rat embryo captures IgG probably by receptor-mediated endocytosis and does not degrade the macromolecule, indicating that IgG is not routed to the lysosomal compartment of the endothelial cell even though the embryo has the capacity to digest BSA . It appears therefore that the embryo is endowed with the capacity to handle the IgG macromolecule well before the macromolecule is introduced into it for passive immunity.

Circulation, 1997 Nov 4, 96(9 Suppl), II - 253-9
Potassium channel opener-augmented cardioplegia: protection of myocyte contractility with chronic left ventricular dysfunction; Dorman BH et al.; BACKGROUND: An increased number of patients with preexisting left ventricular (LV) dysfunction and congestive heart failure (CHF) are undergoing cardiac surgery with a higher risk for decreased LV contractility after hyperkalemic cardioplegic arrest . Activation of adenosine triphosphate-sensitive potassium channels by potassium channel openers (PCO) within the myocyte appears to confer a protective effect in the setting of ischemia . Accordingly, the present study was designed to determine whether PCO supplementation during hyperkalemic cardioplegic arrest would provide protective effects on myocyte contractile function, particularly in the setting of CHF . METHODS AND RESULTS: LV myocytes were isolated from control pigs (n=7) and pigs with CHF (rapid pacing, 240 beats per minute; n=7) and then assigned to the following treatment groups: normothermia (cell culture media, 2 hours, 37 degrees C); cardioplegia (24 mEq/L K+, 2 hours, 4 degrees C; then 10 minutes of reperfusion); or PCO/cardioplegia (cardioplegia supplemented with 100 micromol/L of the PCO aprikalim) . Myocyte velocity of shortening was reduced in both control (66+/-2 versus 33+/-1 microm/s) and CHFmyocytes (32+/-1 versus 22+/-1 microm/s) after hyperkalemic cardioplegic arrest (P<.05) . Contractility after PCO cardioplegia was similar to normothermic values in control (57+/-2 microm/s) and CHF (33+/-1 microm/s) myocytes (P<.05) . Intracellular free Ca2+ increased from normothermia during hyperkalemic cardioplegia in control (81+/-4 to 145+/-7 nmol/L) and CHF (262+/-30 to 823+/-55 nmol/L) myocytes (P<.05) . PCO cardioplegia attenuated the intracellular increase in free Ca2+ during the cardioplegic interval in control (110+/-6 nmol/L) and CHF (383+22 nmol/L) myocytes (P<.05) . CONCLUSIONS: PCO-augmented cardioplegic arrest preserved myocyte contractility and reduced the intracellular free Ca2+ release, which therefore may be of particular benefit in the setting of preexisting LV dysfunction.

Cancer, 1997 Dec 15, 80(12 Suppl), 2660 - 6
Generation of a high-producing clone of a humanized anti-B-cell lymphoma monoclonal antibody (hLL2); Losman MJ et al.; BACKGROUND: LL2 is a murine immunoglobulin (Ig)G2a-kappa anti-B-cell monoclonal antibody with proven targeting and therapeutic efficacy in the management of non-Hodgkin's lymphoma (NHL) . The authors had previously generated a humanized LL2 (hLL2) that demonstrated binding properties identical to those of LL2 . Nevertheless, the productivity of the cell line was insufficient for large-scale production of the antibody for clinical studies . Therefore, the authors chose an amplifiable system for the generation of hLL2 . METHODS: The hLL2 sequences were ligated into the expression vector pdHL2, which has a dhfr amplifiable gene, and were incorporated into the SP2/0 cells by electroporation . A methotrexate (MTX) resistant clone producing hLL2 was identified . Stepwise increases in MTX concentrations, from 0.1 to 5 microM, and subcloning of the cells by limiting dilution were performed . RESULTS: By amplifying the dhfr and hLL2 genes with stepwise increases in the MTX concentration, the antibody production was enhanced from its original 1.4 to 70 +/- 5 mg per liter of culture media . Subsequent subcloning further improved the productivity . Immunoreactivity of the antibody was conserved, as proven by enzyme-linked immunosorbent assay and cell-binding assays . By isoelectrofocusing, the isoelectric point (pI) of the antibody was measured at approximately 9.6 . The productivity of the clone was not affected by culture conditions or storage of the cells in liquid nitrogen . CONCLUSIONS: By means of gene amplification, the authors have generated a high-producing hLL2-IgG clone suitable for production of the quantity of antibody necessary for clinical diagnostic and therapeutic trials of NHL patients.

J Pineal Res, 1997 Oct, 23(3), 156 - 63
Influence of melatonin and serotonin on glucose-stimulated insulin release from perifused rat pancreatic islets in vitro; Peschke E et al.; Insulin plays a key role in the control of glucose homeostasis in mammals . Insulin secretion is regulated by a coordinated interplay of several factors . The role of the indoleamines in the control of insulin secretion has not been fully elucidated yet . The present study was addressed to investigate the function of melatonin and serotonin in the direct control of insulin secretion from the pancreatic islets . Explanted rat Langerhans' islets were treated with melatonin or serotonin while also being exposed to specific (glucose) or non-specific (KCl) stimulus either in a pulsatile or long-term manner in a perifusion system . Insulin content from the effluent tissue culture media was analyzed with RIA . Pulsatile administration of melatonin and serotonin alone did not alter the basal insulin secretion from the explanted islets even at pharmacological (5 microM) level . However, insulin response to specific (glucose) or non-specific (KCl) stimulus was significantly reduced while the islets were treated with melatonin (3 to 12 hr, 10 nM to 5 microM) . This effect was reversible and repeatable . Both the start and end of the effect was rapid, evolving and disappearing within 10 min . On the other hand, under similar experimental protocol, serotonin (at 5 microM concentration) significantly enhanced both glucose and KCl stimulated insulin release . Since the effect of the non-specific stimulation (with KCl) was also altered, melatonin and serotonin seem to alter not only the release but also the synthesis of the insulin . Our data show that melatonin and serotonin have a direct effect on the insulin secretion from the pancreatic islets.

Mol Cell Biochem, 1997 Nov, 176(1-2), 273 - 9
Cardiac hypertrophy: old concepts, new perspectives; Gupta M et al.; Growth of the heart in hypertrophy is accompanied by changes in the phenotypic expression of cardiac genes . To explore the molecular basis of cardiac hypertrophy, we have analyzed the regulation of myosin heavy chain gene (MHC) expression . In one set of experiments, pressure overload on the rat heart was produced by constriction of the abdominal aorta . Changes in the alpha and beta-MHC mRNA were then studied in overloaded hearts and following load removal . Pressure overload resulted in down-regulation of the alpha-MHC with corresponding up-regulation of the steady state level of beta-MHC mRNA . Load removal (debanding) resulted in regression of cardiac hypertrophy and a rapid return of alpha-MHC mRNA to normal values . In contrast, the recovery in beta-MHC mRNA was much slower to the extent that it remained substantially elevated compared to respective sham controls even after 7 weeks of post-debanding . These results suggest that putative load-related signals independently regulate two genes . Several lines of evidence indicate that adrenergic nervous system plays an important role in the induction and maintenance of cardiac hypertrophy and in the redistribution of myosin isoforms . We have analyzed the effect of cAMP inducing agents on the regulation of alpha-MHC gene in primary cultures of the fetal (18 day) rat cardiac myocyte . Inclusion of 8 Br-cAMP in the culture media increased the expression of alpha-MHC promoter/reporter construct comprising of 2.9 kb upstream sequence of the alpha-MHC gene . Several deletion mutations in the alpha-MHC gene promoter defined the cAMP responsive boundaries to be a 32 bp region comprising of -71 to -40 bp sequences . Deletion of this region resulted in loss of cAMP response as well as in basal expression of alpha-MHC promoter/reporter construct . These data suggest a role of beta-adrenergic pathway in the modulation of alpha-MHC gene expression.

J Med Microbiol, 1997 Dec, 46(12), 1043 - 6
PCR identification of Trichophyton mentagrophytes var . interdigitale and T . mentagrophytes var . mentagrophytes dermatophytes with a random primer; Liu D et al.; Dermatophytes are a group of keratinophilic fungi falling within the genera of Epidermophyton, Microsporum and Trichophyton . The genus Trichophyton is particularly important and complex; it comprises at least 15 recognised species . In addition, there are several different variants in the species T . mentagrophytes, which occur both in man and animals . The current methods of determining T . mentagrophytes varieties may require several different culture media and time-consuming procedures, as well as specialist skills . This study used a random primer, 5'-GAGCCCGACT-3', in the arbitrarily primed polymerase chain reaction (AP-PCR) and showed that the two common T . mentagrophytes varieties (var . interdigitale and var . mentagrophytes) can be clearly identified on the basis of their characteristic DNA band patterns . The relative reproducibility, ease of use and precision of this method make the AP-PCR a valuable tool in the laboratory diagnosis of human dermatophytosis.

Transpl Immunol, 1997 Sep, 5(3), 225 - 32
Unexpected augmentation of mycophenolic acid pharmacokinetics in renal transplant patients receiving tacrolimus and mycophenolate mofetil in combination therapy, and analogous in vitro findings; Zucker K et al.; Mycophenolate mofetil (MMF) a potent immunosuppressive agent, has recently been approved for clinical use (CellCept) in renal transplant patients in combination with cyclosporine (CsA) . With the expanded use of tacrolimus (Prograf) as well in renal transplant patients, there is a lack of pharmacokinetic studies clarifying drug interactions between the three agents . A pharmacokinetic study was performed on 18 stable renal transplant patients receiving MMF and tacrolimus together, and four control groups, one receiving tacrolimus alone, two receiving CsA, in combination with MMF (1.0 or 1.5 g bid), and one receiving CsA microemulsion (Neoral) . Area-under-the-curve values were calculated for each drug to assess if there was a reciprocal effect on the respective bioavailability of each . In vitro, the immunosuppressive effect of trough level plasma from each patient group was studied using mixed lymphocyte culture (MLC), as well as MLC reactions spiked with various combinations of each drug . There was a minimal effect of MMF on tacrolimus pharmacokinetics . However, patients receiving tacrolimus and MMF displayed significantly higher levels (Cmin and area under the curve) of mycophenolic acid (MPA) than those receiving CsA (Sandimmune or Neoral) and the same dose of MMF (50.2 +/- 16.5 vs 32.1 +/- 16.7 micrograms h/ml AUC, p < 0.02) . Equivalent MPA levels could be attained in patients receiving CsA if the MMF dose was increased by 50% (1.5 g bid) . There were also significantly lower levels of the glucuronide metabolite of MPA (MPAG) (755 +/- 280 vs 1230 +/- 250 micrograms h/ml AUC, p = 0.02), suggesting a specific inhibition (either direct or indirect) of the conversion of MPA to MPAG in tacrolimus patients, as opposed to those receiving CsA . For each drug combination, there was a positive correlation between the plasma immunosuppressive effect seen in MLC assays and the MMF dose . In addition, trough plasma from patients receiving tacrolimus and MMF was significantly more MLC inhibitory than from those receiving CsA or CsA microemulsion and equivalent-dose MMF . Culture media containing MPA and tacrolimus equal to clinical therapeutic trough concentrations (10 ng/ml) were significantly more MLC inhibitory than CsA at equivalent clinical therapeutic trough concentrations (200 ng/ml) with equivalent MPA levels . These studies in renal transplant patients suggest that tacrolimus in combination with MMF may result in a greater degree of immunosuppression than may be anticipated.

Hum Reprod, 1997 Oct, 12(10), 2263 - 6
Prospective randomized study comparing human serum albumin with fetal cord serum as protein supplement in culture medium for in-vitro fertilization; Laverge H et al.; The use of human serum albumin (HSA) instead of fetal cord serum (FCoS) as protein supplement highly simplifies the preparation of culture medium for human in-vitro fertilization (IVF) but whether they are equivalent in sustaining embryo development is still controversial . We performed a prospective randomized study of patients undergoing IVF or intracytoplasmic sperm injection (ICSI) where embryos were cultured in Earle's balanced salt solution containing either 8% (v/v) FCoS or 0.4% (w/v) HSA as protein source . Fertilization rates, morphological embryonic quality and pregnancy rates were compared . A total of 2189 oocytes from 210 cycles were cultured in medium supplemented with HSA in patient group 1 and 2109 oocytes from 203 cycles in medium supplemented with FCoS in patient group 2 . The fertilization rate, defined as the presence of two nuclei, for microinjected oocytes was similar in both patient groups (77.4 and 76.7%, respectively) . The fertilization rate for inseminated oocyte-cumulus complexes was significantly higher in the HSA group than in the FCoS group (62.9 versus 53.8%, P < 0.025) . The embryonic quality was significantly better after culture in medium supplemented with HSA than with FCoS (13.7 versus 9.9% morphologically excellent embryos, P < 0.001) . Implantation rates per transferred embryo were not significantly different (22.5 versus 18.2%), but there was a significantly higher pregnancy rate per embryo transfer in the HSA group (45.7 versus 35.9%, P < 0.05, respectively) . Non-evolutive pregnancy rates were significantly different (27.4 and 16.7%) . Our data demonstrate that the use of human serum albumin as a protein supplement for culture medium in human IVF programmes is associated with improved embryonic quality and significantly higher pregnancy rates . For this reason as well as the additional benefits of being virus-free and being purified, HSA is preferable to FCoS for the preparation of culture media in human IVF.

Biol Pharm Bull, 1997 Nov, 20(11), 1131 - 5
Inhibitory effect of oversulfated fucoidan on tube formation by human vascular endothelial cells; Soeda S et al.; Fucoidan is a sulfated poly(L-fucopyranose) present in brown marine algae . In this study, we examined the effect of native and chemically oversulfated fucoidans (NF and OSF) on the tube structure formation by human umbilical vein endothelial cells (HUVEC) on the basement membrane preparation, Matrigel . Unlike NF, OSF significantly decreased the tube formation: maximal inhibition (50% of control) was obtained with 25 micrograms/ml . The OSF effect was mediated, at least in part, through the inhibition of HUVEC migration, as determined by the ability to block chemotaxis in a Transwell chamber assay . Quantitative immunoreactive assays for tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) in the culture media indicated that OSF (25 micrograms/ml) increased the accumulation of PAI-1 antigen, but not of t-PA antigen, 2.7-fold compared with control . The release of both antigens by HUVEC was slightly affected by the addition of NF . Determination of the media levels of type IV collagenase activity and tissue inhibitor of metalloproteinase-1 (TIMP-1) antigen showed that OSF (25 micrograms/ml) decreased the collagenolytic activity by 50% compared to the control, without alteration of the TIMP antigen level . However, the collagenase inhibition by OSF was not observed in an assay system using purified enzyme . NF had no effect on collagenase activity or TIMP-1 antigen levels . These results indicate that the introduction of sulfate groups into NF enables it to effectively inhibit the formation of capillary-like structures by HUVEC on Matrigel by reducing the basement membrane destruction and cell migration . It is involved as at least one of the mechanisms by which the OSF-induced increase in HUVEC PAI-1 decreases plasmin formation and suppresses the following pro-collagenase activation.

Immunol Invest, 1997 Aug-Dec, 26(5-7), 561 - 8
In vitro suppression of the normal mitogenic T lymphocyte response by steady state sickle cell disease sera; Taylor S et al.; This study is part of a long term evaluation of sickle cell disease (SCD) as a paradigm for immunosuppression . Serum was obtained from 43 SCD patients during the steady (healthy) state . Peripheral blood mononuclear cells (PBMC), separated by density gradient were obtained from 8 normal healthy donors . PBMC were utilized in assays directly or as a source for obtaining, total T (CD3) and helper T (CD4) cell populations separated by specific T cell columns . Standard in vitro phytohemagglutinin (PHA) stimulation of lymphocyte cultures was done with culture media containing 10% SCD serum, as compared to normal pooled O, Rh+ (O+) serum . Mitogenic responses were expressed as mean counts per minute (cpm) and stimulation index of triplicate cultures . Results revealed PHA responses were positive in all experiments when a standard stimulation index of 10 or greater was used as a test parameter for comparison . Positive results were demonstrated in 43/43 (100%) of triplicate cultures regardless of serum type in all experiments . Conversely, by using mean cpm as the test criterion, suppression of PHA response was shown in SCD serum supplemented cells as follow; 36/43 (84%) of PBMC, 35/43 (81%) of CD3 and 37/43 (86%) of CD4 cultures . The degree of suppression ranged from > 10% to 98% in individual experiments, as compared to O+ serum . Inhibitors of normal T lymphocyte in vitro PHA response appear to be present in a significant percentage of SCD sera even during the healthy state of disease . Type 2 cytokines which suppress cell mediated immunity would seem to be the most likely inhibitory agents.

J Am Podiatr Med Assoc, 1997 Nov, 87(11), 498 - 506
Pathologic and diagnostic considerations in onychomycosis; Lemont H; Understanding the physiology and function of the nail unit and its potential avenues of invasion, and properly identifying invading organisms are two key aspects of using the newer therapies available for the treatment of onychomycosis . This article discusses the most common pathologies of onychomycosis, as classified by the sites of entry of the invading fungi . Susceptibility factors leading to infection are also discussed . Obtaining proper tissue samples, using appropriate tests and culture media, and accurately interpreting test results are all paramount to correct identification of the invading organism and, in turn, to effective prescribing . When fungal-growth results do not support the clinical symptoms, or if a more specific identification of the organism is required, additional diagnostic tests are available and are outlined here.

Pediatr Res, 1997 Dec, 42(6), 788 - 93
The production of macrophage inflammatory protein-1alpha in the cerebrospinal fluid at the initial stage of meningitis in children; Inaba Y et al.; Neutrophils in the cerebrospinal fluid (CSF) increase during the initial stage of meningitis . Some cytokines induce the accumulation of such neutrophils, and we and other investigators have revealed transient increases in the levels of granulocyte-colony stimulating factor (G-csf) and IL-8 in the CSF of patients with meningitis . To explore the coordination of other cytokines with G-csf and IL-8 in the neutrophil accumulation in the CSF, we herein investigated macrophage inflammatory protein-1alpha (MIP-1alpha), which can induce the infiltration of neutrophils . The modulation of MIP-1alpha levels in the CSF in children with bacterial (n = 10) and aseptic (n = 22) meningitis was examined using an ELISA . MIP-1alpha levels in the CSF were detectable at the stage with symptoms of meningitis: 289.9 +/- 270.7 ng/L in the bacterial meningitis group and 16.1 +/- 12.5 ng/L in the aseptic meningitis group . These levels decreased with the improvement of symptoms . MIP-1alpha was not detectable (<6 ng/L) in all of the control patients without meningitis (n = 19) . The MIP-1alpha levels in the CSF showed a significant correlation with the CSF neutrophil counts (r = 0.750, p < 0.0001; n = 80) of meningitis, and the values of MIP-1alpha (log ng/L)/neutrophil counts (log/L) ratio were calculated (1.003 +/- 0.576) . The MIP-1alpha levels in the serum were significantly lower than those in the CSF (p = 0.0464) . We found MIP-1alpha mRNA in the CSF cells by the reverse transcriptase-PCR method, and high levels of MIP-1alpha protein in the culture media from mononuclear cells in the CSF in vitro . In summary, The MIP-1alpha level increases in the CSF at the symptomatic stage of meningitis in children, and its cellular source is, in part, mononuclear cells which have infiltrated the CSF . We propose that MIP-1alpha, in addition to G-csf and IL-8, plays an important role in the accumulation of neutrophils in the CSF of patients with meningitis.

Circulation, 1997 Nov 18, 96(10), 3737 - 44
Significance of ventricular myocytes and nonmyocytes interaction during cardiocyte hypertrophy: evidence for endothelin-1 as a paracrine hypertrophic factor from cardiac nonmyocytes; Harada M et al.; BACKGROUND: In cardiac hypertrophy, both excessive enlargement of cardiac myocytes and progressive interstitial fibrosis are well known to occur simultaneously . In the present study, to investigate the interaction between ventricular myocytes (MCs) and cardiac nonmyocytes (NMCs), mostly fibroblasts, during cardiocytes hypertrophy, we examined the change in cell size and gene expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in cultured MCs as markers for hypertrophy in the neonatal rat ventricular cardiac cell culture system . METHODS AND RESULTS: The size of cultured MCs significantly increased in the MC-NMC coculture . Concomitantly, secretions of ANP and BNP into culture media were significantly increased in the MC-NMC coculture compared with in the MC culture (with the possible contamination of NMC <1% of MC) . Moreover, in the MC culture, enlargement of MC and an increase in ANP and BNP secretions were induced by treatment with conditioned media of the NMC culture . A considerable amount of endothelin (ET)-1 production was detected in the NMC-conditioned media . BQ-123, an ET-A receptor antagonist, and bosentan, a nonselective ET receptor antagonist, significantly blocked the hypertrophic response of MCs induced by treatment with NMC-conditioned media . Angiotensin II (Ang II) (10(-10) to 10(-6) mol/L) and transforming growth factor-beta1 (TGF-beta1) (10(-13) to 10(-9) mol/L), both of which are known to be cardiac hypertrophic factors, did not induce hypertrophy in MC culture, but both Ang II and TGF-beta1 increased the size of MCs and augmented ANP and BNP productions in the MC-NMC coculture . This hypertrophic activity of Ang II and TGF-beta1 was associated with the potentiation of ET-1 production in the MC-NMC coculture, and the effect of Ang II or TGF-beta1 on the secretions of ANP and BNP in the coculture was significantly suppressed by pretreatment with BQ-123 . CONCLUSIONS: These results demonstrate that NMCs regulate MC hypertrophy at least partially via ET-1 secretion and that the interaction between MCs and NMCs plays a critical role during the process of Ang II- or TGF-beta1-induced cardiocyte hypertrophy.

Infect Immun, 1997 Dec, 65(12), 5257 - 61
Calcium dependence and binding in cultures of Histoplasma capsulatum; Batanghari JW et al.; Histoplasma capsulatum is a pathogenic fungus with two distinct morphologies and lifestyles . The saprophytic form of this organism, a mold, thrives in soil and is especially abundant in the Ohio and Mississippi River valleys . Its parasitic counterpart, a yeast, colonizes phagolysosomes of mammalian macrophages . We have observed a major difference in the calcium requirements of the two forms of Histoplasma, potentially implicating the phagolysosome as a calcium-limiting compartment . Deprivation of calcium by the addition of EGTA to culture media inhibited the growth of mycelial H . capsulatum but had no effect on yeast growth in vitro . In addition, yeasts released a calcium-binding protein (CBP) detectable by a 45CaCl2 blotting technique . CBP was a major component of yeast culture supernatant and was also detectable by ruthenium red staining, another assay for calcium-binding activity . Conversely, mycelial H . capsulatum did not produce CBP, a finding that correlates with the dependence of mycelia on calcium for growth . We also describe here the purification of CBP from yeast culture supernatant by reversed-phase high-pressure liquid chromatography.

Genitourin Med, 1997 Aug, 73(4), 297 - 8
A comparison of the sensitivity of the InPouch TV, Diamond's and Trichosel media for detection of Trichomonas vaginalis; Borchardt KA et al.; OBJECTIVE: This study compared the ability of three culture media (InPouch TV, Diamond's, and Trichosel) to support the growth of clinical isolates of Trichomonas vaginalis and their relative sensitivity for detection of the organism . METHODS: The majority of the clinical isolates were obtained from two San Francisco Bay Area clinics . T vaginalis was subcultured in 4 ml of one of the InPouch, Diamond's, or Trichosel media for 24-48 hours before evaluation . Twenty isolates were initially cultured in the InPouch test, 13 with Diamond's, and 10 with Trichosel . A haemocytometer was used to measure the initial concentrations of the organisms . Then serial dilutions were made in saline to yield approximately 2.0 x 10(4), 2.0 x 10(3), and 2.0 x 10(2) motile T vaginalis per ml . A 30 microliter inoculum from each dilution was transferred into 4 ml aliquots of the three media (387 individual tests, 43 x 3 dilutions x 3 media) . Microscopic examinations for viable trichomonads were made at 24, 48, and 96 hours . Microscopy was through the pouch wall for the InPouch medium, and through a cover slipped slide with one drop of Diamond's and Trichosel media . RESULTS: At 24 hours, the InPouch demonstrated 84/129 positive, Diamond's 23/129, and Trichosel 18/129 . At 48 hours, an accumulative positive rate for the InPouch was 98/129, for Diamond's 55/129, and Trichosel 47/129 . At 96 hours the total positives for each test were 112/129 for the InPouch, 78/129 for Diamond's, and 74/129 for Trichosel . CONCLUSIONS: The InPouch TV test was significantly more sensitive than either Diamond's or Trichosel (at 0.01 level of significance, pInPouch > pDiamond's; pInPouch > pTrichosel on all three dilutions at 24, 48, and 96 hours) . This increased sensitivity was the result of either a reduced generation time or the larger volume of media examined microscopically.

Growth Factors, 1997, 14(4), 297 - 306
Regional variability in the time course of TGF-beta 1 expression, cellular proliferation and extracellular matrix expansion following arterial injury; Creighton WM et al.; Transforming growth factor-beta 1 (TGF-beta 1) has been variably associated with the regulation of cellular proliferation and extracellular matrix expansion after arterial injury . We tested these associations in vivo in the rat carotid injury model . At 0, 3, 7, 14 and 28 days following arterial balloon injury, regional expression of TGF-beta 1 mRNA was assessed using in situ hybridization and the results compared to measures of cellular proliferation and extracellular matrix expansion . Both the TGF-beta 1 concentration measured in culture media of explanted carotid arteries and the quantitative in situ hybridization signal for TGF-beta 1 arterial media and neointima were maximal at 14 days after balloon injury . However, medial cellular proliferation was maximal at 3 days whereas neointimal proliferation was maximal at 14 days and significantly greater than medial proliferation . Neointimal cell density declined significantly between 7 and 14 days, indicating the expansion of extracellular matrix; however, medial cell density was unchanged between 3 and 28 days after balloon injury . Thus, differences in the regional arterial wall relationships between the time course of cellular proliferation, extracellular matrix expansion and the level of TGF-beta 1 expression demonstrate in vivo variability in the response to TGF-beta 1.

Arch Dermatol Res, 1997 Sep, 289(10), 585 - 95
Culture of reconstructed epidermis in a defined medium at 33 degrees C shows a delayed epidermal maturation, prolonged lifespan and improved stratum corneum; Gibbs S et al.; In this study we compared human keratinocyte cultures grown at the air-liquid interface on de-epidermized dermis at 33 degrees C or at 37 degrees C in two different culture media: medium I--a fully defined serum- and EGF-free medium; and medium II-a serum- and EGF-containing medium . Cultures grown in medium II were initially hyperproliferative followed rapidly by senescence, and had a high triglyceride content . The hyperproliferation was ascribed to the presence of EGF in the medium . In contrast, cultures grown in medium I at 33 degrees C showed a greatly improved balance between cell proliferation and differentiation . They had a prolonged lifespan of at least 32 days without a significant decrease in the number of living cell layers, a rate of proliferation similar to that of native epidermis and a low triglyceride content . Culturing at 37 degrees C increased the rate of differentiation without affecting the rate of proliferation . Furthermore, both at 33 degrees C and at 37 degrees C, keratin 6 was expressed only in the first suprabasal layer but was expressed in all suprabasal layers in cultures grown in medium II . High keratin 6 expression was not directly linked to hyperproliferation but to deregulated terminal differentiation . Involucrin, transglutaminase and SPRR1 were abnormally expressed irrespective of the culture conditions used, whereas SKALP expression was decreased in cultures grown in medium I . The epidermal lipid profile was better in cultures grown in medium I; the relative amounts of ceramides, free fatty acids and cholesterol being comparable to native epidermis . Small-angle X-ray diffraction showed a slightly improved structural organization of stratum corneum lipids as demonstrated by the appearance of second- and third-order peaks of the 12-nm long phase and a marked reduction in the polycrystalline cholesterol peak.

Osteoporos Int, 1997, 7(4), 323 - 30
Progesterone-mediated stimulation of osteoprogenitor proliferation and differentiation in cell populations derived from adult or fetal rat bone tissue depends on the serum component of the culture media; Ishida Y et al.; We have shown previously that progesterone (Prog) and dexamethasone (Dex) stimulate osteoprogenitor proliferation and differentiation in cell populations derived from adult rat vertebrae and in primary cultures of fetal rat calvariae . In these two in vitro systems, osteoprogenitors can be identified by the appearance of colonies of differentiated osteoblasts producing bone (bone nodule formation) . Culture conditions supporting proliferation and differentiation of osteoprogenitors include a requirement for the presence of serum in the culture media . Our major interest in the present study was to investigate whether Prog- and Dex-mediated osteoprogenitor proliferation and differentiation was observed to the same degree in different lots of fetal bovine serum (FBS) . In addition, we wanted to investigate whether osteoprogenitors present in cell populations derived from fetal calvarial bone and those present in populations derived from adult vertebral bone would respond similarly under the different culture conditions . We found that, in populations derived from adult rat vertebrae, the effects of the serum component of the culture medium on the number of bone nodules induced by Prog and on the dose-dependency of the Prog effect were striking: in culture media containing the most effective serum the number of bone nodules was 22-fold higher than that in the least effective serum . In addition, Prog responses were detectable at 10(-5) M only in some sera but were significant at 10(-7) M in others . The effect of Dex in the adult rat vertebrae-derived populations was much less dependent on the serum used: the number of bone nodules in culture media containing the most effective serum was only 1.3 times greater than that in media containing the least effective serum . In cell populations derived from fetal calvariae, the serum dependence of the Prog response was less pronounced: a 4.3-fold increase over control was observed in the most effective serum, and a 2.4-fold increase in the least effective serum . No effects of the serum component of the culture medium on the Dex response were detectable . Thus, Prog-induced bone nodule formation appears to be strongly dependent on the particular type of FBS used for osteoprogenitors present in bone cell populations derived from adult rat vertebrae but much less so in populations obtained from fetal rat calvariae . Preliminary experiments suggest that the estrogen content of the culture media may be one of the determinants regulating Prog responsiveness of the osteoprogenitors . Dex-induced proliferation and differentiation of osteoprogenitors in bone cell populations derived from both adult rat vertebrae and fetal rat calvariae, on the other hand, did not appear to be strongly dependent on factor(s) present in the FBS component of the culture medium.

Am J Obstet Gynecol, 1997 Oct, 177(4), 918 - 23
Antiphospholipid immunoglobulin G antibodies reduce annexin-V levels on syncytiotrophoblast apical membranes and in culture media of placental villi; Rand JH et al.; OBJECTIVES: The mechanism by which antiphospholipid antibodies are associated with pregnancy loss and thromboembolism has not been established . We previously showed that annexin-V, a phospholipid-binding protein with potent anticoagulant activity, is present on the apical membranes of the syncytiotrophoblasts that line placental villi and that this protein is reduced, by immunohistochemistry, on placentas of patients with antiphospholipid antibodies . We therefore investigated whether annexin-V in apical membranes of placental villi is quantitatively reduced by antiphospholipid antibody immunoglobulin G . STUDY DESIGN: Placentas were obtained from an index patient with antiphospholipid syndrome with intrauterine growth restriction and from a patient with an uncomplicated pregnancy who were both delivered by cesarean section . Apical villous membranes were isolated and annexin-V levels were measured by enzyme-linked immunosorbent assay . We then studied the effects of antiphospholipid immunoglobulin G on placental villous apical annexin-V in vitro . Antiphospholipid immunoglobulin G was isolated from the sera of five different patients with antiphospholipid antibody syndrome along with five paired control immunoglobulin Gs . Short-term cultures were established from normal placental villi and were exposed to the antibodies, after which isolated apical membranes and culture media were immunoassayed for annexin-V levels . RESULTS: Measurements of apical membrane-associated annexin-V from the antiphospholipid placenta showed significantly less apical membrane-associated annexin-V than did the normal placenta (mean +/- SEM: 4.9 +/- 0.4 micrograms/gm villi for antiphospholipid placenta vs 10.2 +/- 0.6 micrograms/gm villi for control, p < 0.001, n = 4) . Exposure of placental villous cultures to five different antiphospholipid immunoglobulin Gs for 24 hours resulted in significant reduction of the levels of apical membrane annexin-V (mean +/- SEM: 3.9 +/- 0.3 micrograms/gm villi) compared with paired controls (5.1 +/- 0.3 micrograms/gm villi, p = 0.02) . Villi incubated with the different antiphospholipid immunoglobulin Gs had significantly less annexin-V in conditioned media (mean +/- SEM: 45.1 +/- 4.9 ng/gm villi) compared with the paired normal immunoglobulin G control levels (72.6 +/- 11.4 ng/gm villi, p = 0.03) . CONCLUSIONS: Antiphospholipid immunoglobulin G reduces the levels of syncytiotrophoblast apical membrane-associated annexin-V in placental villi and the release of annexin-V into surrounding media . Reduction of this anticoagulant protein at the maternal-fetal interface may account for the pregnancy loss observed in patients with antiphospholipid syndrome . Short-term culture of placental villi may offer an in vitro model to further study the mechanism of this effect of antiphospholipid antibodies.

J Biomed Mater Res, 1997 Dec 5, 37(3), 324 - 34
The effects of calcium phosphate particles on the growth of osteoblasts; Sun JS et al.; With advances in ceramics technology, calcium phosphate bioceramics have been applied as bone substitutes for several decades . The focus of this work is to elucidate the biocompatibility of the particulates of various calcium phosphate cytotoxicities . Four different kinds of calcium phosphate powders, including beta-tricalcium phosphate (beta-TCP), hydroxyapatite (HA), beta-dicalcium pyrophosphate (beta-DCP), and sintered beta-dicalcium pyrophosphate (SDCP), were tested by osteoblast cell culture . The results were analyzed by cell count, concentration of transforming growth factor-beta 1 (TGF-beta 1), alkaline phosphatase (ALP), and prostaglandin E2 (PGE2) in culture media . The changes were most significant when osteoblasts were cultured with beta-TCP and HA bioceramics . The changes in cell population of the beta-TCP and HA were quite low in the first 3 days, then increased gradually toward the seventh day . The changes in TGF-beta 1 concentration in culture medium inversely related to the changes in cell population . The ALP titer in the culture media of the beta-TCP and HA were quite high in the first 3 days, then decreased rapidly between the third and seventh days . The concentrations of PGE2 in the culture media tested were quite high on the first day, decreased rapidly to the third day, and then gradually until the seventh day . The changes in the beta-DCP and SDCP were quite similar to those of HA and beta-TCP but much less significant . We conclude that HA and beta-TCP have an inhibitory effect on the growth of osteoblasts . The inhibitins effects of the HA and beta-TCP powders on the osteoblast cell cultures possibly are mediated by the increased synthesis of PGE2.

Brain Res, 1997 Sep 12, 768(1-2), 63 - 70
Melatonin suppression of PC12 cell growth and death; Roth JA et al.; Melatonin has previously been reported to influence cell differentiation and growth in a number of cell culture systems in vitro . In this paper, we describe the effects of high pharmacological and low physiological concentrations of melatonin on cell growth in rat pheochromocytoma cells (PC12 cells) . Melatonin produced a biphasic response with respect to cell growth in PC12 cells . At low concentrations (1-10 nM) melatonin suppressed PC12 cell growth whereas at higher concentration (10 microM) it prevented cell death . Cultures treated with high concentrations of melatonin displayed an increase in cell number and a decreased release of lactic acid dehydrogenase (LDH) into the culture media, indicating that melatonin was enhancing cell survival as opposed to stimulating cell proliferation . Inhibition of cell death by high concentrations of melatonin was both time and concentration-dependent and did not require the continued presence of melatonin throughout the entire time of incubation . These studies suggest melatonin is preventing either apoptosis or programmed cell death . In contrast, concentrations of melatonin (1-10 nM) at or near the binding affinity for the nuclear receptor, RZRbeta, suppressed PC12 cell growth . At these concentrations, melatonin failed to inhibit forskolin-induced cAMP formation and process outgrowth as well as prevent forskolin suppression of cell growth . These data indicate that PC12 cells probably lack functionally active cell surface receptors for melatonin and suggest the interaction of melatonin with the nuclear receptor may be responsible for suppression of PC12 cell growth.

Biol Reprod, 1997 Nov, 57(5), 1016 - 22
Prostaglandin F2alpha induces expression of prostaglandin G/H synthase-2 in the ovine corpus luteum: a potential positive feedback loop during luteolysis; Tsai SJ et al.; The primary role of prostaglandin (PG) F2alpha in regression of the corpus luteum has been clearly demonstrated in many mammalian species . We have used in vivo and in vitro approaches to investigate the possibility that exogenous PGF2alpha induces expression of prostaglandin G/H synthase-2 (PGHS-2; cyclooxygenase-2) and causes production of PGF2alpha in ovine luteal cells . Ewes received infusions into the ovarian artery of 1 ml PGF2alpha (1 micromol) or saline, and corpora lutea were collected at various times and analyzed for PGHS-2 mRNA using quantitative, competitive reverse transcription polymerase chain reaction . PGF2alpha dramatically increased the steady-state concentration of mRNA for PGHS-2 within 1 h, but basal concentration returned at 12 h posttreatment . In vitro studies using isolated ovine large luteal cells indicated that mRNA for PGHS-2 was induced by PGF2alpha, phorbol didecanoate, and ionomycin in a pattern similar to that observed in vivo . PGHS-2 protein was induced by all three treatments 4-12 h later, and accumulation of PGF2alpha in the culture media increased at 12 and 24 h posttreatment . In conclusion, we have provided evidence that PGF2alpha, probably acting through the protein kinase C/free intracellular calcium pathway, can stimulate large luteal cells to express PGHS-2 and produce PGF2alpha . This luteal PGF2alpha is likely to have an autocrine/paracrine function to augment the luteolytic effect of PGF2alpha of uterine origin.

Neurochem Int, 1997 Nov, 31(5), 715 - 22
Kainate excitotoxicity is mediated by AMPA- but not kainate-preferring receptors in embryonic rat hippocampal cultures; Ohno K et al.; We investigated kainate-induced excitotoxicity in embryonic rat hippocampal cells cultured in a chemically defined medium . Treatment with kainate for 24 h resulted in neuronal death, as assessed by the release of lactate dehydrogenase into the culture media . This neurotoxic effect was kainate dose- and culture age-dependent . EC50 of kainate was 127 +/- 11 microM . 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo (f)quinoxaline (NBQX) completely blocked the toxicity, while MK801, an N-methyl-D-aspartate (NMDA) receptor antagonist, also blocked it but not completely . Furthermore, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) attenuated the kainate injury, while the selective and noncompetitive AMPA-preferring receptor antagonist 1-(4-aminophenyl)-4-methyl-7, 8-methylenedioxy-5H-2,3-benzo-diazepine (GYKI 52466) blocked it completely . Concanavalin A (ConA), which potentiates the response to kainate at kainate-preferring receptors, had little effect on kainate toxicity . Further, AMPA alone induced little toxicity, but produced remarkable toxicity when cyclothazide was used to block the desensitization of AMPA-preferring receptors . These results indicate that kainate excitotoxicity in hippocampal cultures is mediated by AMPA- but not kainate-preferring receptors, and that it involves NMDA-receptor-mediated toxicity . The non-desensitizing response at AMPA-preferring receptors may play an important role in kainate-induced excitotoxicity.

Early Pregnancy, 1995 Jun, 1(2), 134 - 40
Production of endometrial placental protein 14 and prolactin by cultured endometrial explants after collagenase and freeze/thaw treatment, and in response to progesterone; Bersinger NA et al.; OBJECTIVE: Investigation of methods for maintaining functional endometrial explants in culture after cryopreservation with or without previous enzymatic dispersion of stromal cells and epithelial glands . Such a standardized culture system is a requirement for the development of a non-invasive bioassay for embryo quality in in vitro fertilization programs, a method that will eventually measure endometrial response to embryo conditioned media . METHOD: Culture of mid-luteal phase endometrial biopsies, in the presence of {35S}methionine, with or without prior collagenase treatment and/or storage in liquid nitrogen in the presence of dimethyl sulfoxide . Determination of released de novo synthesized total protein by trichloroacetic acid precipitation of culture media . Measurement, after culture in absence and presence of progesterone, of prolactin and placental protein 14 (PP14) production by sensitive non-isotopic immunoassays . RESULTS: Production of prolactin, but not PP14, was increased by 200 nmol/l progesterone, 2-8-fold after 4 days and 1.5-700-fold after 7 days in culture . After limited collagenase treatment (but without separation of stromal cells from glands), both marker protein productions were similar compared to untreated explants; however, there was no significant stimulation of prolactin by progesterone . After freezing and thawing, production was markedly reduced, particularly from explants frozen following collagenase treatment . CONCLUSIONS: Both stromal and glandular viability are maintained after collagenase treatment but the response to progesterone is lost . Cryopreservation reduced prolactin and PP14 production in subsequent culture . Therefore, novel freezing protocols should be developed which preserve both endometrial structure and function.

J Heart Lung Transplant, 1997 Oct, 16(10), 1035 - 45
Low multiplicity cytomegalovirus infection of human aortic smooth muscle cells increases levels of major histocompatibility complex class I antigens and induces a proinflammatory cytokine milieu in the absence of cytopathology; Arkonac B et al.; BACKGROUND: Cytomegalovirus has been implicated in the development of allograft vasculopathy in heart transplant recipients . Given that allograft vasculopathy is a form of chronic rejection, it is conceivable that cytomegalovirus somehow alters the allogeneic response to the vasculature . Prior work has demonstrated that smooth muscle cells (SMCs) are highly permissive for cytomegalovirus and exhibit cytopathologic characteristics and alterations in MHC class I antigens in response to cytomegalovirus at a high multiplicity of infection (MOI) . METHODS: To determine whether cytomegalovirus at low, more clinically relevant MOI, can alter SMCs phenotypically, human aortic SMCs were infected with approximately 1 plaque forming units/3000 cells of cytomegalovirus strain AD169 . RESULTS: One week after infection, human aortic SMCs (compared with human foreskin fibroblasts) demonstrated no cytopathologic characteristics (n = 6), released reduced amounts of intact virion into the culture media (assessed by exposing naive monolayers of human foreskin fibroblasts to media and staining for cytomegalovirus immediate-early antigen, n = 3), yet had at least, if not greater detectable total cytomegalovirus vital DNA levels . Infected HASMCs uniformly increased their expression of MHC class I antigen by 55% +/- 21% above constitutive levels (assessed by flow cytometry (n = 5, p < 0.0001) . Cytomegalovirus infection resulted in an increase in interleukin-6 mRNA expression compared to control (297 +/- 63 vs 188 +/- 50, respectively; p = 0.02, n = 6) and reduced the expression of transforming growth factor-beta mRNA (802 +/- 152 vs 1201 +/- 236, respectively; p = 0.05) . CONCLUSIONS: These data suggest that low MOI of cytomegalovirus can infect SMCs without producing cell cytolysis and, in spite of this lack of overt infection, modulate cell surface antigens and cytokine mRNA levels that can influence allogeneic responses.

Photodermatol Photoimmunol Photomed, 1997 Feb-Apr, 13(1-2), 27 - 36
Use of dermal equivalent and skin equivalent models for identifying phototoxic compounds in vitro; Augustin C et al.; Phototoxicity inducing in vivo photoirritation, a reversible inflammatory reaction of the skin after chemical contact and UVA radiation exposure, is increasingly observed as a side effect associated with the use of both cosmetics and systemic drugs . In order to systematically screen for the phototoxic potential of new compounds, we propose two three-dimensional models suitable for in vitro testing: a dermal equivalent (DE) and a skin equivalent (SE) model . The DE model includes a collagen-glycosaminoglycans-chitosan porous matrix populated by normal human fibroblasts . The SE model is made by seeding normal human keratinocytes onto the DE, leading to a fully differentiated epidermis . The objectives of this pilot study are: 1) to compare the deleterious effects of UVA radiation on the two models and 2) to evaluate to what extent the in vitro results can predict the in vivo phototoxicity caused by well-known photoirritant compounds, included in the COLIPA validation phototoxicity reference chemical list . Dilutions of thiourea, sulisobenzone, promethazine, chlorpromazine and tetracycline were applied (20 microliters) onto DEs and SEs (n = 6) and incubated for 1 h (or 15 h) at 37 degrees C . Irradiated samples received 3 J/cm2 UVA . The 24 h post-irradiation residual cellular viability was measured using the MTT test on treated and untreated tissues and IL-1 alpha release measurement in collected SE culture media . A concordance in terms of photoirritant/non-photoirritant was obtained between the in vivo data and the in vitro results, suggesting that the DE and the SE models could be integrated, after a complete validation study, into a protocol for in vitro testing of the photoirritant potential of new molecules.

J Biochem (Tokyo), 1997 Jun, 121(6), 1054 - 60
Effect of unsaturated fatty acids and alpha-tocopherol on immunoglobulin levels in culture medium of rat mesenteric lymph node and spleen lymphocytes; Hung P et al.; Mesenteric lymph node (MLN) and spleen lymphocytes of Sprague-Dawley rats were cultured with 1 mM unsaturated fatty acids (UFAs) with or without 100 microM alpha-tocopherol (Toc), and the immunoglobulin content and thiobarbituric acid (TBA) value of the culture media were measured to clarify the relationship between lipid peroxidation and the IgE level in the culture medium . The increase in the IgE content and TBA value induced by UFAs was alleviated in the presence of Toc in both lymphocytes, and was correlated well with their oxidation rates in most cases . Gamma-linolenic acid enhanced the IgE level much more than would be expected from its oxidation rate in both lymphocytes, and linoleic acid showed similarly high activity only in splenocytes . These results suggest that lipid peroxidation is partly responsible for the enhancement of IgE level induced by UFAs.

J Biol Chem, 1997 Nov 7, 272(45), 28615 - 21
A peptidyl-prolyl cis/trans-isomerase (cyclophilin G) in regulated secretory granules; Takaki Y et al.; A 27-kDa protein (p27) in horseshoe crab hemocyte that cross-reacts with antiserum against a beta-glucan-sensitive protease zymogen was purified to homogeneity, and its cDNA was cloned . The 1.7-kilobase pair cDNA contains an open reading frame of 660 base pairs, encoding a 23-amino acid signal sequence followed by a mature protein of 197 residues . The sequence of p27 exhibits strong similarity to that of cyclophilin B, a peptidyl-prolyl cis/trans-isomerase . p27 exhibits isomerase activity with a kcat/Km of 0.18 microM-1 s-1 for a peptide substrate; this activity is inhibited by cyclosporin A but is not affected by FK506 . Although the p27 precursor possesses an amino-terminal secretory hydrophobic signal sequence, unlike other cyclophilin B molecules, it lacks a conserved carboxyl-terminal endoplasmic reticulum retention signal and it contains a central 8-amino acid insertion . Although p27 is secreted into the culture media of transiently expressed COS cells, it is not detected in horseshoe crab hemolymph plasma but rather is localized to the hemocyte large granules, the regulated secretory granules that are exocytosed upon stimulation . These results indicate that p27 is a new peptidyl-prolyl cis/trans-isomerase in the regulated secretory granules, and is thus designated cyclophilin G . This first report of a cyclophilin homologue in the secretory granule of the horseshoe crab hemocyte suggests that such chaperon-like proteins may constitute a key quality control system for stored proteins in exocytotic granules.

Eur J Clin Chem Clin Biochem, 1997 Sep, 35(9), 655 - 60
Substance P induces the secretion of gelatinase A from human synovial fibroblasts; Hecker-Kia A et al.; We investigated the secretion of the matrix metalloproteinases, interstitial collagenase (matrix metalloproteinase-1), gelatinase A (matrix metalloproteinase-2) and stromelysin-1 (matrix metalloproteinase-3) in human synovial fibroblasts after stimulation with the neuropeptide substance P . Human synovial fibroblasts were stimulated with substance P or interleukin-1 beta (IL-1 beta) . In the cell culture media gelatinase A, interstitial collagenase and stromelysin-1 were identified and their activities towards different substrates were determined . Substance P in synovial fibroblasts induced an increase in the overall matrix metalloproteinase activity towards the dinitrophenyl-labelled peptide by 85%, against an increase of 124% after stimulation with IL-1 beta . In case of substance P stimulation, the increase in activity reflects a significantly enhanced secretion of gelatinase A, whereas no significant increase of stromelysin-1 and collagenase secretion could be observed . The matrix metalloproteinase pattern showing the highest gelatinase A secretion was obtained after stimulation with substance P . This pattern was very pronounced and differed very clearly from the pattern seen after IL-1 beta stimulation which caused a significant rise in collagenase and stromelysin-1 activity . We assume that distinct stimulation pathways are involved and that the neuropeptide (substance P), which is always present in the inflamed joint, plays its own and separate role in proliferative processes leading to the cartilage destruction.

Artif Cells Blood Substit Immobil Biotechnol, 1997 Nov, 25(6), 563 - 75
Effects of perfluorocarbon emulsions on cultured human endothelial cells; Mathy-Hartert M et al.; Perfluorocarbons (PFCs) and their emulsions (PFCEs) were used in organ preservation before transplantation, but not in organ perfusion . Our purpose was to achieve organ perfusion with a PFCE at room temperature or at 37 degrees C, i . e . with oxygenation, to prevent damages related to reoxygenation after hypoxia . Therefore, we first investigated the effect of such emulsions on endothelial cells, the first cells to be in contact with the emulsion . A stem emulsion was prepared from perfluorooctyl bromide (90% w/v), emulsified with egg yolk phospholipids (2% w/v) and stabilized with a mixed fluorocarbon-hydrocarbon "molecular dowel" (1.4% w/v) (droplets of ca 0.2 micron in diameter) . This emulsion was found to be stable when diluted with cell culture media or organ preservation fluids . Endothelial cells from human umbilical vein (HUVECs) were cultured in multiwell plates in M199 medium (with growth factors, 10% foetal calf serum and 5% human serum) . Confluent cells were incubated overnight with 51Cr, washed and overlayed with M199 (control) or the above PFCE diluted 2x or 4x with M199 (test) . After incubation, the cytotoxicity of the PFCEs was estimated by measuring 51Cr release and observing cell morphology by electron and light microscopy . The percentages of released 51Cr were identical to those of the control cells for the 2x, 3x or 4x diluted PFCEs at 4, 25 or 37 degrees C . After return to the M199 medium, the cells grew and multiplied normally . We conclude that the diluted PFCEs were devoid of cytotoxicity . The 2x diluted PFCE was however partially taken up by the cells: by microscopy, we observed intracellular PFC droplets and by density gradient analysis we found a slight increase in cellular density . The diluted PFCEs were compared to classical organ preservation solutions : HUVECs were incubated with UW (University of Wisconsin) or EC (EuroCollins) solutions at +4 and 37 degrees C (3, 17 or 24 h of incubation) . The solutions were observed to be toxic to the cells under these conditions, with cell mortality after return to the M199 medium . This cytotoxicity may be attributed to the high K+ concentration of UW and EC, since similar assays performed on HUVECs with Hank's solution adjusted to 100 mM K+ showed a similar % of 51Cr release . UW and EC are therefore not acceptable as dilution media for PFCEs.

Am J Reprod Immunol, 1997 Oct, 38(4), 279 - 85
The effect of interleukin-1 beta and interleukin-4 on the expression of prostaglandin receptors EP1 and EP3 in amnion WISH cells; Spaziani EP et al.; PROBLEM: Although prostaglandin E2 (PGE2) is believed to modulate biochemical and immunological events leading to parturition, the role of prostaglandin E receptors during labor has not been investigated . METHOD OF STUDY: Amnion WISH cells were incubated in media containing increasing concentrations of either interleukin-1 beta (IL-1 beta) or IL-4 . Increased EP1 and EP3 protein expression was determined by Western blot analysis with peptide-specific antibodies . Concomitant measurements of culture media PGE2 were made by an enzyme immunoassay . RESULTS: Incubation of WISH cells with IL-1 beta or IL-4 caused a two- to three-fold increase in EP1 protein levels . IL-1 beta and IL-4 also caused six- and two-fold increases, respectively, in culture fluid PGE2 concentrations . IL-1 beta or IL-4 had no effect on EP3 protein levels . CONCLUSIONS: Based on these results, it is proposed that IL-1 beta and IL-4 may be involved in the initiation and promotion of labor by inducing EP1 levels and PGE2 production in amnion.

Endocrinology, 1997 Nov, 138(11), 4806 - 11
Corticotropin-releasing hormone (CRH) inhibits steroid biosynthesis by cultured human granulosa-lutein cells in a CRH and interleukin-1 receptor-mediated fashion; Ghizzoni L et al.; The presence of immunoreactive CRH was recently demonstrated in human ovaries . CRH immunoreactivity was localized by immunohistochemistry in the cytoplasm of thecal cells surrounding the ovarian follicles, in luteinized cells of the stroma, and in large granulosa-derived luteinized cells of developing corpora lutea . Also, CRH and its receptors were identified in Leydig cells of the testis where CRH was shown to inhibit testosterone biosynthesis . To examine the role of CRH in the ovary, we studied its effect on estradiol (E2) and progesterone (P4) release by human granulosa cells obtained from women undergoing in vitro fertilization for male factor infertility or uni- or bilateral tubal impatency . In all subjects, superovulation was induced by treatment with gonadotropins . The effects of graded doses of ovine CRH (10{-11}-10{-6} mol/liter) were evaluated in the conditioned medium obtained after 24 h incubation of the cells . All CRH concentrations employed except for the lowest one (10{-11} mol/liter) caused a significant decrease of media E2 and P4 levels . Maximal inhibition for both E2 and P4 production was obtained by 10{-6} mol/liter CRH concentration, which decreased hormone production by 39% and 34%, respectively . The alpha-helical CRH9-41 antagonist at 10(-6) and 10(-7) mol/liter blocked the suppressive effect of 10(-9) mol/liter CRH on both E2 and P4 secretion, while it had no effect when added to the culture media without CRH . Since interleukin (IL-1)-1 mediates certain actions of CRH on leukocytes, we examined whether the CRH effect on ovarian steroidogenesis was IL-1-mediated . Interleukin-1 receptor antagonist at 10(-7) and 10(-6) mol/liter blocked the inhibitory effects of CRH on E2 and P4 secretion, while it had no effect in the absence of CRH . In conclusion, CRH exerts a CRH- and IL-1 receptor-mediated inhibitory effect on ovarian steroidogenesis and might be actively involved in the still enigmatic processes of follicular atresia and luteolysis.

Histochem J, 1997 Aug, 29(8), 593 - 606
The use of Fluoresceincadaverine for detecting amine acceptor protein substrates accessible to active transglutaminase in living cells; Lajemi M et al.; The use of Fluoresceincadaverine as a primary amine donor for detecting the endogenous substrates for active transglutaminase in living cells was studied . Fluoresceincadaverine was found to be suitable for labelling cells in culture as it did not induce cytotoxicity when used at 0.5 mM in culture media and diffused throughout the cell . After appropriate fixation using methanol, Fluoresceincadaverine-labelled cells were observed by direct fluorescence microscopy, allowing visualization of the substrates for active transglutaminase . Simultaneous detection of transglutaminase and of Fluoresceincadaverine incorporated into proteins strongly suggested that cytosolic transglutaminase was inactive in these living cells . However, transglutaminase co-distributed with Fluoresceincadaverine-labelled structures, which resembled a lattice . Fluoresceincadaverine-labelled proteins detected by Western blotting using an anti-Fluorescein antibody showed that, in living cells, the major transglutaminase substrate migrated at an apparent molecular weight of 220 kDa, as does fibronectin . Fibronectin was found to co-distribute with Fluoresceincadaverine-labelled lattice . This confirmed that these lattice structures were extracellular and, therefore, that transglutaminase is in an active form in this compartment . This opportunity to perform morphological and biochemical analyses in the search for transglutaminase substrates in living cells should help in determining the specific function of transglutaminases in a particular cell type as well as in universal cellular events, such as apoptosis or cell growth.

Appl Environ Microbiol, 1997 Oct, 63(10), 3916 - 8
Efficacy of vaporized hydrogen peroxide against exotic animal viruses; Heckert RA et al.; The efficacy of vapor-phase hydrogen peroxide in a pass-through box for the decontamination of equipment and inanimate materials potentially contaminated with exotic animal viruses was evaluated . Tests were conducted with a variety of viral agents, which included representatives of several virus families (Orthomyxoviridae, Reoviridae, Flaviviridae, Paramyxoviridae, Herpesviridae, Picornaviridae, Caliciviridae, and Rhabdoviridae) from both avian and mammalian species, with particular emphasis on animal viruses exotic to Canada . The effects of the gas on a variety of laboratory equipment were also studied . Virus suspensions in cell culture media, egg fluid, or blood were dried onto glass and stainless steel . Virus viability was assessed after exposure to vaporphase hydrogen peroxide for 30 min . For all viruses tested and under all conditions (except one), the decontamination process reduced the virus titer to 0 embryo-lethal doses for the avian viruses (avian influenza and Newcastle disease viruses) or less than 10 tissue culture infective doses for the mammalian viruses (African swine fever, bluetongue, hog cholera, pseudorabies, swine vesicular disease, vesicular exanthema, and vesicular stomatitis viruses) . The laboratory equipment exposed to the gas appeared to suffer no adverse effects . Vaporphase hydrogen peroxide decontamination can be recommended as a safe and efficacious way of removing potentially virus-contaminated objects from biocontainment level III laboratories in which exotic animal disease virus agents are handled.

Ocul Immunol Inflamm, 1997 Sep, 5(3), 181 - 95
Effect of sialodacryoadenitis virus exposure on acinar epithelial cells from the rat lacrimal gland; Wickham LA et al.; Sialodacryoadenitis virus (SDAV), a RNA coronavirus, induces degenerative, necrotic and atrophic alterations in acinar epithelial cells of the rat lacrimal gland . To begin to explore the underlying mechanism(s) of this viral effect, we sought in the present study to: (1) determine whether SDAV invades and replicates in lacrimal gland acinar cells in vitro and (2) assess whether short-term SDAV challenge interferes with the viability or function of acinar cells in vitro . For comparison we also evaluated the relative infectivity of SDAV in acinar epithelial cells from lacrimal, submandibular and parotid glands, given that salivary tissues are known to be highly susceptible to SDAV infection in vivo . Acinar epithelial cells from lacrimal, submandibular or parotid glands were isolated from male rats, exposed briefly to SDAV or control cell antigen and then cultured for four, eight or twelve days . At experimental termination, SDAV titers in both media and sonicated cell extracts were evaluated by plaque assay titration on mouse L2 cell monolayers . To evaluate functional aspects of lacrimal gland acinar cells, SDAV-infected cells were incubated in the presence or absence of dihydrotestosterone and culture media were analyzed by RIA to measure the extent of the androgen-induced increase in secretory component (SC) production . Our results showed that: (1) SDAV invades and replicates in lacrimal gland acinar cells, Viral challenge resulted in a significant, time-dependent increase in SDAV titers, that were primarily cell-associated and greatly exceeded amounts contained in the original inoculum; (2) SDAV infection did not compromise lacrimal acinar cell viability or prevent the cellular SC response to androgens . Viral presence, though, did often attenuate the magnitude of this hormone action; and (3) SDAV infects salivary acinar cells, but the kinetics and magnitude or viral replication in lacrimal, submandibular and parotid cells showed considerable variations . These findings demonstrate that SDAV invades and replicates in acinar epithelial cells from lacrimal and salivary glands . The resulting release of infectious progeny may play a role in the SDAV-induced pathology of exocrine tissues in vivo.

Mol Cell Endocrinol, 1997 Sep 19, 132(1-2), 117 - 26
IFN-tau increases PGE2 production and COX-2 gene expression in the bovine endometrium in vitro; Asselin E et al.; Prostaglandins (PGs) are well known for their role in reproductive processes . At the time of pregnancy recognition, PGF2alpha is luteolytic and PGE2 may be antiluteolytic and luteotropic . During the preimplantation period, interferon-tau (IFN-tau) is produced by the conceptus and plays a crucial role in maternal recognition of pregnancy in domestic ruminants . We have demonstrated previously that recombinant bovine and ovine interferon-tau (rbIFN-tau and roIFN-tau) stimulate PGE2 production in epithelial cells, changing the primary PG produced by these cells from F2alpha to E2 . In stromal cells, where PGE2 is the major PG produced, roIFN-tau induced an increase of both types of PGs . The aim of this paper is to identify the possible involvement of cyclooxygenases (COXs) in the modulation of PG production by trophoblastic interferons . Epithelial and stromal cells cultured in vitro were isolated from bovine endometrium and stimulated with increasing doses (1, 10 and 20 microg/ml) of roIFN-tau . PG levels in the culture media were measured by enzyme immunoassays (EIA) and total RNA was extracted from the cells . Northern blot analysis was performed to quantify cyclooxygenase COX-1 (constitutive), COX-2 (inducible) and phospholipase A2 (PLA2) messenger RNA (mRNA) production in response to treatment . The results indicate that roIFN-tau treatment did not affect COX-1 and PLA2 mRNA production in either cell type, whereas COX-2 expression was upregulated in both . The up-regulation of COX-2 transcript was greater in stromal than in epithelial cells . The increase in COX-2 mRNA levels was concurrent with increased production of PGE2 and PGF2alpha in stromal cells and principally PGE2 in epithelial cells . Furthermore, addition of indomethacin (1 microM) and a specific COX-2 inhibitor (NS-398, 1 microM) blocked the roIFN-tau-stimulation of PG production in both cell types . The mechanism whereby elevated COX-2 expression results in a selective increase of PGE2 in epithelial cells remains to be elucidated . In stromal cells, an increase in COX-2 mRNA levels may explain increased PG production . The overall effect of roIFN-tau in the two cell types is a net increase in PGE2 output.

J Lipid Res, 1997 Sep, 38(9), 1722 - 9
Lipoprotein lipase enhances human monocyte adhesion to aortic endothelial cells; Mamputu JC et al.; Lipoprotein lipase (LPL)-mediated lipolysis of very low density lipoprotein (VLDL) has been demonstrated to increase U937 monocyte adhesion to endothelial cells . In the present study, we evaluated the ability of LPL to enhance human monocyte adhesion to bovine aortic endothelial cells (BAEC) in the absence of exogenous lipoproteins . Exposure of BAEC to 1 microgram/ml LPL at 37 degrees C resulted in a significant increase in monocyte adhesion over control values . Addition of VLDL in the culture media further enhanced the LPL effect . A significant increase in monocyte adhesion was also observed when BAEC were incubated with LPL at 4 degrees C . Heparin or heparinase treatment of BAEC totally abolished the LPL stimulatory effect on monocyte adhesion . In addition, incubation of monocytes with heparinase suppressed the ability of LPL to stimulate monocyte adhesion to endothelial cells . These treatments also markedly decreased LPL binding to the monocyte and endothelial cell surfaces . In contrast to native LPL, heat inactivated or phenylmethylsulfonyl fluoride (PMSF)-treated LPL did not increase monocyte adhesion to BAEC . Finally, incubation of LPL in the presence of the 5D2 antibody resulted in a total suppression of the LPL-induced monocyte adhesion to BAEC . Taken together, these data demonstrate that LPL activity plays an important role in LPL-induced monocyte adhesion and that LPL binding to heparan sulfate proteoglycans expressed on both monocytes and endothelial cells surfaces is required for the enhanced monocyte adhesion . These results suggest a new mechanism by which LPL may promote the development of atherosclerosis, that of facilitating monocyte adhesion to the endothelium.

Clin Exp Metastasis, 1997 Nov, 15(6), 568 - 79
Activation of protein kinase C-alpha isoform in murine melanoma cells with high metastatic potential; La Porta CA et al.; Metastasis is a multistep process in which protein kinase C (PKC) appears to be significantly involved . We analysed the activity and expression of classical (alpha, beta, gamma) and novel PKC epsilon isoforms in B16-F1 and B16-BL6 melanoma cells maintained under different culture conditions in vitro . We used high and low concentrations of tyrosine and phenylalanine in different media (DMEM or RPMI 1640 respectively) that affect the metastatic potential and also the proliferative capacity of the cells . We also tested a weakly metastatic amelanotic B78-H1 melanoma cell line which is unaffected by the different culture conditions . In both B16 melanoma cell lines activation of PKC alpha (without increased expression) occurred under growth conditions permissive of metastasis (DMEM) . In contrast, the weakly metastatic amelanotic B78-H1 cell line showed a substantial inactivation of this isoform in the two different culture media, suggesting a specific involvement of PKC alpha in the metastatic process . Moreover, in B16 melanoma cells, novel PKC epsilon was activated under culture conditions which stimulated growth but not metastasis (RPMI 1640) . In order to define the relationship between PKC activation and the metastatic process we also determined the release of cathepsin B . No correlation between PKC activity and cathepsin B release in either B16 melanoma cell lines could be demonstrated.

J Mol Endocrinol, 1997 Oct, 19(2), 191 - 201
Expression of membrane and soluble intercellular adhesion molecule-1 in Graves' disease; Massart C et al.; We have investigated the in vitro expression of membrane and soluble intercellular adhesion molecule-1 (ICAM-1) by human thyroid cells from 20 patients with Graves' disease and 5 normal subjects . Membrane ICAM-1 was not detected by flow cytometry analysis in non-cultured thyrocytes from either normal or Graves' disease tissues . It appeared on thyroid cells after a 24-h culture in monolayers and showed a regular dose-dependent increase . The same results were obtained with soluble ICAM-1 (sICAM-1) in culture media from cells cultured in monolayers, vesicles or follicles . No change was obtained with different concentrations of fetal calf serum added to the media . Coculture of Graves' disease thyrocytes with autologous peripheral blood lymphocytes (PBL) or intrathyroidal lymphocytes (ITL) enhanced the expression of both membrane and sICAM-1 whatever the culture model . When normal thyrocytes were cocultured with PBL, sICAM-1 increased but with ITL sICAM-1 remained unchanged . High concentrations of gamma interferon induced an increase of both membrane and sICAM-1 in the three culture models . However the increases were greater with vesicles and follicles . Only sICAM-1 levels were raised with 0.1, 1 and 10 microM retinoic acid . These results suggest that ICAM-1 appears in culture, possibly due to mechanical effects such as adherence to plates and cell-to-cell contacts . Moreover, its expression is modulated by several factors such as cytokines or retinoic acid . Further investigations are needed to establish whether ICAM-1 is really involved in the pathogenesis of Graves' disease.

Planta Med, 1997 Oct, 63(5), 441 - 5
Extracellular polysaccharides produced by suspension-cultured cells from Digitalis lanata; Hensel A et al.; Extracellular polysaccharides (ECP) were isolated in yields of up to 4 mg/ml from the culture media of suspension-cultured cells from Digitalis lanata Ehrh . ECP content was increasing continuously over the first ten days of cultivation and then stayed constant until day 20 . ECP were fractionated by ion-exchange chromatography into two neutral and one acidic fractions . Further fractionation was achieved by gel-permeation chromatography (GPC) . One neutral fraction was separated into two distinct fractions with average molecular weights of 160 and 70 kDa, respectively . The second neutral fraction was hetero-disperse in GPC with average molecular masses of 112, 32, and 8 kDa . Polysaccharides of all neutral fractions consisted of glucose, xylose, galactose, and arabinose . Methylation analysis indicated these fractions to contain xyloglucans besides minor amounts of highly branched arabinogalactans . Xyloglucans were, using endo-beta-(1-->4)glucanase, fragmented into subunits which were identified mainly as tri- and pentasaccharides . The acidic fraction eluated as a single peak during gel-permeation chromatography with an average molecular weight of 56 kDa . Analysis of carbohydrate composition and linkage analysis indicated that this polysaccharide is an acidic arabinogalactan . 2,6-Dideoxysugars, the typical carbohydrate components of cardiac glycosides in Digitalis lanata, were not detected in ECP.

Proc Natl Acad Sci U S A, 1997 Oct 28, 94(22), 12041 - 6
Epstein-Barr virus-induced gene 3 and the p35 subunit of interleukin 12 form a novel heterodimeric hematopoietin; Devergne O et al.; The Epstein-Barr virus-induced gene 3 (EBI3) is a novel soluble hematopoietin component related to the p40 subunit of interleukin 12 (IL-12) . When EBI3 was expressed in cells, it accumulated in the endoplasmic reticulum and associated with the molecular chaperone calnexin, indicating that subsequent processing and secretion might be dependent on association with a second subunit . Coimmunoprecipitations from lysates and culture media of cells transfected with expression vectors for EBI3 and/or the p35 subunit of IL-12 now reveal a specific association of EBI3 with p35 . Coexpression of EBI3 and p35 mutually facilitates their secretion . Most importantly, a large fraction of p35 in extracts of the trophoblast component of a human full-term normal placenta specifically coimmunoprecipitated with EBI3, indicating that EBI3 is in a heterodimer with p35, in vivo . Because EBI3 is expressed in EBV-transformed B lymphocytes, tonsil, spleen, and placental trophoblasts, the EBI3/p35 heterodimer is likely to be an important immunomodulator.

Bioelectromagnetics, 1997, 18(7), 524 - 6
Vertical circularly polarized ELF magnetic fields and induced electric fields in culture media; Misakian M; Some properties of induced electric fields in cell culture media produced by vertical circularly polarized magnetic fields are examined . The described geometry is not advantageous for determining effects that may be attributable to induced electric fields or currents.

Microsc Res Tech, 1997 Sep 1, 38(5), 512 - 8
Comparison of MB-check and Löwenstein-Jensen media for recovery of mycobacteria; Palacios JJ et al.; We have compared the rate of recovery of mycobacteria with the MB-Check culture system (liquid phase) and the Lowenstein-Jensen (LJ) medium in 2,907 clinical specimens obtained from 830 patients submitted for mycobacterial culture during 1-year period . Direct smear examination was carried out by auramine-rhodamine staining . All primary isolates from the culture media were confirmed by Ziehl-Neelsen staining and identified by acridinium-ester-labeled DNA probes specific for Mycobaterium tuberculosis complex . A total of 214 isolates were of the M . tuberculosis complex (88 patients) and 54 of "potentially pathogenic environmental mycobacteria" (45 patients) . A total of 117 (54.7%) samples were smear-positive and the remaining 97 (45.3%) were smear-negative . There was a significant difference in the percentage of positive cultures obtained by the MB-Check method (99.1%) as compared with the LJ medium (73.8%) (P < 0.05) . This difference, however, occurred almost exclusively at the expense of the 97 smear-negative samples (positive cultures 97.95% by the MB-Check method vs . 42.3% by the LJ culture, P < 0.05) . The number of patients diagnosed of tuberculosis by the MB-Check was significantly higher as compared with LJ medium (88 {100%} vs . 77 {87.5%}, P < 0.05) . In 11 (12.5%) patients, the diagnosis was only established by the MB-Check system . In smear-positive samples, the mean (+/-SD) detection time for M . tuberculosis complex was 14.8 +/- 8 days with MB-Check and 19.9 +/- 7 days with LJ medium . The corresponding figures in smear-negative samples were 22.8 +/- 3 days and 27.8 +/- 6 days, respectively . DNA probes directly applied to MB-Check liquid medium showed a sensitivity of 98.8% and specificity of 100% . These results indicate that the MB-Check system is more efficient for the recovery of mycobacteria than LJ medium.

Circulation, 1997 Oct 7, 96(7), 2376 - 84
Preservation of myocyte contractile function after hyperthermic cardioplegic arrest by activation of ATP-sensitive potassium channels; Dorman BH et al.; BACKGROUND: Left ventricular (LV) dysfunction can occur after hyperkalemic cardioplegic arrest and subsequent reperfusion and rewarming . Activation of adenosine triphosphate (ATP)-sensitive potassium (KATP) channels within the myocyte sarcolemma has been shown to be cardioprotective for myocardial reperfusion injury and ischemia and may play a contributory role in preconditioning for cardioplegic arrest . Accordingly, the present study tested the hypothesis that cardioplegic arrest and activation of KATP channels by a potassium channel opener (PCO) would attenuate alterations in ionic homeostasis and improve myocyte contractile function . METHODS AND RESULTS: Porcine LV myocytes were isolated and randomly assigned to the following treatment groups: normothermic control, incubation in cell culture media for 2 hours at 37 degrees C (n=60); hyperkalemic cardioplegia, incubation for 2 hours in hypothermic hyperkalemic cardioplegic solution (n=60); or PCO/cardioplegia, incubation in cardioplegic solution containing 100 micromol/L of the PCO aprikalim (n=60) . Hyperkalemic cardioplegia and rewarming caused a significant reduction in myocyte velocity of shortening compared with normothermic control values (33+/-2 versus 66+/-2 microm/s, P<.05) . Cardioplegic arrest with PCO supplementation significantly improved indices of myocyte contractile function when compared with hyperkalemic cardioplegia (58+/-4 microm/s, P<.05) . Myocyte intracellular calcium increased during hyperkalemic cardioplegic arrest compared with baseline values (147+/-2 versus 85+/-2 nmol/L, P<.05) . The increase in intracellular calcium was significantly reduced in myocytes exposed to the PCO-supplemented cardioplegic solution (109+/-4 nmol/L, P<.05) . CONCLUSIONS: Cardioplegic arrest with simultaneous activation of KATP channels preserves myocyte contractile processes and attenuates the accumulation of intracellular calcium . These findings suggest that changes in intracellular calcium play a role in myocyte contractile dysfunction associated with cardioplegic arrest . Moreover, alternative strategies may exist for preservation of myocyte contractile function during cardioplegic arrest.

Korean J Parasitol, 1997 Sep, 35(3), 149 - 54
Development of mass rearing technique of Tyrophagus putrescentiae (Acari: Acaridae) found in house dust; Ree HI et al.; A storage mite, Tyrophagus putrescentiae, is recently known to be widely distributed in Korea, being commonly found in house dust, and may, therefore, be allergenically important . The purpose of this study was to develop mass rearing techniques for supplying a large quantity of allergens . The laboratory mouse food powder gave the highest yield, showing 1,251.5-fold increase in number after 10 weeks, and the mixed powder of laboratory mouse food and yeast (1:1) also gave same level of the production (1,203.1-fold increase in week 10) . Several different combinations of temperature and relative humidity conditions were compared, and the maximum propagation was obtained at 25 degrees C and 64% RH, showing 960-fold increase in number . When the same amount of culture media was used, the size of the culture container did not significantly influence the quantitative yield of T . putrescentiae mites.

J Anim Sci, 1997 Oct, 75(10), 2744 - 8
Mechanisms of action of growth hormone-releasing peptide-2 in bovine pituitary cells; Roh SG et al.; We conducted this study to investigate the mechanisms of action of growth hormone-releasing peptide-2 (D-Ala-D-beta Nal-Ala-Trp-D-Phe-Lys-NH2; GHRP-2) in bovine anterior pituitary primary cell culture . Doses of GHRP-2 from 10(-13) to 10(-7) M) increased (P < .05) GH secretion . The GHRP-2 (10(-7) M) and GH-releasing factor (GRF; 10(-7) M) administered together had an additive effect on the release of GH (P < .05) . Somatostatin (1 microM) decreased GH secretion in response to GHRP-2 and(or) GRF (P < .05) . Secretion of GH in response to GHRP-2 was blocked (P < .01) by a GRF receptor antagonist (.1 microM) . Nifedipine (10 microM), a voltage-dependent Ca2+ channel blocker, inhibited (P < .01) GHRP-2-stimulated GH release . The GH release in response to GHRP-2 and 4 beta-phorbol-12-myristate-13-acetate (10(-7) M), a protein kinase C activator, was additive (P < .01) . Forskolin (30 microM), a cAMP elevating agent, further stimulated (P < .01) the GH release in response to GHRP-2 . Bovine GH concentrations in culture media were assayed by indirect competitive enzyme immunoassay . These results showed that GHRP-2 1) stimulates GH secretion from bovine pituitary cells, 2) may partially act via GRF receptor, 3) has GH secretion activity caused by Ca2+ influx via Ca2+ channels, and 4) may increase GH secretion via protein kinase C and cAMP pathways.

Cell Transplant, 1997 Sep-Oct, 6(5), 447 - 54
Functional recovery of porcine hepatocytes after hypothermic or cryogenic preservation for liver support systems; Naik S et al.; The provision of an immediate supply of isolated porcine hepatocytes for artificial liver support requires preservation techniques that will allow maintenance of cell viability and detoxification functions . By means of a simple and cost-effective cryopreservation system, porcine hepatocytes can be available for both local and distant medical treatment facilities . Additionally, cryopreservation provides an adequate period for quality control testing to be completed prior to use of any specific cell lot . We are reporting a dual approach, namely the preservation of porcine hepatocytes, at 4 degrees C and at -196 degrees C in liquid nitrogen (LN2) . Using a combination of cryoprotectant agents with Chee's modified Eagle's culture media (CEM), collagenase isolated hepatocytes stored at 4 degrees C for 24 h maintained 80% of the initial diazepam metabolism measured in freshly isolated cells and nearly 100% of initial function was preserved in hepatocytes stored up to 6 mo at -196 degrees C . University of Wisconsin solution (UW) was also tested and while adequate for 4 degrees C storage, it certainly did not match the performance of the CEM formulations for preservation of metabolic function of cells stored in liquid nitrogen . Based on our results of viability and detoxification function the combination of CEM with DMSO, polyethylene glycol and serum provided optimal protection for LN2 frozen cells . Other findings in these studies underlined the importance of the gradual introduction of DMSO in the prefreezing process, the period of osmotic equilibration, and the rapid postthaw withdrawal of this agent to minimize cytotoxic effects at these critical stages . Our freezing methodology provides the foundation for further technological developments in the cryopreservation of the large numbers of cells (billions) that are necessary for extracorporeal liver assist devices.

J Reprod Fertil, 1997 Jul, 110(2), 347 - 53
Effect of insulin-like growth factor I (IGF-I) at high concentrations on blastocyst development of bovine embryos produced in vitro; Palma GA et al.; This study was carried out to determine the effects of oestrous cow serum containing insulin-like growth factor I (IGF-I) and supplementation with recombinant IGF-I on subsequent development of bovine embryos produced in vitro . When culture medium was supplemented with oestrous cow serum containing 34.8 ng endogenous IGF-I ml-1, more embryos (P < 0.01) developed to blastocysts by day 9 and more blastocysts hatched on day 13 after insemination (P < 0.01) than in the control group . The effect of the addition of 10, 50 and 100 ng IGF-I ml-1 to culture media containing oestrous cow serum and granulosa cells was also evaluated . Supplementation with 10 ng IGF-I ml-1 did not improve embryo development at any stage . The addition of 50 and 100 ng IGF-I ml-1 did not affect development during the first three cell divisions . However, on day 7 these groups yielded a higher embryo rate than did the control group . Similarly, the proportion of blastocysts on day 9 was enhanced . The addition of 100 ng IGF-I ml-1 also increased the proportion of blastocysts . These data suggest that IGF-I at high concentrations accelerates the development to the blastocyst stage by shortening the transition from the morula to the blastocyst stage . The addition of 100 ng IGF-I ml-1 increased the proportion of hatched blastocysts on day 13 . The addition of oestrous cow serum and IGF-I to TCM 199 free of granulosa cells did not increase the proportion of embryos on day 7 . However, the progress to blastocysts and hatched blastocysts on days 9 and 13 was significantly lower (P < 0.05) . The addition of IGF-I to culture medium without oestrous cow serum but with granulosa cells resulted in significantly lower embryo development than in the control group or in the group supplemented with oestrous cow serum and IGF-I (P < 0.01) . The results support the hypothesis that culture media containing high concentrations of IGF-I combined with oestrous cow serum and granulosa cells can improve the development of embryos produced in vitro.

Am J Physiol, 1997 Sep, 273(3 Pt 1), C1100 - 7
Upregulation of P2Y2 nucleotide receptors in rat salivary gland cells during short-term culture; Turner JT et al.; In contrast to the widespread expression of G protein-coupled P2Y2 receptors for extracellular nucleotides in permanent cell lines of salivary gland origin, there is less evidence for robust P2Y2 receptor activity in normal rat salivary gland cells assayed immediately after isolation . We examined the effect of short-term culture (3 h to 6 days) of normal rat submandibular gland (SMG) cells on P2Y2 receptor activity and mRNA expression . Results indicate that increases in the intracellular free Ca2+ concentration in SMG cells in response to the P2Y2 receptor agonist UTP (100 microM) were detectable after 3 h in culture and that after 3 days in culture the magnitude of the response to UTP was similar to that obtained with maximal muscarinic cholinoceptor activation . The Ca2+ mobilization response exhibited the pharmacological profile (UTP = ATP > 2-methylthioadenosine 5'-triphosphate) typical of the P2Y2 receptor subtype and was accompanied by enhanced production of inositol phosphates, reflecting the activation of phospholipase C ubiquitously associated with P2Y2 receptors . The time-dependent increase in P2Y2 receptor activity was accompanied by an increase in the steady-state level of P2Y2 receptor mRNA, as assessed by reverse transcription-polymerase chain reaction . Other studies revealed that the increased P2Y2 receptor activity was independent of cell proliferation, was similar in serum-containing and defined culture media, and was blocked by inhibitors of transcription and translation . Upregulation of the P2Y2 receptor was observed in both acinar cell- and ductal cell-enriched cultures of the SMG and in cells isolated from rat parotid and sublingual glands but not in cells isolated from the pancreas . These in vitro results were complemented by in vivo studies in which P2Y2 receptor activity and mRNA levels were increased in SMG after ligation of the main excretory duct but were not increased in the contralateral, nonligated gland . These findings suggest that changes in the expression and activity of the P2Y2 receptor in salivary gland cells may be related to pathological challenges to the gland in vivo.

Poult Sci, 1997 Oct, 76(10), 1379 - 86
Development of a highly quantitative, reproducible assay for determination of chicken T cell growth factor biological activity; Pfohl JL et al.; This report examines optimal culture conditions necessary for accurate and sensitive quantification of chicken T Cell Growth Factor (TCGF) activity . With this bioassay, TCGF is quantified by measuring its ability to cause proliferation of splenocytes prestimulated with mitogen . Proliferation is quantified by determining the optical density (OD) or "signal" of test samples in microtiter wells by measuring the incorporation of tetrazolium salt by live cells . To optimize assay conditions, systematic evaluation of the effects of cell culture variables was carried out with the constant aim of increasing signal to noise ratio in the assay . Higher signal to noise ratios were found when using Dulbecco's Modified Eagle's Medium (DMEM) rather than Roswell Park Memorial Institute Medium (RPMI) for basal tissue culture media containing the same supplements . The addition of lipid supplement to the assay system not only increased the proliferation signal, but also decreased the background OD . Incubation temperatures of 41 C rather than 37 C for both the mitogen prestimulation and proliferation phases of the assay also resulted in a higher signal to noise ratio . While incorporating the optimal experimental conditions, a finalized assay procedure employing test sample normalization with an internal assay standard was tested for accuracy . The assay can accurately detect 2 to 15 U/mL of TCGF activity . The within-assay variation ranged from 2 to 13% and the between-assay variation ranged from 11 to 22% depending upon the TCGF preparation being tested . The excellent reproducibility of this assay has facilitated investigations of TCGF production, processing, and purification.

Neurosurgery, 1997 Oct, 41(4), 908 - 15
Antiproliferative effect of c-myc antisense phosphorothioate oligodeoxynucleotides in malignant glioma cells; Broaddus WC et al.; OBJECTIVE: To improve the prognosis for primary malignant tumors of the central nervous system, new therapeutic strategies are needed . Antisense oligodeoxynucleotides (ODNs) offer the potential to block the expression of specific genes within cells . The proto-oncogene c-myc has long been implicated in the control of normal cell growth and its deregulation in the development of neoplasia . We therefore reasoned that a strategy using ODNs complementary to c-myc messenger ribonucleic acid would be a potent inhibitor of glioma cell proliferation . METHODS: A variety of antisense, sense, and scrambled (15-mer) phosphorothioate ODNs targeted to rat and human c-myc messenger ribonucleic acid were synthesized and added to the media of cultured RT-2 cells (a rat glioblastoma cell line) . Cell growth was assessed by 3-{4,5-dimethylthiazol-2yl}-2,5-diphenyltetrazolium bromide dye assay 1 to 5 days after adding the ODNs . c-Myc protein expression was analyzed by Western blot analysis . The stability of the ODNs was confirmed by gel electrophoresis . RESULTS: Compared with cultures containing standard media, two of three antisense ODNs significantly inhibited the growth of glioma cells, whereas sense and scrambled sequence ODNs did not significantly affect cell growth at the concentrations tested . A human c-myc antisense sequence, which differed from the rat sequence by one base substitution, also had an inhibitory effect on RT-2 cells . Western blot analysis demonstrated that expression of immunoreactive c-Myc protein was also greatly reduced in the rat antisense ODN-treated cells (and not in sense-, scrambled-, or control-treated cells) . The degree of reduction of c-Myc protein expression correlated well with the decrease in cell growth observed with several antisense ODNs . Phosphorothioate ODNs were stable in cell culture media for at least 5 days . CONCLUSION: These results suggest that c-Myc plays a critical role in glioma cell proliferation and demonstrate that antisense ODNs can suppress proto-oncogene expression and inhibit the proliferation of glioma cells . Our results indicate that the antiproliferative activity of these ODNs was mediated predominantly through sequence-specific antisense mechanisms, but that sequence-specific nonantisense effects may also contribute to the strongest effects demonstrated . These findings support a potential role for antisense strategies designed to inhibit c-myc expression in the treatment of malignant gliomas.

Biochemistry, 1997 Oct 7, 36(40), 12355 - 63
Mutants of human choriogonadotropin lacking N-glycosyl chains in the alpha-subunit . 1 . Mechanism for the differential action of the N-linked carbohydrates; Purohit S et al.; Analogs of human choriogonadotropin (hCG) lacking N-glycosyl chains at alpha52Asn and alpha78Asn were purified from the culture media of insect cells by immunoaffinity chromatography using a monoclonal antibody column . As previously reported, while analogs lacking carbohydrate at alpha52Asn and alpha78Asn had similar receptor binding activities compared with the wild type recombinant hCG (hCGwt), they differed in their signal transduction properties . The mutant lacking carbohydrate at alpha78Asn had 20% less cAMP-stimulating activity than hCGwt, but the absence of glycosylation at alpha52Asn resulted in the reduction of cAMP accumulation by 90-95% . A similar effect of the mutations was observed on the stimulation of steroidogenesis . Circular dichroism spectra of the two mutants showed significant differences . The mutant lacking carbohydrate at alpha52Asn had a much higher negative mean residue ellipticity (MRE) at 200 nm and a lower negative MRE at 220 nm than that lacking carbohydrate at alpha78Asn and hCGwt . The dissociation rates of the alpha52Asn and alpha78Asn carbohydrate deficient mutants at pH 3 and room temperature, measured by using 1-anilino-8-naphthalenesulfonate, were 9.4 x 10(-5) and 3.8 x 10(-5) s-1, respectively, as compared with 1.5 x 10(-5) s-1 for hCGwt . The results of both CD measurements and dissociation studies strongly suggest that the absence of carbohydrate at alpha52Asn results in conformational changes in the mutant which might explain the loss in its signal transduction function . This is further supported by indirect evidence from two other lines of experimentation . Unlike the mutant lacking carbohydrate at alpha78Asn, the one lacking carbohydrate at alpha52Asn cross-reacted with the two subunit specific monoclonal antibodies, anti-hCGalpha and anti-hCGbeta, which normally did not cross-react with the native or the hCGwt . Also, polyclonal anti-hCGbeta but not anti-hCGalpha was able to restore the cAMP-producing activity of the alpha52Asn carbohydrate deficient mutant . From all the data taken together, it appears that the loss of second messenger-producing activity of hCG with the absence of the glycosyl chain at alpha52Asn was probably due to a conformational change in the heterodimer rather than due to the loss of the alpha52Asn-carbohydrate-receptor interaction.

Hypertension, 1997 Sep, 30(3 Pt 1), 449 - 54
11BetaOH-progesterone affects vascular glucocorticoid metabolism and contractile response; Brem AS et al.; Vascular smooth muscle (VSM) contains a bidirectional isoform of 11beta-hydroxysteroid dehydrogenase (11beta-HSD), the enzyme that can metabolize endogenous glucocorticoids to their respective 11-dehydro derivatives . 11BetaOH-progesterone (11betaOH-P), a compound that can be produced in vivo, is as potent or more potent than licorice derivatives in inhibiting renal and hepatic 11beta-HSD . When studied in homogenates prepared from primary cultures of rat VSM, 11betaOH-P and its derivative, 11-keto-progesterone (11-keto-P), proved to be potent, directionally specific inhibitors of vascular 11beta-HSD . 11BetaOH-P selectively inhibited the forward dehydrogenase reaction (corticosterone-->11-dehydrocorticosterone), whereas 11-keto-P selectively blocked the reverse oxidoreductase reaction . To test the physiological effects, vascular rings were prepared from rat aorta . Rings were incubated in culture media containing either a submaximal concentration of corticosterone (10 nmol/L), 11-dehydrocorticosterone (100 nmol/L), 11betaOH-P (1 micromol/L), 11-keto-P (1 micromol/L), or a combination of glucocorticoid and inhibitor for 24 hours . After the 24-hour incubation, rings were briefly stimulated sequentially with phenylephrine (10 nmol/L to 1 micromol/L) and angiotensin II (1 micromol/L) . The immediate contractile response in rings incubated with both corticosterone and 11betaOH-P was greater than in rings previously incubated with either the corticosterone or 11betaOH-P alone (eg, response to 100 nmol/L phenylephrine in milligrams of force, mean+/-SE: corticosterone, 728+/-56, n=9; 11betaOH-P, 325+/-105, n=4; both, 1132+/-122, n=8; corticosterone versus both, P<.01) . In contrast, the immediate contractile responses to phenylephrine and to angiotensin II were attenuated in rings exposed previously to both 11-dehydrocorticosterone and 11-keto-P . Thus, 11betaOH-P and 11-keto-P (and possibly structurally similar compounds) alter the vascular effects of glucocorticoids and may play a role in glucocorticoid-induced hypertension.

Mol Med, 1997 Aug, 3(8), 519 - 29
Construction and characterization of a replication-deficient adenovirus expressing rat-soluble interleukin-6 receptor; Thibault V et al.; BACKGROUND: The pleiotropic cytokine interleukin-6 mediates its multiple effects at the cell level through a multimeric receptor consisting of a binding protein (gp80) and a signal transducer (gp130) . A soluble form of gp80 (sIL-6R or gp55) is found released from the surface of cells and appears to possess interleukin-6 (IL-6) agonist activity . Increases in circulating levels of sIL-6R have been reported in different pathological conditions but the precise role of this protein in vivo remains unknown . MATERIALS AND METHODS: The cDNA encoding the extracellular domain of the rat IL-6R (sIL-6R) with an appropriate leader sequence has been cloned into the E1 region of an adenovirus vector under the control of the hCMV promoter (Ad5.sIL-6R) . RESULTS: Infection of different human or rodent cell lines with Ad5.sIL-6R leads to extended production of recombinant sIL-6R protein into the culture media . The kinetics of transgene expression depends both on the cell type and the species . sIL-6R produced in this manner is biologically active as it confers responsiveness of human hepatoma cells (HepG2) to rat IL-6 stimulation . Adenovirus vectors have been shown to be highly effective for transient delivery of cytokines in vivo . Antibodies against recombinant rat soluble IL-6R were generated and an ELISA developed that allowed us to quantify sIL-6R concentrations . The sIL-6R expressing adenovirus vector has been instilled intratracheally into rats and induced an increase in lung sIL-6R concentration from Day 1 up to Day 10 . We demonstrate the potency of our system to deliver in vivo or in vitro soluble cytokine receptors in a prolonged but transient manner.

Cornea, 1997 Sep, 16(5), 537 - 40
Impact of growth factors on morphometric corneal endothelial cell parameters and cell density in culture-preserved human corneas; Barisani-Asenbauer T et al.; PURPOSE: Donor corneas can be preserved for < or = 4 weeks in organ culture (31 degrees C) by using modified minimal essential medium (MEM) . About one fifth of them have to be discarded, however, as disintegration of the endothelial cell monolayer-enhanced polymegethism, cell loss-occurs . The objective of this study was to investigate whether addition of insulin, dextran, and epidermal growth factor (EGF) makes corneal endothelial cells more viable, stable, and homogeneous . METHODS: Sixteen paired human donor corneas were cocultured in media supplemented with EGF and insulin-like growth factor (IGF) or in conventional modified MEM for 4 weeks . Endothelial parameters were evaluated at the outset and at days 7, 14, 21, and 28 of culture by using an automated digital image-analysis system . RESULTS: No significant differences were observed in the first 2 weeks of culture . Beginning with day 14, however, stabilization of endothelial cell patterns was evident for corneas cultured in supplemented culture media . CONCLUSION: Our data indicate that the addition of growth factors to culture media might increase the percentage of corneas available for transplantation and would also allow a significantly longer period of preservation.

Mol Pathol, 1997 Jun, 50(3), 153 - 9
"Aggrecanase" activity is implicated in tumour necrosis factor alpha mediated cartilage aggrecan breakdown but is not detected by an in vitro assay; Buttle DJ et al.; AIMS: To develop an in vitro assay for the putative glutamyl endopeptidase, "aggrecanase", which is thought to degrade cartilage aggrecan, and to examine the role of the enzyme in tumour necrosis factor stimulated aggrecan cleavage . METHODS: Aggrecan fragments released by bovine nasal cartilage explants, with and without exposure to tumour necrosis factor alpha, were purified and analysed by western blotting and N-terminal sequencing . Intact bovine aggrecan was incubated with extracts of cartilage, lysed chondrocytes, or cartilage explant conditioned culture medium under a variety of conditions . Deglycosylated aggrecan was incubated with nasal cartilage explants . Proteoglycan breakdown was assessed by metachromatic assay of fragments in culture media, and cleavage of the substrate at the aggrecanase cleavage site was detected and measured using the antibody BC3, which recognises a neoepitope produced by aggrecanase cleavage of aggrecan . RESULTS: Aggrecan fragments generated from explants treated with tumour necrosis factor had N-terminal sequences consistent with cleavage of aggrecan at a restricted number of glutamyl bonds . Aggrecanase generated fragments were found in cartilage explant culture medium and chondrocyte monolayers . However, no aggrecanase activity could be detected in extracts of cartilage, or chondrocytes from which endogenous aggrecan fragments had been removed, under a variety of assay conditions . Deglycosylated aggrecan, added to explant cultures, efficiently inhibited endogenous aggrecan breakdown . CONCLUSIONS: Aggrecanase is active in cartilage and in chondrocyte monolayers, and its action is stimulated by tumour necrosis factor alpha . However, activity due to this enzyme could not be detected in vitro under our assay conditions, although a deglycosylated version of the substrate inhibited aggrecan breakdown in explant cultures.

Placenta, 1997 Sep, 18(7), 521 - 6
Tumour necrosis factor-alpha induces cyclo-oxygenase-2 gene expression in first trimester trophoblasts: suppression by glucocorticoids and NSAIDs; Imseis HM et al.; Tumour necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine which stimulates the synthesis and release of prostaglandins (PGs) in several in vitro and in vivo models of preterm labour . While TNF-alpha simulated PG production has been described in decidual, amnion and myometrial cells, to date no studies have focused on the role of TNF-alpha in the stimulation of arachidonic acid metabolism in placental trophoblast cells . Cyclo-oxygenase-2 (COX-2) is the rate-limiting enzyme in PG biosynthesis and is expressed de novo during cellular activation by cytokines . To test whether TNF-alpha alters expression of COX-2, trophoblasts from first trimester chorionic vili were cultured as a continuous cell line and treated with TNF-alpha alone or with TNF-alpha and dexamethasone (Dex) . Total RNA and protein were extracted from the trophoblasts and subjected to Northern and immunoblot analysis, respectively . Northern blots were hybridized with a 32P-labelled probe encoding the COX-2 cDNA and immunoblots were incubated with anti-COX-2 antibodies . There was a time- and dose-dependent increase in COX-2 mRNA and protein expression in cells stimulated with TNF-alpha . The effect of TNF-alpha on COX-2 mRNA and protein expression was inhibited by dexamethasone (Dex) . To examine the production of PGE2 and PGF(2 alpha), specific RIAs were performed on culture media from similarly stimulated cells . PG accumulation after TNF-alpha stimulation occurred in a time- and dose-dependent fashion with a similar inhibition of PG accumulation after Dex exposure . To be certain that TNF-alpha stimulated PGE2 production was, indeed, a result of COX-2 induction, RIAs were carried out with the COX-2-selective inhibitor NS-398 . Cells stimulated with the NS-398 after TNF-alpha exposure demonstrated suppression of TNF-alpha-stimulated PGE2 formation . The results suggest that TNF-alpha elicits part of its pathophysiologic effects in preterm labour via alterations in COX-2 gene expression within the placental microenvironment.

Biol Reprod, 1997 Sep, 57(3), 552 - 60
Ratio of inner cell mass and trophoblastic cells in blastocysts derived from porcine 4- and 8-cell embryos and isolated blastomeres cultured in vitro in the presence or absence of protein and human leukemia inhibitory factor; Eckert J et al.; In this study we investigated effects of developmental stage at onset of individual in vitro culture on the progress of development in intact embryos and individual blastomeres derived from 4-cell and 8-cell porcine embryos (referred to as 1/4 and 1/8, respectively), the necessity of serum or BSA supplementation in culture media for embryos and blastomeres (in contrast to development in a defined medium), and the role of two concentrations of human leukemia inhibitory factor (hLIF) on development of blastomeres and embryos . More (p < 0.05) 1/4 blastomeres developed to the blastocyst stage than did 1/8 blastomeres . In the serum-supplemented medium, the percentage of inner cell mass (ICM)/total cells in 8-cell- and 1/8-derived blastocysts was higher (p < 0.05) than that in 4-cell- and 1/4-derived embryos . Development to blastocysts was similar in BSA-supplemented and defined medium as compared to that in serum-enriched medium in intact 4-cell and 8-cell embryos, and 1/4 blastomeres . More (p < 0.05) 1/8 blastomeres developed to the blastocyst stage in serum-supplemented medium than in defined medium . The high hLIF concentration (1000 IU/ml) decreased (p < 0.05) blastocyst development in 1/4 blastomeres in defined medium, but fewer blastocysts (p > 0.05) lacked an ICM (blastocyst-like vesicles) than in defined medium without hLIF . It is concluded that 1) porcine intact embryos and isolated blastomeres can be cultured individually in defined medium up to the blastocyst stage from the 4-cell stage onwards; 2) more 1/4 isolated blastomeres develop to blastocysts with more total cells but with a lower ratio of ICM to total cells than blastocysts derived from 1/8 blastomeres; 3) the effects of hLIF are dependent on proteins present in the culture medium and on the embryonic stage; and 4) in defined medium, high concentrations of hLIF are inhibitory to blastocyst formation, but fewer blastocyst-like vesicles are formed . The defined culture system employed in this study allows examination of the effects of growth factors or cytokines in porcine early embryonic development.

Biol Reprod, 1997 Sep, 57(3), 525 - 31
Immunologic and molecular characterization of an estrogen-dependent glycoprotein in the rhesus (Macaca mulatta) oviduct; Verhage HG et al.; The objective of this study was to detect and characterize a secreted oviduct-specific glycoprotein (OGP) in the rhesus macaque (Macaca mulatta) and to compare the characteristics of this OGP to those previously characterized in baboons and women . Oviducts were obtained from untreated ovariectomized rhesus and from ovariectomized rhesus either treated with estradiol (E2) for 14 days or treated sequentially with E2 for 14 days and then with E2 plus progesterone (P4) for an additional 14 days . Segments of oviducts were either fixed for morphological analysis, cultured for OGP synthesis and release, or frozen for RNA analysis . The proteins present in the culture media were separated by one-dimensional SDS-PAGE, and OGP was detected on Western blots using polyclonal antibodies generated against the reduced form of baboon OGP or a 17-amino acid segment of the baboon core protein . Cross-reacting antigens were present in the 120-kDa region, identical to what was observed for baboon and human OGP . Indirect immunogold localization of OGP on thin sections demonstrated specific clustering of gold particles over the apical secretory granules of the secretory cells of the oviductal epithelium . A cDNA was generated using RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE), and sequenced . The total transcript was 2237 nucleotides in length plus a poly(A) tail . The largest open reading frame was 624 amino acids, which would produce a protein of 69.3 kDa . The nucleotide sequence was more than 95% identical to the nucleotide sequences of baboon and human OGP . Northern blots revealed a single message at 2.4 kilobases (kb) in oviduct samples obtained from E2-treated rhesus . This message was absent in oviducts obtained from untreated ovariectomized and from sequential E2 plus P4-treated rhesus macaques . In summary, the rhesus oviduct synthesizes and secretes an OGP in the presence of E2 that is immunologically and structurally similar to the baboon and human OGP . The presence of a highly homologous glycoprotein in several primates suggests a similar function for OGP in the reproductive process.

J Clin Microbiol, 1997 Sep, 35(9), 2430 - 1
A new experimental in vitro culture medium for cultivation of Leishmania species; Limoncu ME et al.; A new liquid culture medium prepared with chemicals that can be obtained economically and commercially was tested in in vitro cultivation of Leishmania promastigotes to obtain a large number of organisms to use in serological studies . The number of Leishmania infantum and Leishmania tropica promastigotes taken from Novy-MacNeal-Nicolle (NNN) medium reached 1 x 10(7)/ml at the end of the 8th day in our new medium, though in NNN medium the number of organisms reached only 5 x 10(6)/ml . After 10 subsequent passages, the culture medium prepared was evaluated as being quite inexpensive, simple, and successful compared with other commercially available liquid culture media.

Arch Androl, 1997 Sep-Oct, 39(2), 119 - 25
Assessment of the quality of frozen serum by spectrophotometric analysis and sperm bioassay; Hossain AM et al.; Serum is an integral part of media used for in vitro fertilization (IVF) and andrology work . Previous studies showed that the IVF results could benefit if sera were screened for deleterious effects before use . Such screening is impractical when fresh sera are used but may be feasible if the serum is frozen prior to use . This study assessed the impact of freezing on the quality of serum . A total of 158 serum samples, prepared in a university-based andrology-IVF center, were included in the study . The frozen sera were thawed in batches to be used in a series of laboratory experiments . Serum quality was evaluated by spectrophotometric analysis and sperm bioassay under several defined conditions: fresh, frozen, pre- and postfiltration, pre- and postcentrifugation, and the patients' fertility condition . Although all sera were filtered through 0.22-micron filter, more than 10% frozen sera required 0.4- or a combination of 0.8- and 0.4-micron filters before they could be passed through the 0.22-micron filter . Frozen sera that were directly filtrable with a 0.22-micron filter lost 13% turbidity upon filtration . The turbidity of the frozen sera were higher compared to fresh ones as revealed by optical density (OD) and relative light scattering (RLS) spectrophotometry . The freeze/storage-induced spectrophotometric changes did not correlate with the storage time . The centrifugation caused precipitation of sera components . The rate of precipitation of the serum components correlated with the duration of freezing . Spectrophotometric analysis and sperm bioassay did not differentiate the sera of pregnancy-positive and pregnancy-negative subjects . The sperm bioassay failed to detect any biological impact of freezing-induced spectrophotometric changes in the sera, suggesting that the freezing-induced changes did not significantly diminish the serum's capability of supplementing the culture media.

Proc Soc Exp Biol Med, 1997 Sep, 215(4), 412 - 7
Insulin-like growth factor-I stimulates proliferation of mouse uterine epithelial cells in primary culture; Shiraga M et al.; Estrogens stimulate proliferation and differentiation of uterine epithelial cells in vivo . Mitogenic action of estrogens may be mediated by growth factors such as insulin-like growth factor-I (IGF-I) . This study was designed to determine whether IGF-I and insulin affect proliferation of uterine epithelial cells obtained from 3- to 4-week-old immature female mice in a serum-free culture system . The epithelial cell number on Day 5 in culture was significantly increased by adding IGF-I (10 and 100 ng/ml) or insulin (100 and 1000 ng/ml) to the culture media, indicating that IGF-I is more effective than insulin in inducing the epithelial growth . The epithelial DNA synthesis was significantly stimulated by IGF-I (1 and 10 ng/ml), suggesting that both the epithelial proliferation and their detachment from substratum are stimulated by 1 ng/ml of IGF-I, but that the former is more accelerated than the latter by 10 ng/ml of IGF-I . These results demonstrate that both IGF-I and insulin directly stimulate the growth of uterine epithelial cells, and suggest that insulin may act via IGF-I receptors . IGF-I immunoreactivity was detected in the cytoplasm of the cultured cells, indicating that the cells synthesize IGF-I . Estradiol-17 beta (E2) at lower concentrations (0.001-0.1 nM) tended to increase the number of epithelial cells, while E2 at higher concentrations (1 to 100 nM) did not affect it . It is highly probable that IGF-I produced in endometrial cells induces their proliferation by an autocrine or paracrine mechanism.

J Neurooncol, 1997 Sep, 34(2), 111 - 22
Lectin histochemistry of astrocytic tumors and in vitro characterization of lectin-induced modifications on the proliferation of the SW1088, U373 and U87 human astrocytic cell lines; Camby I et al.; The role of lectins as biosignalling molecules or as markers of human astrocytic tumors remains relatively unexplored . The aim of the present work is to investigate (1) whether or not human astrocytic tumors express specific glycans, evidenced experimentally by means of lectin histochemistry, and (2) whether, in turn, these lectins can significantly modulate astrocytic tumor cell proliferation . Using a cell image processor, we therefore began by quantitatively measuring the histochemical binding pattern of 5 lectins (WGA, PNA, PHA-L, GSA-IA4 and Con A) in 5 astrocytomas, 5 anaplastic astrocytomas and 5 glioblastomas . Secondly, we measured the influence of these 5 lectins on the proliferation of 3 astrocytic tumor cell lines (SW1088, U373 and U87) growing in vitro as monolayers . Cell proliferation was assessed by means of the colorimetric MTT assay . The histochemical lectin staining markedly varied intra- and inter-group . However, some constant results were obtained . Indeed, the staining increased markedly from GSA-IA4 and PHA-L through WGA and PNA to ConA in the three histopathological groups . The assessment of cell proliferation demonstrated that WGA, Con A and PHA-L very significantly decreased proliferation in the 3 astrocytic cell lines in a dose-dependent manner . Astrocytic tumor cells in the confluent growth phase were less sensitive to the WGA, Con A and PHA-L lectin-induced effects than cells in the log growth phase . The GSA-IA4 and PNA lectins had globally very weak effects on the proliferation of the astrocytic tumor cell lines . Increasing the fetal calf serum from 1% to 10% in the culture media significantly antagonized the WGA-, Con A- and PHA-L-induced cell proliferation decrease in the 3 astrocytic cell lines . In conclusion, the present data strongly suggest that some lectins (including WGA, Con A and PHA-L) significantly influence the proliferation of astrocytic tumor cells.

Arch Biochem Biophys, 1997 Aug 15, 344(2), 404 - 12
Inhibition of cartilage degradation and changes in physical properties induced by IL-1beta and retinoic acid using matrix metalloproteinase inhibitors; Bonassar LJ et al.; Bovine cartilage explants were treated with 100 ng/ml recombinant human interleukin-1beta (IL-1beta) or 1 microM all-trans retinoic acid (RA) and changes in biochemical, biomechanical, and physicochemical properties were assessed . Additionally, samples cultured with IL-1beta or RA were treated with 4 microM recombinant human tissue inhibitor of metalloproteinases-1 (TIMP-1) or a synthetic metalloproteinase inhibitor (L-758,354) to inhibit this degradation . Treatment with IL-1beta or RA each resulted in >90% GAG loss after 8 days in culture . Addition of TIMP or L-758,354 to the culture media inhibited IL-1beta-induced loss of tissue GAG by 40 and 65%, respectively, and inhibited RA-induced GAG loss by 35 and 65%, respectively . Analysis of degradation products in the culture media using a G1 antibody indicated that IL-1beta- and RA-treated plugs released 68-kDa fragments of aggrecan, corresponding to a segment of the aggrecan core protein from the G1 domain to the C-terminus NITEGE, consistent with "aggrecanase" activity . Release of the G1 fragment was inhibited by treatment with L-758,354 . Both IL-1beta and RA induced significant loss of hyaluronan from cartilage explants after 8 days of exposure and HA loss was also inhibited by addition of L-756,354 to the culture media . IL-1beta, but not RA, induced a significant increase in swelling ratio (wet weight in 0.01 M NaCl normalized to wet weight in DMEM) after 8 days in culture, consistent with degradation of the collagen network, and the increase in tissue swelling was inhibited by treatment with TIMP-1 or L-758,354 . Exposure to IL-1beta or RA resulted in significant changes in cartilage physical properties including streaming potential, equilibrium modulus, hydraulic permeability, and electrokinetic coupling coefficient after 8 days in culture, and these changes were inhibited by 40-90% by exposure to TIMP and by 50-90% by exposure to L-758,354 . Measurement of dynamic streaming potential showed that changes due to treatment with IL-1beta alone were highly dependent in compression frequency, with dramatic changes seen at high frequency prior to changes in mechanical properties, and little initial change seen at low frequency . Streaming potential and equilibrium modulus of explants treated with RA decreased to 10% of their initial values after 8 days in culture, but decreased to only 40 and 90%, respectively, when treated with RA plus TIMP-1.

J Clin Invest, 1997 Aug 15, 100(4), 829 - 38
Interferon gamma and interleukin 4 stimulate prolonged expression of inducible nitric oxide synthase in human airway epithelium through synthesis of soluble mediators; Guo FH et al.; Human respiratory epithelium expresses inducible nitric oxide synthase (iNOS) continuously in vivo, however mechanisms responsible for maintenance of expression are not known . We show that IFNgamma is sufficient for induction of iNOS in primary human airway epithelial cells (HAEC) in vitro, and IL-4 potentiates IFNgamma-induced iNOS expression in HAEC through stabilization of iNOS mRNA . IFNgamma/IL-4- induced iNOS expression in HAEC was delayed in onset and prolonged with expression up to 1 wk . Removal of overlying culture media resulted in loss of expression, while transfer of conditioned media induced iNOS mRNA in other HAEC . IFNgamma and IL-4 stimulation activated STAT1 and STAT6 in HAEC, but conditioned media transfer to HAEC produced even higher levels of STAT1 activation than achieved by direct addition of cytokines . Although cytokine induction of iNOS was dependent on new protein synthesis, conditioned media induction of iNOS in HAEC was not . Further, removal of overlying culture media from cells at different times after cytokine stimulation demonstrated that mediator synthesis and/or secretion important for induction and maintenance of iNOS occurs early after cytokine stimulation . In conclusion, a combination of IFNgamma/ IL-4, which occurs naturally in the lung epithelial lining fluid, leads to maintenance of iNOS expression in human airway epithelium through production of soluble mediators and stabilization of mRNA.

J Assist Reprod Genet, 1997 Aug, 14(7), 398 - 403
Nonessential amino acids and glutamine decrease the time of the first three cleavage divisions and increase compaction of mouse zygotes in vitro; Lane M et al.; PURPOSE: The objective of this study was to determine the effect of supplementing embryo culture media with amino acids on the duration of the first three cell cycles of mouse zygotes in vitro . METHODS: Zygotes were cultured in the presence of different groups of amino acids and cleavage assessed every 30 min . RESULTS: Culture of zygotes with Eagle's nonessential amino acids and glutamine significantly reduced the time of cleavage divisions to the eight-cell stage compared to culture without amino acids . Beneficial effects of amino acids were found to be cumulative over time . Nonessential amino acids and glutamine also increased the percentage of eight-cells that compacted after 57 hr of culture compared to embryos in medium devoid of amino acids . CONCLUSIONS: The present data suggest that media for the development of cleavage-stage embryos, such as in clinical IVF, should be supplemented with Eagle's nonessential amino acids and glutamine.

Am J Physiol, 1997 Aug, 273(2 Pt 2), R731 - 8
Central noradrenergic system modulates plasma interleukin-6 production by peripheral interleukin-1; Huang QH et al.; The role of the central noradrenergic system in systemic interleukin-6 (IL-6) production induced by intravenously administered recombinant human interleukin-1 beta (IL-1 beta) was examined in rats . Pretreatment of rats intracerebroventricularly with 6-hydroxydopamine (6-OHDA, 100 or 200 micrograms/rat) significantly attenuated the increase in plasma IL-6 levels caused by IL-1 beta (2 micrograms/kg i.v.) . A modest inhibition of the IL-1 beta-induced plasma IL-6 production was observed following pretreatment with prazosin (20 micrograms/rat i.c.v.) but not after administration of idazoxan or propranolol . There were no significant increases in the IL-6 content in the hypothalamus, medulla oblongata, and cortex of the brain after intravenous IL-1 beta . Adrenalectomy produced an augmented plasma IL-6 response to intravenous IL-1 beta, whereas chemical sympathectomy with intraperitoneal injection of 6-OHDA (50 or 100 mg/kg) decreased the IL-1 beta-induced plasma IL-6 levels . Nor-epinephrine (NE), in the dose range 10(-6)-10(-4) M, significantly increased the IL-6 levels in the rat spleen lymphocyte culture media . At doses of 10(-9)-10(-7) M, NE enhanced the effect of IL-1 beta on the IL-6 release by spleen lymphocytes in a dose-dependent manner . These findings suggest that the plasma IL-6 response to intravenous IL-1 beta is partially mediated through the activation of the central noradrenergic system and a consequent increase in the sympathetic outflow to the peripheral tissues and that the NE released from the sympathetic terminals may function as a mediator and/or modulator to facilitate the synthesis/release of IL-6 in the sympathetic nerve-innervated organs.

Am J Reprod Immunol, 1997 Aug, 38(2), 129 - 39
TGF-beta 2 and PGE2 in rabbit blastocoelic fluid can modulate GM-CSF production by human lymphocytes; Fortin M et al.; PROBLEM: During normal pregnancy, major changes occur in the production of Th2/Th1 cytokines at the feto-maternal interface . Th2 cytokines such as interleukin-4 (IL-4) or interleukin-10 (IL-10) are predominantly produced locally in the uterine and placental tissues, whereas the production of Th1 cytokines such as tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) are decreased . Because these modulation might be induced by the embryo, the current study was carried out to test the effect of rabbit blastocoelic fluid on the production of Th2/Th1 cytokines by lymphocytes, and to investigate the possible implication of transforming growth factor beta 2 (TGF-beta 2) prostaglandin E2 (PGE2) as modulators of the production of these cytokines . METHOD OF STUDY: Human peripheral blood lymphocytes (PBL) were cultured along with ConcanavalinA(Con A), and rabbit blastocoelic fluid was collected on day 12 of gestation (BF d-12) . Concentrations of cytokines in culture media were determined by enzyme-linked immunoadsorbent assay (ELISA) . RESULTS: Addition of BF d-12 in the culture medium induced a strong inhibition of IL-2, TNF-alpha, IL-10, and granulocyte-macrophage colony-stimulating factor (GM-CSF) production . However, an initial pretreatment of the lymphocytes with BF d-12, followed by a Con A stimulation, led to a marked increase in GM-CSF production, whereas IL-2, TNF-alpha, and IL-10 secretions were inhibited . It was also demonstrated, for the first time, that a pretreatment of the lymphocytes with TGF-beta 2 and PGE2 increased GM-CSF production to the same level reached after the addition of BF d-12 . Furthermore, removal of TGF-beta 2 and PGE2 from BF d-12 by affinity chromatography reduced the effect of BF d-12 on GM-CSF production . CONCLUSIONS: Taken together, these findings suggest that the embryo, in modulating harmful and beneficial cytokine production locally, plays an active role in its protection against maternal immune cellular assault . These results also emphasize the importance of growth factors for successfully maintaining pregnancy.

Am J Respir Cell Mol Biol, 1997 Aug, 17(2), 227 - 34
Induction of cystine transport and other stress proteins by disulfiram: effects on glutathione levels in cultured cells; Deneke SM et al.; Disulfiram (Antabuse) (DSF) has been reported to protect rats and other animals from the effects of hyperbaric hyperoxia at 4 to 6 ATA (atmospheres) . In contrast, DSF and diethyldithiocarbamate (DDC), its metabolite, accelerate the toxic effects in rats of 100% oxygen at 1 to 2 ATA . We have examined the effects of DSF and DDC on glutathione (GSH) levels in bovine pulmonary artery endothelial cells and Chinese hamster ovary cells . Increases in intracellular GSH occurred 8 to 24 h after addition of DSF to the culture media . These increases in intracellular GSH were associated with increases in the rate of uptake of cystine into the cells . DDC was a less effective inducer of cystine uptake and increased intracellular GSH levels than was DSF . At the concentrations used, neither DDC nor DSF caused significant decreases in intracellular superoxide dismutase levels . Exogenous sulfhydryl compounds including GSH and cysteine partially blocked the induction of cystine transport by DSF or DDC, suggesting that the induction might be mediated through a sulfhydryl reaction between DSF and some cellular components . The increases in GSH in the cultured cells were not significant by 4 h of exposure . In contrast, other stress proteins including heme oxygenase are induced by 2 to 4 h after DSF addition . In previously reported in vivo studies, DSF treatment protected against hyperbaric oxygen damage after as little as 1 to 4 h pre-exposure . This suggests that effects of DSF exposure other than GSH augmentation may be responsible for the protective effects seen in vivo.

Thromb Haemost, 1997 Aug, 78(2), 827 - 33
Homocysteine metabolism in endothelial cells of a patient homozygous for cystathionine beta-synthase (CS) deficiency; van der Molen EF et al.; Homocystinuria due to cystathionine beta-synthase (CS) deficiency is the most common inborn error of methionine metabolism . Patients with CS-deficiency have an extremely high risk of vascular disease . The underlying mechanism is still unsolved . Dysfunction of endothelial cells could be the trigger in the formation of atherosclerosis and thrombosis . Therefore, differences in cell function were studied between normal and CS-deficient human umbilical endothelial cells (HUVECs) . Total homocysteine (tHcy) concentrations in culture media as a measure of homocysteine export increased in all cell lines, including the cell line with CS-deficiency, with constant amounts of approximately 2.5 microM every 24 h . von Willebrand factor (vWF), tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI-1) in culture media were used as markers of endothelial function and increased also with progression of culture time . The effects of additions of folate, vitamin B6 and methionine to the culture medium were studied . The homocysteine export and the markers of endothelial function did not differ between the control and the CS-deficient HUVECs under various test conditions . These data show that CS-deficient endothelial cells have normal homocysteine export and normal endothelial cell function . In CS-deficient patients the very high blood levels of homocysteine, probably due to deficient CS function in liver and kidney, seems to be the hazardous factor to endothelial cells, thus promoting atherosclerosis and thrombosis in CS-deficient patients.

J Bone Miner Res, 1997 Aug, 12(8), 1210 - 22
Characterization of native and recombinant bone sialoprotein: delineation of the mineral-binding and cell adhesion domains and structural analysis of the RGD domain; Stubbs JT 3rd et al.; Bone sialoprotein is a small, sulfated, and phosphorylated integrin-binding glycoprotein apparently found only in tissues that eventually mineralize . Nondenatured bone sialoprotein (BSP) purified from rat osteosarcoma cell line (UMR 106-01 BSP) culture media is shown to have a hydroxyapatite Kd approximately 2.6 x 10(-9) M, perhaps the strongest affinity for this mineral of any of the matrix proteins . Both native BSP and a 47 kD fragment of UMR-BSP (Fragment 1 approximately 133A- approximately 265Y) are more potent inhibitors of seeded hydroxyapatite crystal growth than recombinant human BSP fragments lacking post-translational modifications . The recombinant proteins, however, do show reproducible inhibitory activity, suggesting that at least some of the strong mineral-binding properties are encoded directly within the protein sequence itself . BSP facilitates the adhesion of several cell types through its integrin binding (RGD) tripeptide sequence . Nuclear magnetic resonance (NMR) analysis of a 15N-enriched 59 amino acid recombinant domain containing the RGD tripeptide shows that the structure of this isolated domain is highly flexible with or without 5 mM calcium . Previous work has also shown that an endogenous fragment of UMR-BSP (Fragment 1) supports cell adhesion in the absence of the RGD sequence . In this report, non-RGD cell adhesion sites are localized within conserved amino- and carboxy-terminal tyrosine-rich domains of recombinant human BSP . Given the proximity of the latter non-RGD cell adhesion site to the RGD tripeptide, a model of BSP-receptor interactions is presented.

Blood Cells Mol Dis, 1997 Aug, 23(2), 169 - 76
Co-transactivation of the 3' erythropoietin hypoxia inducible enhancer by the HIF-1 protein; Varma S et al.; Erythropoietin (Epo) is a glycoprotein hormone that is the primary regulator of red blood cell production . Epo production increases in response to tissue hypoxia . This increase occurs primarily at the transcriptional level . Hypoxia inducible factor (HIF-1) is a DNA binding protein that binds to a hypoxia inducible enhancer in the 3' flanking sequence of the Epo gene . HIF-1 is a heterodimer that consists of an alpha and beta subunit . HIF-1 DNA binding activity is induced in response to hypoxia . In order to determine if one or both HIF-1 subunits is capable of ligand binding, subsequently leading to Epo production we performed co-transactivation experiments . Transfections were performed in Hep 3B, an Epo producing human hepatoma cell line and Cos-7, a non-Epo producing monkey kidney cell line . Cells were co-transfected with the 38 bp Epo enhancer fragment bearing the HIF-1 binding motif, subcloned in the luciferase reporter plasmid and either the HIF-1alpha cDNA, HIF-1beta cDNA, HIF-1alpha and HIF-1beta cDNAs or pREP-4 respectively . Cells were incubated in an hypoxic (1%O2) or normoxic (21%O2) environment and assayed for luciferase activity . Epo levels were measured in the culture media from the transfected plates by an ELISA assay . Under hypoxic conditions Hep 3B cells transfected with the HIF-1alpha cDNA alone showed a 2.2 fold increase in luciferase activity, HIF-1beta showed a 3.4 fold increase and cells transfected with HIF-1 alpha and beta showed a 6 . 9 fold increase in activity over cells transfected with pREP-4 . The baseline luciferase activity in transfected 3B cells incubated in normoxia was very low . However, a similar fold increase in luciferase activity in cells transfected with both HIF-1alpha and beta was noted . Under normoxic or hypoxic conditions in Cos-7 cells, a 1.5 fold increase was obtained with the HIF-1alpha and beta constructs transfected independently and a 3.5 fold increase was noted in cells transfected with both constructs . Epo levels increased several fold in all Hep 3B cells that were incubated in hypoxic conditions . However, there was no additional increase in Epo levels in transfected Hep 3B cells . We therefore conclude that although the HIF-1alpha and beta subunits can independently co-transactivate the Epo enhancer, binding of both subunits and a hypoxic environment is necessary for maximal transactivation . Overexpression of the HIF-1 protein alone in normoxic or hypoxic conditions is insufficient for an increase in Epo secretion . Activation/inactivation and interaction of other tissue specific factors is necessary for an increase in Epo gene expression in response to hypoxia.

J Neurooncol, 1997 Aug, 34(1), 5 - 22
Glial ontogeny and glial neoplasia: the search for closure; Linskey ME; The last ten years have seen rapid progress in both our understanding of the normal progression and control of gliogenesis and in the laboratory techniques necessary to sustain and study most glial cell types, including progenitor cells of both type-1 astrocyte (T1A) and oligodendrocyte-type-2 astrocyte (T2A) lineage . These studies have direct relevance for the lineage analysis of human gliomas, optimizing in vitro glioma models, and suggesting potentially fertile new grounds for glioma biology research . We do not yet known whether malignant transformation occurs only in mature glia that then 'de-differentiate' into cells with glial precursor phenotypes and behavior characteristics, whether neoplastic transformation occurs in O-2A progenitor cells, or whether both mechanisms may occur in different patients . However, preliminary results suggest that astrocytomas can arise from two different glial lineages, that oligodendrogliomas and mixed oligo-astrocytomas arise exclusively from the O-2A lineage, and that medulloblastomas may also have a connection with the O-2A lineage . An ontogeny-based glioma classification system may lead to better prognostic patient data and better predict patient response to treatment than currently available classification systems . Available data from the study of developmental glial biology raises serious doubts about the fidelity and relevance of in vitro glioma models that rely on culture media supplemented with animal serum and suggest that relying on chemically-defined media conditioned by astrocytes may be the better research strategy . Findings from the study of normal gliogenesis also suggest that growth factors are likely to act as much more than simple mitogens in glioma biology . Potentially fertile areas of research for glioma biology include studying the cooperative effect of multiple growth factors, potential growth factor effects as survival factors, inhibitors of differentiation, and differentiation inducers, and studying potential positive humoral feedback loops between glioma cells and normal glial cells, as well as normal non-glial cells, within and surrounding each glioma.

Arch Biochem Biophys, 1997 Jul 15, 343(2), 243 - 8
Ascorbate and alpha-tocopherol prevent apoptosis induced by serum removal independent of Bcl-2; Barroso MP et al.; Cells require serum to maintain growth in vitro . Serum provides growth and survival factors and its removal causes an oxidative stress that induces peroxidations in membrane lipids and development of programmed cell death (apoptosis) in some cells . Cells containing Bcl-2 are partially protected against both lipid peroxidation and apoptosis and some cell lines, such as Daudi, which lack this protein, are very sensitive to serum removal . Thus, cells are grown for 48 h in the absence of fetal calf serum and apoptotic cells are scored . HL-60 cells containing a moderate amount of Bcl-2 show 30% apoptosis, while 55% cells are apoptotic of the Bcl-2-negative Daudi cell population . Apoptosis is reduced to 15% in the transiently transfected Daudi/Bcl-2 cells . Ascorbate (Asc) and alpha-tocopherol (alphaTOH) can prevent lipid peroxidation and apoptosis caused by serum withdrawal, when added to culture media, even in the absence of Bcl-2 . Also, these two antioxidants increase survival of cells grown in the absence of serum independent of their Bcl-2 content . Immunostaining and quantification of Bcl-2 show that HL-60 cell line is a heterogeneous population relative to the expression of Bcl-2 . When these cells are grown in the presence of serum, cells lacking Bcl-2 survive, but no Bcl-2-negative cells survive without serum . Part of this population of Bcl-2-negative cells is rescued by Asc and alphaTOH . Antioxidants effective at the plasma membrane such as Asc and alphaTOH can protect cells from oxidative damage and prevent apoptosis independent of Bcl-2 content.

Neurosci Lett, 1997 Jul 11, 230(1), 9 - 12
Nerve growth factor inhibits the expression of nitric oxide synthase in neurones in dissociated cultures of rat dorsal root ganglia; Thippeswamy T et al.; In dissociated cultures of DRG derived from 15-day-old rats the numbers of neurones expressing immunocytochemically detectable quantities of the neuronal isoform of nitric oxide synthase (nNOS) was determined and the effects of different culture media examined . The availability of NGF in the cultures was found to be a critical determinant of nNOS expression . In a serum-rich media (SRM) supplemented with NGF, 24% of the neurones expressed nNOS compared with 72% in the absence of added NGF and the presence of an antibody to NGF (t-test, P < 0.0001) . Cultures grown in a defined media (DM) developed poorly and many neurones died, these cultures also showed poor growth of other cell types . Immunostaining for NGF revealed that some of the non-neuronal cells produce NGF and that this would be predicted to contribute to the survival of the neurones . In cultures in which neurones were dying most of the surviving neurones expressed nNOS suggesting it may have a survival promoting function.

Biochemistry, 1997 Jul 8, 36(27), 8377 - 83
Specific increase in amyloid beta-protein 42 secretion ratio by calpain inhibition; Yamazaki T et al.; Cerebral deposition of amyloid beta-protein (Abeta) as senile plaques is a pathological hallmark of Alzheimer's disease (AD) . Abeta falls into two major subspecies defined by their C-termini, Abeta40 and Abeta42, ending in Val-40 and Ala-42, respectively . Although Abeta42 accounts for only approximately 10% of secreted Abeta, Abeta42 is the predominant species accumulated in senile plaques in AD brain and appears to be the initially deposited species . Its secretion level has recently been reported to be increased in the plasma or culture media of fibroblasts from patients affected by any of early-onset familial AD (FAD) . Thus, inhibition of Abeta42 production would be one of the therapeutic targets for AD . However, there is little information about the cleavage mechanism via which Abeta40 and Abeta42 are generated and its relationship to intracellular protease activity . Here, we examined by well-characterized enzyme immunoassay the effects of calpain and proteasome inhibitors on the levels of Abeta40 and Abeta42 secretion by cultured cells . A calpastatin peptide homologous to the inhibitory domain of calpastatin, an endogenous calpain specific inhibitor, induced a specific increase in secreted Abeta42 relative to the total secreted Abeta level, a characteristic of the cultured cells transfected with FAD-linked mutated genes, while a proteasome specific inhibitor, lactacystin, showed no such effect . These findings suggest that the Abeta42 secretion ratio is modulated by the calpain-calpastatin system and may point to the possibility of exploring particular compounds that inhibit Abeta42 secretion through this pathway.

FEBS Lett, 1997 Jul 7, 411(1), 83 - 6
beta-Adrenergic stimulation of interleukin-1alpha and interleukin-6 expression in mouse brown adipocytes; Burysek L et al.; Mouse brown adipocytes in primary culture were shown to contain high levels of mRNA for interleukin-1alpha (IL-1alpha) which could be further stimulated up to 9-fold by norepinephrine (NE) . Even higher stimulation by NE, up to 40-fold, was found in case of interleukin-6 (IL-6) . Time-course of activation of both genes was biphasic, but the response of IL-6 gene was slower than of IL-1alpha gene . IL-1alpha mRNA level reached the maximum after 1 h and the second, lower increase, occurred after 8 h . IL-6 mRNA level showed first maximum after 2 h, but the highest level was found after 8 h . Similarly to NE, the expression of IL-1alpha and IL-6 genes was stimulated by selective beta-adrenergic agonist isoproterenol, beta3-selective agonist CGP-12117, forskoline and db-cAMP . The activation of both genes by CGP-12177 was dose-dependent with the optimum at 100 nM concentration . Stimulation of alpha-adrenergic receptors by cirazoline and oxymetazoline was without any effect . When the expression of IL-6 was studied at the protein level, the stimulation of IL-6 gene via beta3-receptors resulted in secretion of IL-6 up to the concentration 10 ng/ml culture media in 24 h . The results indicate a new type of regulation of expression of IL-1alpha and IL-6 genes in brown adipocytes by catecholamines acting via beta3-adrenergic receptors . The resulting increase in IL-6 production by brown adipocytes could significantly contribute to systemic levels of IL-6.

Mol Cell Biochem, 1997 Jul, 172(1-2), 195 - 8
A simple method for preparation of cultured cardiac fibroblasts from adult human ventricular tissue; Agocha AE et al.; Cardiac fibroblasts constitute greater than 90% of non-myocyte cells in the heart . Because they are responsible for synthesis of components of the extracellular matrix, growth factors and cytokines in the myocardium, they play an important role in normal and pathologic performance of the heart . An understanding of their biology requires in depth studies in a stable and reliable system in which the biological responses of cardiac fibroblasts to various stimuli can be determined . With the exception of few, all studies have been performed on cardiac fibroblasts obtained from rodent hearts . We present a method for isolation and subsequent culture of viable cardiac fibroblasts from ventricular tissue of adult human . This method allows rapid and reliable isolation and subsequent culture of cardiac fibroblasts from adult heart tissue without the need for cumbersome isolation techniques and complex nutrient-enriched and hormone-supplemented culture media for maintenance.

Neuromuscul Disord, 1997 Jul, 7(5), 361 - 6
Canine fucosidosis: a model for retroviral gene transfer into haematopoietic stem cells; Ferrara ML et al.; Severe progressive fatal neurological degeneration occurs in fucosidosis, a storage disease . Bone marrow transplantation into affected dogs has shown that haematopoietic stem cells can provide enzyme producing daughter cells to the central nervous system, altering disease course . This makes canine fucosidosis an ideal large animal model for gene therapy . Fucosidosis affected allogeneic or autologous canine marrow was transduced ex vivo by cocultivation, then transplanted into fucosidosis affected dogs conditioned with total lymphoid irradiation . The vectors were Moloney murine leukaemia virus based . Transduction efficiency was increased with multiple cytokines in short term marrow culture . Despite high levels of transduction, proviral sequence was detected 2 months post transplant in only one dog . Early or total graft failure occurred in all transplants . We believe lack of engraftment could be caused by differentiation or change of repopulating ability of marrow cells occurring with multiple cytokine mixes in culture media.

Hum Reprod, 1997 Jul, 12(7), 1525 - 30
Reduced pregnancy rates following the transfer of human embryos frozen or thawed in culture media supplemented with normal serum albumin; Warnes GM et al.; Over a 26 month period 17% of couples having treatment in our clinical programmes selected a commercially available protein (normal serum albumin, NSA) prepared from pooled human sera instead of using their own serum as a supplement for their embryo culture media . In a retrospective analysis of >2000 gonadotrophin-stimulated cycles and 1000 cycles where frozen/thawed embryos were transferred, fertilization, embryo quality and pregnancy rates following in-vitro fertilization (IVF), gamete intra-Fallopian transfer (GIFT) or intracytoplasmic sperm injection (ICSI) were unaffected by the type of protein used to supplement the culture medium . When embryos were thawed in medium containing NSA, both pregnancy (PR) and implantation rates (IR) were significantly lower (P <0.05) than if the medium was supplemented with serum (PR 8.3% and 17.5%; IR 4.6% and 10.5%) . Inclusion of NSA before freezing reduced the IR of thawed embryos . To further test this observation all cycles where embryos were cultured and frozen in medium containing NSA (173 cycles) were matched to cycles where serum was used and the outcome was compared . At the end of 1995 just over half of the embryos in both groups had been thawed . No statistical difference was noted in the pregnancy rates (NSA, 5.6% versus serum, 11.3%) but the IR per embryo was significantly lower when embryos were cultured and frozen in medium supplemented with NSA (2.2%) than when serum was used as the supplement (6.6%).

Hum Reprod, 1997 Jul, 12(7), 1500 - 7
Placental protein 14 production by human Fallopian tube epithelial cells in vitro; Saridogan E et al.; We studied the in-vitro secretory function of non-polarized and polarized cultured Fallopian tube epithelial cells by measurement of the placental protein 14 (PP14) secretion in primary cultures and subcultures from Fallopian tubes obtained from eight premenopausal women in different phases of the ovarian cycle . Primary cultures were established in minimal essential medium in Earle's salts supplemented with fetal bovine serum and the cells were subcultured for six passages, in the polarized cell cultures, the cells being seeded on an extracellular matrix system . Cell freezing was carried out using 10% dimethyl sulphoxide . PP14 secretion into the culture media was measured by a radioimmunoassay using 125I-PP14 as label and rabbit anti-human PP14 serum . There was a large amount of PP14 secretion into the culture media in primary cultures, the secretion decreasing considerably after subculture 1 . PP14 secretion after subculture 2 was not different from the control values . Polarized and non-polarized cells secreted similar amounts of PP14 and frozen-thawed cells did not appear to secrete PP14 . Epithelial cells from Fallopian tubes obtained at different phases of the ovarian cycle did not appear to show any difference in PP14 secretion rates . Our data suggest that the in-vitro secretion of PP14 by human Fallopian tube epithelial cells is adversely affected by cell ageing and freezing.

Br J Rheumatol, 1997 Jul, 36(7), 735 - 43
Selective induction of the secretion of cathepsins B and L by cytokines in synovial fibroblast-like cells; Lemaire R et al.; We have investigated the potent influence of some cytokines, tumour necrosis factor-alpha (TNF-alpha), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF) and interferon-gamma (IFN-gamma), on the secretion of cysteine proteinases (cathepsins B and L) by cultured synovial fibroblast-like cells from patients with rheumatoid arthritis (RA) . After treatment of synovial fibroblast-like cells with cytokines, culture media were evaluated for cathepsins B and L by enzyme immunoassays, and for cathepsin B and L activities using the enzymatic substrates . Z-Phe-Arg-AMC and Z-Arg-Arg-AMC, and specific inhibitors . Treatment of synovial fibroblast-like cells with TNF-alpha or PDGF resulted in a marked increase in cathepsin B secretion . Moreover, after prolonged PDGF treatment, the amount of secreted cathepsin B returned to the low control level . In contrast, bFGF led to increased cathepsin L secretion . IFN-gamma induced both cathepsin B and L secretion . Our results show that cytokines induce a selective secretion of cathepsins B and L by synovial fibroblast-like cells . This selective effect of cytokines on the secretion of cysteine proteinases suggests that synovial fibroblast-like cell-mediated articular degradation is a highly regulated process.

Placenta, 1997 Jul-Aug, 18(5-6), 451 - 8
Influence of hypoxia on vascular endothelial growth factor and chorionic gonadotrophin production in the trophoblast-derived cell lines: JEG, JAr and BeWo; Taylor CM et al.; Growth of trophoblast tissue in early pregnancy is rapid and accomplished in an unusually hypoxic environment . Hypoxia has been reported to upregulate mRNA production of vascular endothelial growth factor (VEGF), and VEGF receptors have been found on trophoblast cells . These observations suggest that VEGF may have an important role in early placentation . This study examines the influence of hypoxia on both the production of the VEGF message and protein and on the production of human chorionic gonadotrophin (hCG) protein by the cell lines JEG, JAr and BeWo . Cells were grown under normoxic and hypoxic conditions for 72 h . The average oxygen tension in the culture media of the hypoxic cultures (6-7 kPa) was significantly less than in the normoxic cultures (19-21 kPa) . RNA was extracted and message for VEGF(121), VEGF(165) and VEGF(189) found in all cell lines by reverse transcription and the polymerase chain reaction (RT-PCR) . These messages were upregulated by hypoxia; findings confirmed by competitive PCR for VEGF and expression of the house keeping gene GAPDH . hCG and VEGF were measured by immunoassay . Hypoxia resulted in an increase in VEGF production (P<0.05) but had inconsistent effects on hCG production . In some experiments the absolute concentrations of hCG and VEGF in the culture media were noted to be significantly correlated (r>0.5, P<0.05) . In addition to its role in angiogenesis, VEGF may have direct effects on trophoblast cells encouraging proliferation and invasion . These effects may be regulated in part through oxygen supply and hCG.

J Mol Cell Cardiol, 1997 Jul, 29(7), 1947 - 58
Angiotensin II stimulated expression of transforming growth factor-beta1 in cardiac fibroblasts and myofibroblasts; Campbell SE et al.; Angiotensin II (Ang II) stimulates pathologic myocardial fibrosis . Cardiac fibroblasts (CFb) and myofibroblasts mediate this response, perhaps in part by indirect production of specific cytokines . We sought to determine if Ang II could stimulate transforming growth factor-beta1 (TGF-beta1) gene expression and protein production in adult rat CFb and two cardiac myofibroblast cell types, scar myofibroblasts (MyoFb) and valvular interstitial cells (VIC) . Confluent CFb, MyoFb, and VIC in serum-deprived (0.4% FCS) media were treated with Ang II (10(-7) m for CFb; 10(-9) m for MyoFb, VIC) for 24 h . Untreated cells served as controls . Culture media was collected and TGF-beta1 levels determined in triplicate using a sandwich ELISA . Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed to determine TGF-beta1 mRNA expression . Ang II increased CFb (P<0.02) and VIC (P<0.04) TGF-beta1 mRNA expression, while the increase in MyoFb was not statistically significant . MyoFb produced the highest TGF-beta1 levels under control conditions compared to VIC and CFb . Ang II stimulated further TGF-beta1 secretion in VIC and CFb, but not MyoFb . The AT1 receptor antagonist Losartan (10(-7) m) greatly attenuated Ang II-stimulated TGF-B1 secretion and decreased TGF-beta1 immunostaining in VIC . The AT2 receptor antagonist PD123177 (10(-7) m) also decreased secretion and immunostaining of TGF-beta1 in VIC, but to a lesser extent than Losartan . TGF-beta1 secretion by MyoFb was unaffected by Losartan and PD123177, although TGF-B1 immunostaining was absent or greatly decreased, respectively, compared to Ang II-treated MyoFb . Ang II stimulates TGF-beta1 gene expression and/or protein production in cardiac fibroblast-like cells which may act as an autocrine/paracrine stimulus to collagen formation . Furthermore, TGF-beta1 production and secretion in these cells can be modulated by specific Ang II receptor antagonists, suggesting a potential benefit in preventing/attenuating pathologic myocardial fibrosis.

J Neurosci Res, 1997 Jul 1, 49(1), 9 - 18
Association of human, rat, and rabbit apolipoprotein E with beta-amyloid; LaDu MJ et al.; In humans, apolipoprotein E (apoE) has three major isoforms, E2 (Cys112, Cys158), E3 (Cys112, Arg158), and E4 (Arg112, Arg158) . While epsilon4 is a genetic risk factor for Alzheimer's disease (AD), epsilon2 may protect against late-onset AD . Using native preparations of apoE from conditioned tissue culture media or plasma lipoproteins, we have previously shown that when equivalent amounts of apoE3 or E4 were incubated with beta-amyloid (A beta), apoE3 formed 20 times as much SDS-stable complex with the peptide as apoE4 . This preferential binding of A beta to apoE3 was abolished when apoE was purified by a process which includes delipidation and denaturation . Here we expand these observations to include A beta binding to lipoprotein-associated and purified apoE2 . Lipoproteins isolated from the plasma of individuals homozygous for either epsilon2 or epsilon3 were incubated with A beta(1-40) . SDS-stable complex formation was analyzed by a non-reducing gel shift assay, followed by immunoblotting with either A beta or apoE antibodies . ApoE2:A beta complex formation was comparable to apoE3:A beta in both native and purified preparations of apoE . In addition, lipoprotein-associated rat apoE (Arg112, Arg158), like human apoE4, did not form complex with A beta, while lipoprotein-associated rabbit apoE (Cys112, Arg158) did bind the peptide . These binding studies provide one possible explanation for protective effects of both apoE2 and E3 against the development of Alzheimer's disease.

Kidney Int, 1997 Jul, 52(1), 120 - 9
Identification of promoter activity and differential expression of transcripts encoding the murine stromelysin-1 gene in renal cells; Yee J et al.; Stromelysin-1, matrix metalloproteinase-3 (MMP-3), is an important endopeptidase selectively expressed by somatic cells in organ tissues . The renal tubulointerstitium, for example, comprises tubular epithelium and interstitial fibroblasts forming the principal mass of the kidney . We observed that mRNA encoding stromelysin-1 is detectable in murine renal fibroblasts, but not in proximal tubular epithelium . Transcripts measured by RNase protection assay in renal fibroblasts increase following exposure to phorbol ester, and thereafter, activated stromelysin-1 protein can be detected in culture media by Western blotting . A 6.4 Kb genomic clone containing the putative stromelysin-1 promoter was isolated and a relevant 2.1 Kb PstI restriction fragment including 2.1 Kb of the immediate 5'-flanking region was sequenced on both strands . Two transcriptional start sites were identified by primer extension; the major start site corresponded to a previously established position in the rat promoter, and a second undescribed minor transcriptional start site was located 16 bp upstream of the primary site . A HiNF-A chromatin-activating element at -106 bp was found in the early promoter region of pR336 and an active AP-1 site at -72 bp with an Ets/PEA-3 motif at -203 bp was suggested by transient transfection of luciferase minigenes into renal fibroblasts responsive to phorbol ester . This Ets element was identical to a site in the early promoter of the fibroblast-specific gene FSP1 . A baseline enhancement in activity of pR336 in fibroblasts was further observed with the addition of 5' flanking sequence out to -1980 bp . This additional region of flanking sequence contains two modular regions: one of multiple PEA-3 elements between -684 bp and -1955 bp and a second region between -1929 bp and -1980 bps containing a second AP-1 site at -1929 bp, a MBF-1/ MEP-1 metal binding site, and a PPAR peroxisome proliferator element at -1950 bp . Our findings implicate a gene structure with expected activity in a mesenchymal phenotype . The PKC-dependent regulation of the stromelysin-1 gene supports the notion that it may be modulated during inflammation or tissue remodeling.

J Med Virol, 1997 Jul, 52(3), 320 - 5
Low rate shedding of HSV-1 DNA, but not of infectious virus from human donor corneae into culture media; Garweg JG et al.; Fluid samples derived from 451 organ cultured corneae were tested for the presence of HSV-1 DNA after electroseparation and amplification for fragments of the glycoprotein D- and thymidine kinase-encoding genes . Of the culture media, 134 were processed immediately after withdrawal (Group 1); 100 were stored at ambient temperature for 6 to 60 weeks (Group 2); 90 were stored at -8 degrees C for 4 to 9 weeks (Group 3); and 127 were stored at -20 degrees C for 2 to 30 weeks (Group 4) . The degradation of human DNA (marker gene, betaglobin) under these different storage conditions and of human and HSV-1 DNA as a sequential function of time at ambient temperature was gauged by the loss of a detectable signal for the respective component . Endothelial cell density within each of the corneal discs was determined before and after organ culture . In 7/451 culture fluid samples, HSV-1 DNA corresponding to either the glycoprotein D- or thymidine kinase-encoding genes was detected . In culture fluid samples derived from Group 2 at ambient temperature, for 6 to 60 weeks) and 3 (at -8 degrees C, for 4 to 9 weeks), complete degradation precluded the detection of human DNA, and hence probably also of HSV-1 DNA; only at -20 degrees C did DNA remain stable for protracted periods of time . Even so, HSV-1 DNA was detected in only 2% of those media in which no degradation was to be expected; additionally, there existed no correlation between its presence in culture fluid samples and the loss of endothelial cells or cytopathic changes . DNA can be extracted successfully and concentrated twenty-fold from high-volume samples by electroseparation . When shed into culture fluid, it is remarkably prone to a time and temperature dependent degradation, which may lead to false negative results . It is concluded that there is no infectious virus to be expected in the specimens; the occurrence of HSV-1 DNA in donor corneae would not appear to be an important factor influencing their biological quality during the period of organ culture.

Fertil Steril, 1997 Jul, 68(1), 65 - 71
Hydrosalpinx fluid enhances human trophoblast viability and function in vitro: implications for embryonic implantation in assisted reproduction; Sawin SW et al.; OBJECTIVE: To assess the effects of hydrosalpinx fluid on human cytotrophoblast viability and function in vitro . DESIGN: Human cytotrophoblasts obtained from third-trimester placentas were cultured in vitro with hydrosalpinx fluid, and cell viability and protein production were assayed . SETTING: A university hospital . PATIENT(S): Ten hydrosalpinx fluid samples obtained from seven women with clearly diagnosed hydrosalpinges . INTERVENTION(S): Recovery of hydrosalpinx fluid by transvaginal aspiration or at the time of surgery . MAIN OUTCOME MEASURE(S): Cell viability was assessed by the XTT assay . Secretion of trophoblast oncofetal fibronectin (tropho-uteronectin) and beta-hCG by cultured trophoblasts was determined by Western blot and ELISA of the culture media . RESULT(S): With increasing concentrations of hydrosalpinx fluid from 0% to 20%, there was a significant increase in trophoblast cell viability (1.63-fold increase in 20% hydrosalpinx fluid) . Likewise, both Western blot and ELISA assays demonstrated a significant increase in tropho-uteronectin production by trophoblasts with increasing hydrosalpinx fluid concentrations (3.76-fold increase in 20% hydrosalpinx fluid) . beta-Human chorionic gonadotropin production also increased significantly in the presence of hydrosalpinx fluid (3.31-fold increase in 20% hydrosalpinx fluid) . CONCLUSION(S): These findings suggest that hydrosalpinx fluid improves human trophoblast viability in vitro and enhances the production of tropho-uteronectin and beta-hCG by these cells.

Cell Tissue Res, 1997 Jul, 289(1), 155 - 61
Thrombin regulates nerve growth factor secretion from vascular, but not bladder smooth muscle cells; Sherer TB et al.; The production of nerve growth factor (NGF) in peripheral organs may play a role in the pathophysiology of hypertension and in obstructive disorders of the bladder outlet . We have been examining the cellular processes of NGF delivery and secretion in smooth muscle . NGF secretion from vascular smooth muscle cells (VSMCs) cultured from genetically hypertensive (WKHT), hyperactive (WKHA), and a control Wistar rat strain were assayed using a two-site ELISA of the culture media . Bladder smooth muscle cells (BSMCs) from the Wistar strain were also studied . The serine protease, thrombin, increased NGF secretion from all types of VSMCs but had no effect on Wistar BSMCs . The thrombin-mediated increase in NGF secretion was prevented by actinomycin D and cycloheximide, suggesting that RNA transcription and protein synthesis are required . The effect of thrombin was additive with a phorbol ester-induced elevation in NGF secretion rates from 4 to 6 h and was attenuated by a 24-h downregulation of protein kinase C . These results suggest that extracellular protease activity may regulate NGF secretion in smooth muscle . Thrombin may act in response to vascular injury, increasing NGF secretion from VSMCs, initiating VSMC migration, and preparing the VSMCs for reinnervation following an insult.

Cell Tissue Res, 1997 Jul, 289(1), 109 - 17
Differentiation-induced changes in the content, secretion, and subcellular distribution of lysosomal cathepsins in the human colon cancer HT-29 cell line; De Stefanis D et al.; Enterocyte-like differentiated HT-29 colon carcinoma cells were shown to contain far higher intracellular levels of activity of lysosomal cathepsins B, D, and L than their undifferentiated counterparts . In the latter, inhibition of lysosomal functions by leupeptin or ammonium chloride led to a marked increase in the cell-associated activity of the three cathepsins . High levels of pro-cathepsins B, D, and L were found in the culture media of both HT-29 cell populations . Ammonium chloride and chloroquine, which are known to impair the mannose-6-phosphate-dependent trafficking of lysosomal-targeted proteins, did not increase the secretion of the three cathepsins in either undifferentiated or differentiated cultures of HT-29 cells . Analyses by cell fractionation revealed heterogeneities with regard to the density and the content of lysosomal cathepsins between the two cell populations . Leupeptin induced the accumulation of mature lysosomal cathepsins B and L in light density organelles in undifferentiated HT-29 cells . Altogether, these data demonstrate that (1) the expression and subcellular distribution of cathepsins B, D, and L in HT-29 cells are influenced by their state of enterocytic differentiation, (2) the segregation of lysosomal cathepsins is largely inefficient in this tumor cell line and does not increase upon differentiation, and (3) the mannose-6-phosphate-receptor-dependent pathway plays a minor role in the sorting of the three cathepsins, both in undifferentiated and enterocytic-differentiated HT-29 cells.

J Chromatogr B Biomed Sci Appl, 1997 Jun 20, 694(1), 83 - 92
Simultaneous analysis of retinol, all-trans- and 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in plasma by liquid chromatography using on-column concentration after single-phase fluid extraction; Teerlink T et al.; A reversed-phase high-performance liquid chromatographic method for the simultaneous analysis of retinol, all-trans-retinoic acid, 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in human plasma and cell culture medium is described . Sample preparation involves precipitation of proteins and extraction of retinoids with 60% acetonitrile . After centrifugation, the acetonitrile content of the supernatant is reduced to 45%, allowing on-column concentration of analytes . Injection volumes up to 2.0 ml (equivalent to 0.525 ml of sample) can be used without compromising chromatographic resolution of all-trans-retinoic acid and 13-cis-retinoic acid . Retinoids were stable in this extract and showed no isomerization when stored in the dark in a cooled autosampler, allowing automated analysis of large series of samples . Recoveries from spiked plasma samples were between 95 and 103% . Although no internal standard was used, the inter-assay precision for all retinoids was better than 6% and 4% at concentrations of 30 nM and 100 nM, respectively . The method is a valuable tool for the study of cellular metabolism of all-trans-retinoic acid, as polar metabolites of this compound can be detected with high sensitivity in cell culture media.

Int J Cancer, 1997 Jun 11, 71(6), 993 - 9
Human pancreatic cancer cells (MPanc-96) recognized by autologous tumor-infiltrating lymphocytes after in vitro as well as in vivo tumor expansion; Peiper M et al.; A human tumor line designated MPanc-96 has been established from a poorly differentiated primary pancreatic adenocarcinoma . MPanc-96 has a doubling time of 27 hr and grows as a confluent monolayer in various culture media . Cytogenetic analysis of in vitro-cultured tumor cells revealed a large number of clonal chromosomal aberrations, confirming their neoplastic origin . MPanc-96 grows in SCID mice when injected s.c . Xenografts established from the tumor line had a similar histology as the primary tumor . Tumor-infiltrating lymphocytes (TILs) were isolated from the primary tumor, and cytotoxic T lymphocytes (CTLs) were generated after activation on immobilized anti-CD3 monoclonal antibody (MAb) for 48 hr, expansion in low-dose IL-2 and repeated stimulation with irradiated MPanc-96 tumor cells . The generated CTLs lysed fresh autologous tumor cells as well as in vitro and in vivo expanded tumor cells from passages 9-53, suggesting that one or more tumor-associated antigens (TAAs) are stably expressed . CTLs lysed tumor cells in an HLA-class I-restricted fashion but showed no significant cytotoxicity against autologous fibroblasts, several allogeneic pancreatic cancer cell lines or K562 . Our findings may be significant for the design of an animal model for studying the mechanisms of immunotherapy in human pancreatic cancer or for the identification of TAAs in pancreatic cancer.

J Chromatogr B Biomed Sci Appl, 1997 Jun 6, 693(2), 415 - 21
Sensitive high-performance liquid chromatographic assay for aminoglycosides in biological matrices enables the direct estimation of bacterial drug uptake; Stead DA et al.; Following the development of a sensitive high-performance liquid chromatographic (HPLC) assay for gentamicin in biological matrices, the utility of this assay for the determination of other clinically important aminoglycosides (neomycin, netilmicin and sisomicin) in bacterial culture media or plasma is demonstrated . The high sensitivity of the assay enables direct measurement of the aminoglycoside content of bacterial cells cultured in the presence of unlabelled drug.

J Chromatogr B Biomed Sci Appl, 1997 Jun 6, 693(2), 359 - 66
Isocratic high-performance liquid chromatographic method for the separation of isradipine and its main metabolites . Application to in vitro metabolization by h3A4/OR cells; Bidouil S et al.; A reversed-phase HPLC method was developed for the study of isradipine oxidation in vitro . The drug and its main metabolites were determined after extraction from culture media and from h3A4/OR cells having the cytochrome P450 isoenzyme involved in the xenobiotic metabolization . The HPLC assay was fully validated and was used to follow the biotransformation kinetics of isradipine.

J Biol Chem, 1997 Jun 6, 272(23), 14516 - 22
Interactions of the amino-terminal noncollagenous (NC1) domain of type VII collagen with extracellular matrix components . A potential role in epidermal-dermal adherence in human skin; Chen M et al.; Type VII collagen, the major component of anchoring fibrils, consists of a central collagenous triple-helical domain flanked by two noncollagenous domains, NC1 and NC2 . The NC1 domain contains multiple submodules with homology to known adhesive molecules including fibronectin type III-like repeats and the A domain of von Willebrand factor . In this study, we produced the entire NC1 domain of human type VII collagen in the stably transfected human kidney 293 cell clones and purified large quantities of the recombinant NC1 protein from serum-free culture media . The recombinant NC1 formed interchain disulfide-bonded dimers and trimers and was N-linked glycosylated . Tunicamycin inhibited the cellular secretion of NC1, suggesting that N-linked glycosylation may play a role in NC1 secretion . The recombinant NC1 was indistinguishable from the authentic NC1 obtained from human amnions or WISH cells with respect to N-linked sugar content, electrophoretic mobility, rotary shadow imaging, and binding affinity to type IV collagen . Purified recombinant NC1, like authentic NC1, also bound specifically to fibronectin, collagen type I, and a laminin 5/6 complex . Both monomeric and trimeric forms of NC1 exhibited equal affinity for these extracellular matrix components, suggesting that the individual arms of NC1 can function independently . The multiple interactions of NC1 with other extracellular matrix components may support epidermal-dermal adhesion.

Endocr J, 1997 Jun, 44(3), 409 - 17
Profiles of insulin-like growth factor binding proteins and the protease activity in the maternal circulation and its local regulation between placenta and decidua; Sakai K et al.; Insulin-like growth factors (IGF) and their specific binding proteins (IGFBPs) are believed to be important regulators of fetal growth . IGFBP protease which proteolyzes IGFBPs and changes the biochemical properties of IGFBPs is also presumed to be involved in fetal growth . The aim of this study is to elucidate the physiological significance of IGFBP protease in fetal growth and regulators of protease in placenta and decidua . The intact IGFBP-3 was proteolyzed into fragments when pregnant serum was incubated with 125I-IGFBP-3 . IGFBP-3 protease activity showed a marked increase at 5 weeks of gestation and reached a plateau in maternal circulation at 15 weeks of gestation . These changes in protease activity correlated with the profiles of IGFBPs in the maternal circulation analyzed by Western ligand blot, where the IGFBP-1 is only the dominant IGFBP . The intact IGFBP-3 was proteolyzed when culture media of decidual cells were incubated with 125I-IGFBP-3, but was not proteolyzed when culture media of trophoblast cells were incubated with 125I-IGFBP-3 . Decidual protease activity was slightly increased by IGF-I and completely inhibited by progesterone . The protease activity was more increased in the mothers with growth retarded infant than in those in the mothers with normal growth infants, suggesting that the protease activity is elevated in compensation for the impaired fetal growth . These results suggest that increased protease activity in maternal blood may be involved in the fetal growth indirectly by reducing the binding activity of IGFBP-3 to IGF-I, and that protease activity in maternal blood may be derived from decidua that is regulated by placental hormones.

Nihon Rinsho Meneki Gakkai Kaishi, 1997 Jun, 20(3), 159 - 65
{Serum levels and in vitro production of IL-5 in children with bronchial asthma}; Ikeda N et al.; IL-5 play important roles in inflammatory responses in bronchial asthma, but little is known about serum levels and in vitro production of IL-5 in childhood bronchial asthma . We further examined serum IL-5 levels in children with bronchial asthma and the controls . IL-5 in serum was detected in all of asthmatic and disease-free individuals . Its values during asthma exacerbation were significantly higher than during remission of asthma . Serum IL-5 values did not significantly differ among groups divided by asthma severity . We studied IL-5 production by peripheral blood mononuclear cells cultured with or without Dermatophagoides farinae (Df) in children with mite allergy . IL-5 concentrations in culture supernatant from after stimulation with Df were significantly higher than those from asthmatic patients without stimulation and from the control subjects . In contrast, IL-5 levels in culture media from the controls were not significantly different between with and without stimulation with Df . Our results suggest that IL-5 may play roles in the pathogenesis of bronchial asthma.

J Vet Med Sci, 1997 Jun, 59(6), 479 - 81
Improved in vitro cultivation of Babesia caballi; Avarzed A et al.; Babesia caballi infected erythrocytes were collected from the blood of an experimentally infected horse and could be continuously cultivated in vitro with parasitemia ranging from 2-4% in RPMI 1640 medium supplemented with 2 mM L-glutamine, 20 mM HEPES and 40% adult horse serum in a low oxygen atmosphere (2% O2, 5% CO2 and 93% N2) . All attempts to increase parasitemia failed using other culture media, serum concentrations and culture vessels . However, parasite growth was enhanced by transfer of cultures from a low oxygen to 5% CO2 in air, with parasitemia ranging from 8-10%.

Diabetes Metab, 1997 Jun, 23(3), 219 - 27
Bioartificial pancreas containing porcine islets of Langerhans implanted in low-dose streptozotocin-induced diabetic mice: effect of encapsulation medium; Delaunay C et al.; Two encapsulation culture media without animal serum were compared for development of a bioartificial pancreas . Porcine islets were suspended in Hams F10 medium supplemented with 2% Ultroser (US) or in Ultraculture medium (UC) and encapsulated in hollow fibres composed of AN69 copolymer . The function of encapsulated islets was assessed by intraperitoneal transplantation of two fibres in streptozotocin-induced diabetic mice . In both groups of transplanted mice (US, n = 26; UC, n = 18), a significant decrease in plasma glucose concentration was observed three days after fibre implantation (from 21.9 +/- to 14.4 +/- 0.8 mmol/l for US fibres and from 22.7 +/- 0.8 to 13.3 +/- 1.3 mmol/l for US fibres and from 22.7 +/- 0.8 to 13.3 +/- 1.3 mmol/l for UC fibres) . Graft survival 17 days after implantation was 61% for mice with UC fibres and 35% for those with US fibres (P = 0.0001) . Intramuscular glucose tolerance tests were performed in these animals (US, n = 5; UC, n = 10), and a normal glucose pattern was observed in both groups of transplanted mice . The results show that a complete normalisation of blood glucose and glucose tolerance can be achieved by implantation of a bioartificial pancreas . Moreover, UC appears to be a more suitable encapsulation culture medium for porcine islets in vivo.

J Neuroendocrinol, 1997 Jun, 9(6), 467 - 78
Differential LHRH secretion, dye coupling, and protein expression in two morphologically distinct cell types identified in GT1-7 cultures; Matesic D et al.; The immortalized neuronal cell line, GT1-7, has been shown to secrete LHRH in a pulsatile manner and to possess many other characteristics of hypothalamic LHRH neurons in vivo, and thus provides a potential model system for studying biochemical and physiological mechanisms regulating LHRH secretion . In the present study, two morphologically and functionally distinct types of cells have been identified in GT1-7 cultures and each type purified to over 95% homogeneity . One type (N cells) appeared more neuronal with extended neurites and somewhat rounded cell perikarya, while the other type (G cells) had flatter cell perikarya that contained filopodia but no neurites . Growth properties of the two cell types also differed . The doubling time for proliferation of N cells was nearly two-fold shorter than that for G cells and N cells displayed 'piling up' whereas G cells exhibited contact inhibition . Functionally, N cells, but not G cells, were dye-coupled as measured by a fluorescence photobleaching assay . While both cell types expressed LHRH, N cells released significantly higher levels of LHRH into the culture media and exhibited more intense LHRH immunostaining . The two cell types also showed differences in immunostaining for other proteins . N cells, unlike G cells, immunostained positive for neuron-specific enolase (NSE), whereas G cells, unlike N cells, stained immunopositive for vimentin . Both cell types expressed SV-40 T antigen protein, indicating that they were derived from the same transgenic mouse hypothalamic tumour . The physiological significance of these two cell types in GT1-7 cultures remains to be determined, but elucidation of their morphological and biochemical properties is intended to contribute to better understanding and application of this experimentally important neuroendocrine cell line.

Eur J Endocrinol, 1997 Jun, 136(6), 640 - 8
Expression of parathyroid hormone-related peptide (PTHrP) and PTH/PTHrP receptor in newborn human calvaria osteoblastic cells; Lomri A et al.; We examined the expression of parathyroid hormone-related peptide (PTHrP) and its receptor in normal newborn human calvaria osteoblastic (NHCO) cells . Northern blot analysis showed that NHCO cells express a single 1.6 kb transcript of PTHrP, which was increased within 1 h (2x) and peaked at 6 h (7x) after serum treatment . In the culture media, the release of PTHrP peptide was maximally increased (4x) 24 h after the addition of serum, as determined by immunoradiometric assay . NHCO cells exhibited a cytoplasmic immunostaining for PTHrP in the presence of serum, and most PTHrP-positive cells were alkaline phosphatase-negative, suggesting that PTHrP was expressed in undifferentiated cells . Furthermore, RT-PCR analysis showed that both PTHrP and PTH/PTHrP receptor were expressed in NHCO cells in basal conditions or after stimulation with serum . The maximal PTHrP expression induced by serum suppressed PTH/PTHrP receptor expression, suggesting that PTHrP down-regulated its receptor in NHCO cells . Treatment with 10 nM human PTH(1-34) which binds to PTH/PTHrP receptors, increased intracellular cAMP levels and alkaline phosphatase activity, and decreased cell growth, indicating that ligand binding to PTH/PTHrP receptors regulates NHCO cell proliferation and differentiation . The expression and synthesis of PTHrP and the presence of functional PTH/PTHrP receptors suggest a possible paracrine mechanism of action of PTHrP in normal human calvaria osteoblastic cells.

Am J Respir Cell Mol Biol, 1997 Jun, 16(6), 724 - 31
Regulation of the secretory phenotype of human airway epithelium by retinoic acid, triiodothyronine, and extracellular matrix; Yoon JH et al.; The purpose of our studies was to identify factors which regulate the composition of airway secretions produced by normal human tracheobronchial epithelial (NHTBE) cells . Individual factors were removed from the culture media of NHTBE cells grown in air-liquid interface (ALI) cultures (which support mucociliary differentiation) and the effects on mucin, lysozyme (LZ), and secretory leukocyte protease inhibitor (SLPI) secretion and gene expression were examined . Deletion of hydrocortisone, epinephrine, transferrin, or gentamycin-amphotericin from the media had no reproducible effects; deletion of insulin was incompatible with culture growth . We identified 3 factors, namely retinoic acid (RA), triiodothyronine (T3) and collagen gel substratum, which had a major impact on the profile of NHTBE secretions . Removal of RA from the media caused a drastic decrease in mucin secretion and a decrease in expression of the mucin genes MUC2 and MUC5AC.LZ and SLPI secretions were increased in these cultures . Paradoxically LZ mRNA was decreased, while SLPI mRNA levels were increased . Removal of T3 selectively increased mucin secretion, MUC2 gene expression was not affected, but MUC5AC mRNA levels reproducibly increased, suggesting that the expression of these two mucin genes is differentially regulated . LZ and SLPI secretion levels were not significantly affected by deletion of T3 from the culture media; however, LZ mRNA levels were increased in the absence of T3 while SLPI transcript levels were not affected . Omission of the attachment substratum, type I collagen gel, resulted in significant increases in all 3 secretory products . MUC2 and MUC5AC steady state mRNA levels were not consistently affected . In contrast LZ and SLPI gene expression were reproducibly increased . Our studies show that individual factors in the epithelial environment can regulate expression of specific secretory cell gene products in a highly selective manner.

J Am Soc Nephrol, 1997 Jun, 8(6), 935 - 42
P-Cresol, a uremic compound, enhances the uptake of aluminum in hepatocytes; Abreo K et al.; In the end-stage renal disease patient, certain uremic compounds could influence the cellular accumulation of aluminum (Al) . In this study, we examined the effect of 15 uremic ultrafiltrate fractions obtained by HPLC on the uptake and toxicity of Al in mouse hepatocytes (MH) in culture, a model system in which Al is taken up bound to transferrin (Tf) . Uremic fractions 4 to 8, 12, 14, and 15 increased cellular Al uptake and aspartate aminotransferase release and decreased cell growth when Tf-Al, not Al citrate, was added to culture media . Compounds that have been extracted previously from these ultrafiltrate fractions (p-cresol, xanthine, tryptophan, hippuric acid, and o-hydroxyhippuric acid) were then tested for their effect on Al uptake and toxicity in MH at concentrations found in uremic serum . Significant Al uptake by MH was observed only when p-cresol was added together with Tf-Al . Time-response curves showed increased Al uptake and toxicity at p-cresol concentrations of 3 mg/dl in culture media . Dose-response curves confirmed that Al uptake and cell toxicity were proportional to p-cresol from 1.5 mg/dl to 3 mg/dl in culture media . p-Cresol was not toxic to MH in the absence of Tf-Al in media . p-Cresol increased Tf-associated Al uptake only because there was no effect on Al uptake when Al citrate was substituted, and studies with Tf-I125-Al in the presence of this compound showed increased Tf-I125 taken up by MH . p-Cresol did not increase Tf saturation with Al . p-Cresol also increased Tf-Al uptake in Friend erythroleukemia and neuroblastoma cells in culture . Our studies suggest that p-cresol and uremic fractions 4 to 8, 12, 14, and 15 increase the uptake and toxicity of Al in cultured MH . These compounds may play a role in the accumulation and toxicity of Al in the liver of end-stage renal disease patients and possibly in all cells that express Tf receptors.

Kidney Int, 1997 Jun, 51(6), 1710 - 8
Phosphate depletion diminishes Mg2+ uptake in mouse distal convoluted tubule cells; Dai LJ et al.; Hypophosphatemia caused by phosphate depletion is associated with renal magnesium wasting . The cellular mechanisms of phosphate depletion were investigated in an immortalized mouse distal convoluted tubule (MDCT) cell line . Intracellular free Mg2+ concentration . {Mg2+}i was determined by microfluorescence . Mg2+ transport was assessed as a function of change in {Mg2+}i with time following placement of Mg(2+)-depleted cells into a buffer containing 1.5 mM magnesium . The uptake rate of Mg2+ into Mg(2+)-depleted cells cultured in normal phosphate, 1.0 mM, was 175 +/- 21 nM/second . Depletion of phosphate in the culture media was associated with a significant decrease in Mg2+ uptake, which was dependent on the degree of phosphate depletion and on the time cultured in phosphate-deficient media . Cells cultured for 16 hours in 0.3 mM and 0 mM phosphate possessed Mg2+ uptake rates of 105 +/- 18 nM/second and 15 +/- 12 nM/second, respectively . Diminished Mg2+ uptake was rapidly induced following placement in low phosphate and was fully reversed following readdition of phosphate to the culture media . The effects of phosphate depletion on Mg2+ uptake was post-translational in nature as fully up-regulated MDCT cells with maximal Mg2+ uptake was associated with a rapid decrease (within 30 min) in Mg2+ transport when placed in phosphate-deficient media . Although Mg2+ uptake is altered by the transmembrane voltage, diminished Mg2+ uptake associated with phosphate depletion was not dependent on changes in membrane voltage . Further, it was not associated with a sustained increase in intracellular Ca2+ concentration . Chlorothiazide, probably through hyperpolarization of the plasma membrane, stimulates Mg2+ uptake in normal . 283 +/- 23 nM/second, and phosphate-depleted cells, 203 +/- 29 nM/second, but failed to entirely correct the defective transport . These studies demonstrate that magnesium wasting associated with hypophosphatemia and phosphate depletion is due, in part, to diminished Mg2+ transport in the distal convoluted tubule . The evidence is that the actions of phosphate deficiency are through alterations of Mg2+ transport across the luminal membrane of the distal convoluted tubule cell.

Arch Biochem Biophys, 1997 Jun 1, 342(1), 38 - 47
A 33.5-kDa heat- and protease-resistant NADH oxidase inhibited by capsaicin from sera of cancer patients; Chueh PJ et al.; Sera from patients with a variety of cancers, including solid carcinomas, leukemias, and lymphomas, contain a ca . 33.5-kDa protein absent from sera of healthy volunteers or patients not diagnosed as having cancer . The protein exhibits an NADH oxidase activity inhibited by 8-methyl-N-vanillyl-6-noneamide (capsaicin) . The activity and the protein are resistant to digestion by proteases (trypsin, chymotrypsin, proteinase K, subtilisin) and to heat . Following protease digestion to reduce the content of major serum proteins, the 33.5-kDa protein could be detected on Western blots of SDS-PAGE transferred to nitrocellulose membranes using polyclonal antisera to a corresponding partially purified 33.5-kDa protein shed into culture media conditioned by growth of HeLa cells . No corresponding protein was seen with control sera . The findings confirm the capsaicin-inhibited NADH oxidase activity of cancer sera as a circulating marker potentially specific to sera of cancer patients and identify a ca . 33.5-kDa protein resistant to proteases and heat as the source of the circulating capsaicin-inhibited NADH oxidase activity.

J Clin Invest, 1997 Jun 1, 99(11), 2581 - 7
A synthetic peptide inhibitor for alpha-chemokines inhibits the growth of melanoma cell lines; Hayashi S et al.; Melanoma growth stimulatory activity (MGSA/GROalpha) is a 73 amino acid peptide sharing sequence characteristics with the alpha-chemokine superfamily . MGSA/GROalpha is produced by diverse melanoma cell lines and reported to act as an autocrine growth factor for the cells . We tested the binding of MGSA/GROalpha to melanoma cell lines, Hs 294T and RPMI7951, and found that these cells could bind to MGSA/GROalpha but not to interleukin-8 . Recently, we defined a novel hexapeptide, antileukinate, which is a potent inhibitor of binding of alpha-chemokines to their receptors on neutrophils . When antileukinate was added to melanoma cells, it inhibited the binding of MGSA/ GROalpha . The growth of cells from both melanoma cell lines was suppressed completely in the presence of 100 microM peptide . The cell growth inhibition was reversed by the removal of the peptide from the culture media or by the addition of the excess amount of MGSA/GROalpha . The viability of Hs 294T cells in the presence of 100 microM peptide was > 92% . These findings suggest that MGSA/GROalpha is an essential autostimulatory growth factor for melanoma cells and antileukinate inhibits their growth by preventing MGSA/GROalpha from binding to its receptors.

Endocrinology, 1997 Jun, 138(6), 2615 - 20
Adrenomedullin as an autocrine/paracrine apoptosis survival factor for rat endothelial cells; Kato H et al.; Adrenomedullin is a potent vasorelaxant/hypotensive peptide recently isolated from human pheochromocytoma . We demonstrate here a novel role of this peptide as an apoptosis survival factor for rat endothelial cells . When rendered quiescent by serum deprivation, a fraction of endothelial cell cultures showed morphological and biochemical features characteristic of apoptosis . Adrenomedullin significantly suppressed apoptosis without inducing cell proliferation . Rat endothelial cells that contained high affinity binding sites for adrenomedullin expressed adrenomedullin gene and released the peptide into culture media . Addition of preimmune rabbit serum prevented apoptosis, whereas rabbit antiadrenomedullin antiserum partially, but significantly, abrogated the protective effect of the preimmune serum, suggesting its autocrine/paracrine role . Although adrenomedullin induced intracellular cAMP formation, other cAMP-elevating agonists, such as prostaglandin I2 and forskolin, did not affect apoptosis . Furthermore, adenosine 3',5'-cyclicmonophosphothioate Rp-isomer, a cAMP antagonist, did not block the cell survival effect of adrenomedullin . Adrenomedullin neither increased intracellular Ca2+ concentrations nor inositol-1,4,5-trisphosphate levels in rat endothelial cells . These results demonstrate that adrenomedullin suppresses serum deprivation-induced apoptosis of rat endothelial cells via cAMP-independent mechanism.

J Biol Chem, 1997 May 30, 272(22), 14159 - 65
Retinyl ester hydrolysis and retinol efflux from BFC-1beta adipocytes; Wei S et al.; Adipose tissue is an important storage depot for retinol, but there are no data regarding retinol mobilization from adipose stores . To address this, dibutyryl cAMP was provided to murine BFC-1beta adipocytes and its effects on retinol efflux assessed . High performance liquid chromatography analysis of retinol and retinyl esters in adipocytes and media indicated that cAMP stimulated, in a time- and dose-dependent manner, retinol accumulation in the culture media and decreased cellular retinyl ester concentrations . Study of adipocyte retinol-binding protein synthesis and secretion indicated that cAMP-stimulated retinol efflux into the media did not result from increased retinol-retinol-binding protein secretion but was dependent on the presence of fetal bovine serum in the culture media . Since our data suggested that retinyl esters can be hydrolyzed by a cAMP-dependent enzyme like hormone-sensitive lipase (HSL), in separate studies, we purified a HSL-containing fraction from BFC-1beta adipocytes and demonstrated that it catalyzed retinyl palmitate hydrolysis . Homogenates of Chinese hamster ovary cells overexpressing HSL catalyzed retinyl palmitate hydrolysis in a time-, protein-, and substrate-dependent manner, with an apparent Km for retinyl palmitate of 161 microM, whereas homogenates from control Chinese hamster ovary cells did not.

Int J Cancer, 1997 May 29, 71(5), 724 - 31
Tumor-associated lympho-monocytes from neoplastic effusions are immunologically defective in comparison with patient autologous PBMCs but are capable of releasing high amounts of various cytokines; Mantovani G et al.; We studied several in vitro activities of tumor-associated lympho-monocytes (TALMs) and the concentrations of interleukin (IL)-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)alpha, interferon (IFN)gamma and soluble IL-2 receptor (slL-2R) in neoplastic effusions and in the serum of advanced stage cancer patients . Comparisons were made with autologous peripheral blood mononuclear cells (PBMCs) . Autologous PBMCs were compared with PBMCs from normal subjects used as controls . TALMs were collected from 13 peritoneal and 18 pleural neoplastic effusions, secondary to primary tumors of different sites . After PHA stimulation, concentrations of IL-1alpha, IL-1beta and TNF alpha in culture media of TALMs both from peritoneal and pleural effusions were lower than those of autologous PBMCs and, similarly, concentrations of IL-4 and IL-10 in culture media of TALMs from peritoneal effusions were lower than those of autologous PBMCs, whereas concentrations of IL-4 and IL-10 in culture media of TALMs from pleural effusions were in the same range as those of autologous PBMCs . On the contrary, IL-2, IL-6 and IFN gamma amounts (only from pleural effusions) were significantly higher . IL-1alpha, IL-1beta, IL-2, IL-6 and TNF alpha production from patient PBMCs was lower than that of control PBMCs, whereas production of IL-4, IL-10 and IFN gamma was higher than that of control PBMCs . Both in peritoneal and in pleural effusions concentrations of IL-1alpha, IL-1beta and IL-4 were not different from those measured in autologous serum, whereas those of IL-6, IL-10, TNF alpha, IFN gamma and sIL-2R were significantly higher . The amounts of IL-2 in pleural effusions were not different from those of autologous serum, but in peritoneal effusions they were higher than those of autologous serum . The amounts of IL-1alpha, IL-1beta, IL-2, IL-6, TNF alpha and sIL-2R were higher in patient than in control sera, whereas those of IL-4, IL-10 and IFN gamma were in the same range in patient and in control sera . Cell cycle analysis of cultured TALMs and PBMCs (from 3 patients) showed a significant accumulation of TALMs in the non-cycling G0/G1 cell population compared with autologous PBMCs.

J Neurosci Methods, 1997 May 16, 73(2), 169 - 76
Organotypic cultures of the rat anterior pituitary: morphology, physiology and cell-to-cell communication; Guerineau NC et al.; Organotypic cultures, prepared from young rats, were used to investigate the neuroendocrine properties of anterior pituitary cells . Pituitary cells maintained the features of endocrine cells, up to 7 weeks in vitro . Secretory granules could be seen with electron microscopy, and cells contained immunocytochemically detectable levels of adenohypophyseal hormones . Significant levels of prolactin (PRL), growth hormone and luteinizing hormone were present in the culture media after several weeks in vitro and PRL release could be modulated by dopaminergic agonists or forskolin . The electrophysiological properties of pituitary cells were investigated with both intracellular and patch-clamp recordings after 2 to 7 weeks in vitro . Cellular resting membrane potentials were approximately -50 mV, and spontaneous or depolarization-induced action potentials were found in approximately 50% of cells . Records of voltage-dependent outward membrane currents showed that cells expressed functional voltage-gated channels . Cells remained responsive to hypothalamic neuropeptides, as shown by the outward membrane current triggered by thyrotropin-releasing hormone . Intracellularly injected Lucifer Yellow readily diffused between neighboring cells, suggesting the presence of gap junctions . These data confirm the viability of organotypic cultures of the anterior pituitary gland, and demonstrate that the characteristic properties of this excitable endocrine tissue are conserved . This neuroendocrine preparation is suitable for studying the mechanisms regulating cell-to-cell communication under conditions resembling the in vivo tissue organization.

J Biol Chem, 1997 May 2, 272(18), 11952 - 8
Interferon regulatory factor-1 up-regulates angiotensin II type 2 receptor and induces apoptosis; Horiuchi M et al.; The expression of the angiotensin II type 2 (AT2) receptor is developmentally and growth regulated . In cultured R3T3 cells, expression of this receptor is markedly induced at the confluent state and with serum deprivation . In this study we demonstrated that the removal of serum from culture media resulted in the induction of apoptosis in these cells and the addition of angiotensin II further enhanced apoptosis . We have previously identified an interferon regulatory factor (IRF) binding motif in the mouse AT2 receptor gene promoter region . In this report, we observed that serum removal increased IRF-1 expression, with a rapid and transient decrease of IRF-2 . To prove that the changes in IRFs after serum removal mediated apoptosis and up-regulated AT2 receptor, we transfected antisense oligonucleotides for IRF-1 or IRF-2 into R3T3 cells and observed that IRF-1 antisense oligonucleotide attenuated apoptosis and abolished the up-regulation of AT2 receptor . IRF-2 antisense oligonucleotide pretreatment did not affect the onset of apoptosis after serum removal; instead, it increased AT2 receptor binding and enhanced angiotensin II-mediated apoptosis . Taken together, these results suggest that increased IRF-1 after serum starvation contributes to the induction of apoptosis and that increased IRF-1 up-regulates the AT2 receptor expression after serum starvation, resulting in enhanced angiotensin II-mediated apoptosis.

Cell Biol Int, 1997 May, 21(5), 303 - 14
Treatment of rat proximal and distal colonic cells with sodium orthovanadate enhances their adhesion and survival in primary culture; Kaeffer B et al.; We have studied the effect of sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, on primary cultures of colonocytes and stromal cells . Everted proximal and distal colonic tissue of adult rats were disintegrated by a collagenase/dispase solution for 60 min at 37 degrees C to prepare viable gland fragments and isolated cells . Cell preparations were inoculated onto plastic substratum or cytodex-3 microcarriers in a defined maintenance medium or in 1% fetal calf serum media . Incorporation of sodium orthovanadate (> or = 50 microM) in these media constantly enhanced the survival (cell enumeration and trypan blue exclusion P < 0.05) and the adhesion (up to four-fold by crystal violet staining, P < 0.01) of colonocytes (characterized by cytokeratin-18, transforming growth factor-alpha or alkaline phosphatase expression) and stromal cells . Removal of sodium orthovanadate from culture media restored cellular death processes . Incorporation of 10 mM n-butyric acid did not promote cell adhesion and survival except for distal cells exposed to 2 mM sodium orthovanadate . Besides studies in the regulation of anoikis in primary culture, the model will help to assay the influences of dietary and growth factors on the biology of non-cancerous colonic cells.

Cell Biol Int, 1997 May, 21(5), 265 - 71
Alzheimer's soluble amyloid beta protein is secreted by HepG2 cells as an apolipoprotein; Koudinov AR et al.; Recently we reported that the soluble form of amyloid beta protein (sA beta) in normal human plasma and cerebrospinal fluid is associated with lipoprotein (LP) particles . In this paper we tested the sA beta secretion by cells in association with LP in the model of the human hepatoma HepG2 cell line . These cells secreted sA beta to the culture media and expressed intracellular sA beta immunoreactivity . Soluble A beta in the cell supernatant was detected in 200-300 kDa LP complexes in association with apoA-I, apoJ, transthyrethin and phospholipids, triglycerides and free and esterified cholesterol . This was assessed by size exclusion HPLC, immunoprecipitation with corresponding antibodies and by analysis of sA beta associated metabolically-labeled lipids, respectively . Our results suggest that sA beta to LP association represents a unique mechanism, governing the normal biology of sA beta.

Osteoarthritis Cartilage, 1997 May, 5(3), 161 - 72
The effect of strenuous versus moderate exercise on the metabolism of proteoglycans in articular cartilage from different weight-bearing regions of the equine third carpal bone; Little CB et al.; Articular cartilage degeneration in the middle carpal joint is a common problem in racing horses . This study evaluated the effect of exercise on the in-vitro synthesis of the large aggregating proteoglycans (aggrecan) and two small proteoglycans, biglycan and decorin, in articular cartilage taken from three weight bearing regions of the third carpal bone of horses which were subjected to moderate or strenuous exercise . Twelve Standardbred horses free from clinical and radiographic disease of the middle carpal joint were subjected to an 8 week moderate exercise program . The horses were then randomly assigned to two groups: group A--continued moderate exercise and group B--strenuous exercise for 17 weeks . Horses were then rested for 16 weeks . Full-depth articular cartilage explants from the dorsal radial facet (DRF), dorsal intermediate facet (DIF) and palmar condyle (PC) of the third carpal bone were collected and cultured . Cartilage proteoglycan content and release into culture media were measured . Newly synthesized proteoglycans were labeled with 35SO4(2-) for 48 h and analyzed by size exclusion and hydrophobic chromatography, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and autoradiography . Histologic sections of adjacent osteochondral regions were evaluated for evidence of arthritic change . No histologic abnormalities or differences in proteoglycan content were detected in any of the articular cartilage regions examined . There was however, a significant reduction (P < 0.05) in aggrecan synthesis and a concomitant increase in decorin synthesis (P < 0.05) in articular cartilage from the DRF of group B animals . There was no change in biglycan synthesis, aggrecan hydrodynamic size or ability to aggregate in any articular cartilage region . This study has demonstrated that strenuous exercise in horses can lead to a disturbance in the biosynthesis of proteoglycans in articular cartilage regions subjected to high contact stresses (DRF) . These metabolic abnormalities, which persisted for 16 weeks after cessation of exercise, could have deleterious effects on the biomechanical properties of the tissue . We suggest that the observed alteration in articular cartilage metabolism in CRF cartilage of strenuously exercised horses could represent a predisposing factor for cartilage degeneration and osteoarthritis at a later stage.

Anticancer Res, 1997 May-Jun, 17(3C), 2009 - 17
In vitro characterisation of soft tissue tumor chemosensitivity; Remmelink M et al.; BACKGROUND: The benefit of performing chemotherapy on soft tissue sarcomas remains controversial . The present study deals with the in vitro characterisation of the influence of 3 antitumoral agents on the growth of 8 sarcoma cell lines . MATERIALS AND METHODS: Cell growth was monitored by means of the MTT colorimetric assay, which was further validated by a direct cell counting method . The three drugs tested included doxorubicin (ADR), cisplatin (DDP) and dacarbazine (DTIC) . ADR was tested at 10(-5) M, 10(-6) M and 10(-7) M; DDP at 10(-5) M, 10(-6) M and 10(-7) M; and DTIC at 10(-3) M, 10(-4) M and 10(-5) M . A combination of the three drugs was also tested in order to ascertain whether a synergistic effect on cell growth inhibition could be obtained . A potential antineoplastic agent-induced influence on cell growth was determined 3 days after the addition of the diverse drug(s) to the culture media . The cell concentration was specifically adapted to each cell line . The 8 cell lines included 3 leiomyosarcomas, 1 malignant mixed Mullerian tumour, 3 rhabdomyosarcomas and 1 fibrosarcoma . RESULTS: The results show that of the three drugs tested, ADR was the most efficient in terms of the level of cell growth inhibition obtained and the number of cell lines whose growth was significantly inhibited . Of the three drugs, the least active was DDP . A significant synergistic effect was observed when the three drugs were added together to the culture medium . This synergistic effect was evident at the lowest doses tested for each drug . Whatever the histopathological type, the 8 cell lines exhibited a wide range of response to chemotherapy . CONCLUSIONS: The present study shows that the inhibition induced by 10(-7) M ADR, 10(-7) M DDP and 10(-5) M DTIC on sarcoma cell line growth is significantly more efficient than if each agent is tested individually . The in vitro methodology used here fits in with clinical reality because it enables sarcoma cell heterogeneity to be taken into account.

Exp Nephrol, 1997 May-Jun, 5(3), 194 - 200
Potential uremic toxins modulate energy metabolism of cardiac myocytes in vitro; Weisensee D et al.; In the present study we investigated the direct effects of potential uremic toxins on the energy metabolism of cultured cardiac myocytes . High-energy phosphates were extracted with perchloric acid and determined by high performance liquid chromatography . Energy charge (calculated from the ratio of {ATP}, {ADP} and {AMP} was significantly reduced by 20 mM urea and the combination of creatinine (5 mM) plus urea (200 mM) . On the other hand, perfusion with culture media containing clinically relevant amounts of urea (20 mM) or creatinine (1 mM) increased the PCr/ATP ratio . This effect was more pronounced after application of an artificial uremic medium (consisting of uremic serum, urea, creatinine and cytokines) or high amounts of creatinine (5 mM) plus urea (200 mM) . As contractility of myocytes is reduced due to application of uremic compounds or uremic serum, we attribute changes in contraction frequency or inotropy to dysregulation of calcium availability within the cell . In fact, the cardiodepressive action of uremic serum (2.5%) could be completely reversed by the calcium agonist, Bay K 8644, thus indicating disturbances in myocardial calcium homeostasis in uremia . Altered calcium regulation by uremic toxins might therefore be responsible for the observed changes in myocardial energy metabolism . These results might contribute to the understanding of the pathogenesis of cardiac damage in end-stage renal disease.

J Mol Cell Cardiol, 1997 May, 29(5), 1375 - 86
Cultured myofibroblasts generate angiotensin peptides de novo; Katwa LC et al.; Scar tissue found at the site of myocardial infarction (MI) contains phenotypically transformed fibroblast-like cells termed myofibroblasts (myoFb) . In injured cardiac tissue, autoradiography and immunolabeling have localized high density angiotensin (Ang) converting enzyme (ACE) and Ang II receptor binding to these cells, suggesting that they may regulate local concentrations of Ang II and transduce signals at this site . Ang II is known to modulate type I collagen gene expression of fibroblasts and myoFb, and to promote fibrous tissue contraction, each of which may contribute to tissue repair . It is unknown whether myoFb themselves generate Ang peptides de novo via expression of angiotensinogen (Ao), an aspartyl protease needed to convert Ao to Ang I, and ACE . We therefore isolated and cultured myoFb from 4-week-old scar tissue of the adult rat left ventricle with transmural MI . In cultured myoFb we found: (a) immunoreactive membrane-bound ACE, cytosolic cathepsin D (Cat-D), and AT, receptors by immunofluorescence and confocal microscopy, (b) mRNA expression for Ao, ACE, and Cat-D, but not renin, by reverse transcriptase-polymerase chain reaction, (c) production of Ang I and II in serum-free culture media; (d) absence of renin activity; (e) a time-dependent conversion of Ao to Ang I by myoFb cytosol, which was inhibited by pepstatin A, but not by renin inhibitor; and (f) significant increase in Ang II production (P < 0.05) by exogenous Ao and Ang I (10 nM), which was significantly blocked by lisinopril (0.1 microM: P < 0.05) . Thus, cultured myoFb express requisite components and are able to generate Ang I and II de novo . In an autocrine and/or paracrine manner, Ang II may regulate myoFb collagen turnover and fibrous tissue contraction.

Int J Parasitol, 1997 May, 27(5), 573 - 9
Inhibition of spleen cell proliferative response to mitogens by excretory-secretory antigens of Fasciola hepatica; Cervi L et al.; The effect of Fasciola hepatica excretory-secretory antigen (ESA) on the proliferative response of spleen mononuclear (SpM) cells of normal rats to stimulation with mitogens has been examined . When ESA was added to normal SpM cells, there was a decrease in the proliferative response to concanavalin A (Con A) or lipo-polysaccharide (LPS) in a dose-dependent manner . The addition of indomethacin, which blocks prostaglandin synthesis, or N omega-nitro-L-arginine methyl ester (L-NAME) a specific inhibitor of nitric oxide (NO) synthase, had no effect on the ability of ESA to suppress the proliferative response to Con A . However, supplementation of the culture media with catalase, which degrades hydrogen peroxide (H2O2) or superoxide dismutase (SOD) to remove superoxide anion (O2), resulted in a restoration of proliferation to Con A . When LPS was used as mitogenic stimulus no inhibitor added to the culture restored the proliferation . These results suggest that H2O2 and O2- are involved in the suppressor phenomenon induced by ESA in the T-cell proliferative events.

J Virol Methods, 1997 May, 65(2), 201 - 7
Construction of hepatitis C-SIN virus recombinants with replicative dependency on hepatitis C virus serine protease activity; Cho YG et al.; An in vivo assay system was developed for the serine protease of hepatitis C virus (HCV) using the sindbis (SIN) viral replication system in which HCV serine protease activity is essential for the replication of the HCV-SIN chimeric virus . Two chimeric viral cDNA clones were constructed by inserting the NS3/4A region and NS3/4A region with the putative helicase deleted, into the N-terminal region of SIN core protein . The constructs were named Tpro CT and Tpro T, respectively . BHK-21 cells transfected with the in vitro transcribed RNAs from Tpro CT and Tpro T showed specific cytopathic morphology and produced chimeric viruses, Vpro CT and Vpro T . In contrast, in vitro transcribed RNAs from Tpro CTI and Tpro TI, in which serine of catalytic triad of HCV protease was changed to alanine, were not infectious . When the chimeric viruses were passaged in BHK-21 cells at about 0.1 multiplicity of infection (MOI), Vpro T, but not Vpro CT, stably expressed HCV protease for up to five passages . Surprisingly, the cell culture media of BHK-21 cells infected with Vpro T, compared to wild-type sindbis virus, showed rapid pH changes by more than 0.8 pH degree at 72 h post-infection . HCV-SIN hybrid viruses could be used in screening the HCV protease-inhibitor in cell culture systems.

Leukemia, 1997 May, 11(5), 701 - 8
Cytokine response profiles of human myeloid factor-dependent leukemia cell lines; Drexler HG et al.; Research in cytokine biology has grown exponentially in recent years as cytokines (often also termed growth factors) are now known to be involved in a wide range of pathological and physiological processes . Continuous human leukemia cell lines represent powerful tools to investigate these mechanisms . Most cell lines grow autonomously in standard culture media (containing fetal bovine serum) independent of externally added growth stimuli . Over the last 5-10 years a battery of myeloid leukemia-derived cell lines has been established that is constitutively dependent on the addition of cytokines to the culture . Such factor-dependent cell lines die rapidly by apoptosis when deprived of the appropriate growth factor . We determined the cytokine response profiles of 19 absolutely growth factor-dependent leukemia cell lines with myelomonocytic, erythroid or megakaryocytic phenotypes with regard to enhanced or suppressed cellular proliferation . Cells were incubated in liquid culture with optimal concentrations of various recombinant human cytokines known to have effects on the growth of hematopoietic cells . A proliferative or anti-proliferative response to these 41 cytokines was assessed by the short-term 3H-thymidine uptake assay . A proliferative response was considered as positive when the stimulation index (SI) was >2; inhibition was regarded as significant with an SI <0.5 . The response profile of each cell line to these 41 cytokines was different and individual . None of the cell lines responded to one or two factors only (minimum to at least five cytokines) . Proliferation of most (n = 13-17), but not of all cell lines was significantly enhanced by GM-CSF, IL-3, PIXY-321, SCF and IFN-gamma . TGF-beta1 consistently inhibited proliferation (in 11/19 cell lines) . IFN-alpha, IFN-beta, TNF-alpha and TNF-beta had either stimulatory or inhibitory effects . The cell lines responding most often proliferatively (to 15-19 different cytokines) were UCSD/AML1, HU-3, TF-1 and M-07e . In summary, these factor-responsive human leukemia cell lines represent extremely useful model systems for the analysis of cytokine effects on hematopoietic cells . The cytokine response profiles of the individual cell lines provide guidelines for the selection of the appropriate cell culture for such experiments.

Exp Cell Res, 1997 May 1, 232(2), 430 - 4
Exposure of phosphatidylethanolamine on the surface of apoptotic cells; Emoto K et al.; In the early stages of apoptosis, phosphatidylserine (PS) is translocated from the inner side of the plasma membrane to the outer layer, which allows phagocytes to recognize and engulf the apoptotic cells . In this study we have analyzed the cell surface exposure of phosphatidylethanolamine (PE) in apoptotic CTLL-2 cells, a cytotoxic T cell line, using a tetracyclic polypeptide of 19 amino acids (Ro09-0198) which specifically recognizes the structure of PE and forms a tight equimolar complex with the phospholipid . Fluorescence microscopic analysis showed that the peptide, conjugated with fluorescence-labeled streptavidin (FL-SA-Ro), bound effectively to the cell surface of cells undergoing apoptosis in response to withdrawal of interleukin-2 from the culture media, but not to nonapoptotic cells . The binding of FL-SA-Ro to apoptotic cells was not uniform and the intense staining was observed on surface blebs of apoptotic cells . The FL-SA-Ro binding was inhibited specifically by liposomes containing PE, suggesting that PE is mainly exposed on the surface blebs of apoptotic cells . The specific binding of FL-SA-Ro to the apoptotic cells was also confirmed using a flourescence-activated cell sorter and the time-dependent cell surface exposure of PE correlated well with the exposure of PS, as detected by the binding of annexin V . This study provides the first direct evidence that PE as well as PS is exposed on the cell surface during the early stages of apoptosis, resulting in the total loss of asymmetric distribution of aminophospholipids in the plasma membrane bilayer.

J Endocrinol, 1997 May, 153(2), 231 - 40
Insulin-like growth factor-I (IGF-I) production by bovine granulosa cells in vitro and peripheral IGF-I measurement in cattle serum: an evaluation of IGF-binding protein extraction protocols; Gutierrez CG et al.; Insulin-like growth factor-binding protein (IGFBP) extraction protocols were tested for their efficacy in removing IGFBPs from bovine plasma and bovine granulosa cell culture medium compared with standard acid exclusion chromatography . Traditional extraction methods, acidification, Sep-Pak, ethanol:acetone:acetic acid (EAA) and EAA-cryoprecipitation (EAA-C), failed to remove all the IGFBPs from both granulosa cell culture medium and plasma . However, EAA and EAA-C treatment of plasma samples did give values similar to those obtained by acid exclusion HPLC, when corrected for extraction efficiency . There was an inverse relationship between insulin-like growth factor-I (IGF-I) concentration in plasma samples, as measured using HPLC chromatography, and IGF-I concentration after EAA extraction . Furthermore, the interference caused by residual IGFBPs differed between samples taken from animals given various treatment that altered peripheral IGF-I concentrations . As for plasma samples, EAA was the most effective extraction method for culture media, but residual IGFBPs caused an overestimation of IGF-I concentrations . In culture media, but not plasma, it was possible to block the interference of IGFBPs in the IGF-I assay, in both extracted and non-extracted culture samples, by the addition of excess IGF-II . Using this assay procedure, no IGF-I production by bovine granulosa cells was detected . This was confirmed by HPLC acid chromatography . It is concluded that HPLC extraction is needed for the accurate measurement of peripheral IGF-I concentrations . For granulosa cell culture media it is possible to measure IGF-I concentration in non-extracted samples if the IGFBPs are blocked by adding IGF-II . Using either this assay, or after HPLC acid chromatography, no IGF-I was detected in culture media, suggesting that IGF-I is not produced by non-luteinised bovine granulosa cells.

J Endocrinol, 1997 May, 153(2), 221 - 30
Development, validation and application of a two-site enzyme-linked immunosorbent assay for activin-AB; Evans LW et al.; Monoclonal antibodies, specific for the beta A and beta B subunits of activin, were used to develop a new two-site ELISA for activin-AB . The assay had a detection limit of 0.19 ng/ml . High concentrations of activin-AB were found in bovine, ovine and porcine follicular fluids (FF), with less in human FF (1310, 1730, 688 and 7 ng/ml respectively) . Recovery of spiked activin-AB standard from human, bovine and ovine FFs and from homogenized human placental extracts averaged 91%, 115%, 115% and 94% respectively . Within-plate coefficients of variation for different concentration of activin-AB were between 1.3% and 2.67% . The between-plate coefficient of variation was 5.5% . Cross-reactivity experiments showed the high specificity of the assay for activin-AB, with inhibin-A, inhibin-B, follistatin, activin-A and activin-B all cross-reacting < 0.2% . Incubation with high concentrations of follistatin (500 ng/ml) prior to assay did not affect the recovery of activin-AB . Samples of bovine, porcine, ovine and human FF gave dose responses parallel to that of the standard, as did bovine granulosa cell-conditioned media . In human and porcine FF, levels of activin-A and activin-AB were similar whereas, in bovine and ovine FF, activin-A levels were approximately threefold higher than activin-A, nearly all of the endogenous activin-AB in bovine FF was detected in the eluate from gel permeation chromatography with an M(r) of > 700000 indicating its association with higher molecular weight binding protein(s) . By contrast, after denaturation, immunoreactive activin-AB was detected with an M(r) of approximately 25000 consistent with the complete dissociation from binding proteins . Activin-A was detected in relatively high concentrations in human FF (approximately 5 ng/ml), homogenized placental extracts (4.35-95.5 ng/g), sera from pregnant women (> 4 ng/ml) and amniotic fluid (3-13 ng/ml), and in much lower concentrations in postmenopausal serum (500 pg/ ml), normal cycle serum (100-200 pg/ml), serum from gonadotrophin-treated women (200 pg/ml), and normal adult male serum (225 pg/ml) . Activin-A was also found in the culture media from explants of human amnion, chorion, maternal decidua and placenta . In marked contrast, activin-AB was undetectable (< 0.19 ng/ml) in all of these samples with the exception of human FF (approximately 7 ng/ml) . In conclusion, we have developed a sensitive and specific ELISA to measure total (bound+free) activin-AB . Preliminary results show a more restricted distribution of this isoform compared with activin-A . The presence of high levels of both activin-A and activin-AB in FF suggests a function for both isoforms in the developing ovarian follicle.

Curr Genet, 1997 May, 31(5), 447 - 54
Cloning and characterisation of glutamine synthetase from Colletotrichum gloeosporioides and demonstration of elevated expression during pathogenesis on Stylosanthes guianensis; Stephenson SA et al.; Experiments were designed to clone and identify genes of the fungal phytopathogen Colletotrichum gloeosporioides expressed at high levels during growth on the compatible host Stylosanthes guianensis when compared with expression in axenic culture . A cDNA clone (pCgGS) that hybridised preferentially to a cDNA probe prepared from infected leaves was isolated by the differential screening of a cDNA library from a nitrogen-starved axenic culture of C . gloeosporioides . The DNA sequence of pCgGS is highly homologous to genes for glutamine synthetase (GS) in other organisms . pCgGS contained all of the conserved regions assigned as catalytic domains in GS enzymes . Comparison with genomic sequences indicated that in C . gloeosporioides the GS gene is present as a single copy with three introns . To our knowledge this is the first report of the cloning of a GS from a filamentous fungus . A second clone (pCgRL1) was also isolated and represented a partial cDNA of the 25s rRNA of C . gloeosporioides . Because pCgRL1 did not hybridise to plant rRNA under high-stringency hybridisation conditions, it was used as a reference to quantify the expression of fungal GS mRNA during pathogenesis in S . guianensis compared to fungal growth in axenic culture . The results indicated that elevated expression of GS occurred during pathogenesis of C . gloeosporioides on S . guianensis, particularly at early stages of infection where expression was about six-times higher than during growth in rich culture media . This work also demonstrates that fungal-specific 25s rRNA fragments, such as pCgRL1, have considerable utility as a reference for quantifying pathogen gene expression in infected plants.

Int Arch Allergy Immunol, 1997 May-Jul, 113(1-3), 307 - 11
Expression of cytokines on human bronchial epithelial cells induced by influenza virus A; Adachi M et al.; Bronchial epithelial cells play an important role in the pathogenesis of some inflammatory diseases of bronchial mucosa . Epithelial-cell-derived cytokines are important in the elucidation of the mechanism by which airway inflammation occurs, especially in respiratory virus infection, because these cells are the primary sites of viral infection . We infected bronchial epithelial cells, NCI-H292, with influenza virus A (H3N2) and examined the concentrations of cytokines, interleukin-6 (IL-6), IL-8 and regulated on activation, normal T cells, expressed and secreted (RANTES), in the culture media of infected cells using the enzyme-linked immunosorbent assay system and gene expression of RANTES on epithelial cells by the reverse-transcriptase-polymerase chain reaction method . We found that significant amounts of IL-6, IL-8 and RANTES were released . RANTES mRNA was also detected in infected bronchial epithelial cells . It is suggested that cytokine production in human bronchial epithelial cells may contribute to the pathogenesis of airway inflammatory disorders.

Proc Natl Acad Sci U S A, 1997 Apr 29, 94(9), 4664 - 8
Ultrarapid, highly efficient viral gene transfer to the heart; Donahue JK et al.; Gene therapy for common myocardial diseases will require effective and homogeneous gene delivery throughout the intact heart . We created two experimental models to identify and optimize parameters important for adenovirus-mediated cardiac gene transfer . In cultured rabbit ventricular myocytes, the percentage of infected cells increased with higher absolute numbers of virus particles, longer durations of virus exposure, physiological temperatures, and specific culture media compositions . Simulating the in vitro conditions, we delivered adenovirus to intact rabbit hearts by intracoronary perfusion . The percentage of infected cells increased with higher coronary flow rates, longer virus exposure times, and higher virus concentrations . Under optimal conditions, nearly 100% of myocytes expressed the reporter gene beta-galactosidase after ex vivo infection . This novel delivery method, the first to demonstrate virtually complete transduction of any intact organ, could be adapted to achieve widespread gene transfer in vivo.

Early Hum Dev, 1997 Apr 25, 48(1-2), 71 - 80
Interleukin-1 beta levels in human embryo culture supernatants and their predictive value for pregnancy; Ines Baranao R et al.; Interleukin 1 (IL-1) is possibly one factor produced by the embryo that might have a role in the maternal immunological recognition of pregnancy . The purpose of this study was to identify an embryo-related factor suitable for prediction of pregnancy during IVF procedures . For this purpose, IL-1 beta levels were measured in 21 samples of human embryo culture-conditioned media . The average number of embryos per sample was 5 +/- 1 . Simultaneously, 16 cell culture media containing 10% autologous serum but no embryos were tested as controls . IL-1 beta levels were measured using the ELISA technique, and the biological activity of IL-1 was measured by means of a C3H/HeJ mice thymocyte proliferation assay . The average IL-1 beta level +/- S.E.M . was 49 +/- 7 pg/ml in embryo culture-conditioned media and 12 +/- 2 pg/ml in controls (P < 0.001) . The average IL-1 beta level in embryo culture-conditioned media from viable pregnancy cycles was 82 +/- 6 pg/ml (n = 8), while in those cases that did not result in viable pregnancies the IL-1 beta level was significantly lower (28 +/- 4 pg/ml, n = 13, P < 0.001) . The IL-1 activity of embryo culture-conditioned media, measured by {3H}thymidine incorporation in thymocytes was increased, compared with control media (442 +/- 51 counts/min vs . 337 +/- 13 counts/min, P < 0.05), and the highest values corresponded to media containing those embryos that resulted in pregnancies (589 +/- 41 counts/min, P < 0.01 vs . controls) . We conclude that the determination of the levels of this cytokine in embryo culture-conditioned media might be a predictive parameter for pregnancies in patients undergoing IVF-ET.

Biochim Biophys Acta, 1997 Apr 24, 1356(2), 171 - 84
Overproduction of urokinase-type plasminogen activator is regulated by phospholipase D- and protein kinase C-dependent pathways in murine mammary adenocarcinoma cells; Aguirre Ghiso JA et al.; Urokinase-type plasminogen activator (uPA) initiates a proteolytic cascade with which invasive cells eliminate barriers to movement . The signaling pathways regulating uPA production in tumor cells remain unclear . We first studied the effects of n-butanol, a phospholipase D (PLD) and protein kinase C (PKC) inhibitor, on the production of uPA in murine mammary adenocarcinoma cells . Tumor cell monolayers treated during 24 h with 0.3% v/v n-butanol, secreted 45-50% less uPA to the culture medium than control monolayers (P < 0.001) as determined by radial caseinolysis, zymography and western blot . This inhibition occurred also with 5-h treatments and remained up to 5 h after the removal of the alcohol . Treatment with the phorbol ester PMA or with EGF, strongly increased uPA production (P < 0.001) . Interestingly, a mild inhibition of uPA production was observed when PMA stimulation was assayed in cotreatments with n-butanol . In contrast EGF was unable to reverse the inhibition induced by n-butanol . H7 significantly inhibited uPA activity (P < 0.001) secreted to the culture media . Furthermore, phosphatidic acid significantly stimulated uPA production meanwhile propranolol, which blocks phosphatidic acid availability, reduced it, suggesting a main regulatory role for this intermediary metabolite . These results suggest for the first time that uPA production is regulated by PLD and PKC signal transduction pathways in murine mammary adenocarcinoma cells.

Brain Res, 1997 Apr 18, 754(1-2), 65 - 71
The inhibitory effects of beta-amyloid on glutamate and glucose uptakes by cultured astrocytes; Parpura-Gill A et al.; beta-Amyloid is the primary protein component of neuritic plaques, which are degenerative foci in brains of patients with Alzheimer's disease (AD) . The effects of this naturally occurring beta-amyloid on the cells of the central nervous system have not been completely understood . beta-Amyloid increases the vulnerability of cultured neurons to glutamate-induced excitotoxic damage . Because astrocytes play a key role in uptake of extracellular glutamate and glutamate uptake is ATP-dependent, we studied the effect of beta25-35 on glutamate and glucose uptake in cultured hippocampal astrocytes following 7 days of exposure to beta25-35 . Astrocytic glutamate uptake was studied at 1, 5, 10, 15, 20, and 60 min following the addition of {3H}glutamate (5 nM) to the culture media, and astrocytic glucose uptake was assessed at 60 min after the addition of {14C}glucose (600 and 640 nM) to the media . Glutamate uptake by control astrocytes was time-dependent . Astrocytes exposed to beta25-35, however, showed significantly lower glutamate uptake at all sampling times . Similarly, {14C}glucose uptake by astrocytes was inhibited by beta25-35 . When glucose uptake was blocked by phloretin (10 mM), astrocytic {3H}glutamate uptake was also blocked, suggesting that the inhibitory effect of beta-amyloid on glutamate uptake is caused by diminished glucose uptake . Thus, our present study suggests a possible link between two proposed mechanisms of pathogenesis of the Alzheimer's disease: glutamate neurotoxicity and global defect in cerebral energy metabolism.

J Neurosci Res, 1997 Apr 15, 48(2), 83 - 94
Multipotential and lineage restricted precursors coexist in the mammalian perinatal subventricular zone; Levison SW et al.; Developmental studies have shown that both neurons and glia arise from the subventricular zone (SVZ) but there have been no clonal analyses to determine whether a single progenitor can produce both . Therefore, we used replication deficient retroviral vectors to analyze the clonal progeny of single rat SVZ cells that were maintained in culture media permissive or non-permissive for neuronal differentiation . When maintained in medium supplemented with 5% fetal bovine serum, all surviving progenitors generated glial cell clones . Within these glial clones we often observed both type 1 astrocytes and O-2A lineage cells . When SVZ cells were maintained in medium permissive for neurogenesis approximately 50% of the total clones contained at least one antigenically defined neuron . Of those clones that contained neurons, 60% contained neurons and glia . The other 50% of the total clones were either comprised of only astrocytes, astrocytes and oligodendrocytes, or were unidentifiable . Since the culture environment permitted multilineage clone formation, yet many homogeneous neuronal or astrocytic clones were obtained, some progenitors must become developmentally restricted while they are in the germinal zone . Therefore, we conclude that the perinatal SVZ is a mosaic of multipotential, bipotential, and lineage restricted precursors, and that the lack of postnatal neocortical neurogenesis is not due to the absence of potential neuroblasts.

Int J Pancreatol, 1997 Apr, 21(2), 157 - 64
Dissociated secretion of islet amyloid polypeptide and insulin in serum-free culture media conditioned by human pancreatic adenocarcinoma cell lines; Wang F et al.; CONCLUSION: The cosecretion of insulin and islet amyloid polypeptide (IAPP) is altered when isolated rat pancreatic islets are incubated in culture media conditioned by human pancreatic cancer cells . BACKGROUND: Pancreatic cancer is usually associated with impaired glucose tolerance . This study investigates the tumor-derived influence on beta-cell secretion of pancreatic islets . METHODS: Four conditioned media were prepared from two human pancreatic cancer cell lines (Panc-1 and HPAF), a hamster pancreatic cancer cell line (PC-1), and a fibroblast cell line (Ag1523) . Isolated rat pancreatic islets were incubated first in the conditioned media or nonconditioned control medium for 24 h, then in the same kind of media containing 100 microM carbamylcholine for 90 min . Insulin and IAPP secretion were measured by radioimmunoassay . RESULTS: Islets in media conditioned by Panc-1 and HPAF cells demonstrated dissociation of insulin and IAPP secretion . During 24-h incubation, the dissociation was expressed as selectively decreased insulin secretion . With addition of 100 microM carbamylcholine, the dissociation was expressed as normal secretion of insulin and hypersecretion of IAPP . As a result, the IAPP/insulin molar ratios were increased in both groups during both time periods . The islets in PC-1 and Ag1523 media did not show any significant changes in insulin and IAPP secretion.

Methods Find Exp Clin Pharmacol, 1997 Apr, 19(3), 153 - 9
Oxidized low density lipoprotein acts on endothelial cells in culture to enhance endothelin secretion and monocyte migration; Achmad TH et al.; Monocyte deposition on the endothelium is the initial step in atherogenesis . Oxidized low density lipoprotein (Ox-LDL) is involved in the development of the fatty streak which progresses to the atherosclerotic lesion . Our interest focussed on the question, does the endothelium react to Ox-LDL to produce humoral substances that might influence the migration of human blood monocytes? Chemotaxis of monocytes was assessed by the modified membrane-filter technique based on the Boyden chamber principle . Exposure of porcine aorta endothelial cells (ECs) to Ox-LDL (100 micrograms/ml) increased the directional migration of monocytes by 25% (p < 0.01) over that of ECs in the absence of Ox-LDL . Radioimmunoassay of the EC culture media revealed the presence of immunoreactive endothelin-1 (ir-ET-1) . The endothelin converting enzyme inhibitor, phosphoramidone (10 microM), when incubated together with ECs and Ox-LDL, suppressed the synthesis of ir-ET-1 by 53% (p < 0.05) and the migration decreased by 12% (p < 0.05) . Preincubation of monocytes with the ETA receptor-selective antagonist, BQ-123 (1 microM), followed by exposure to ECs plus Ox-LDL, lead to a decrease in their migration by 12% (p < 0.05) compared to monocytes not treated with BQ-123 . These results show that Ox-LDL acts on ECs to enhance the synthesis of ir-ET-1 which in turn increases the directional migration of monocytes . Phosphoramidone decreased the synthesis of ir-ET-1 but migration was affected only modestly; monocyte ETA receptor blockade by BQ-123 also suppressed migration toward EC chemoattractants to a small extent . Both results suggest that in addition to ir-ET-1 other chemotactic factors are being released by the ECs; Ox-LDL appears to enhance their release or synthesis.

J Surg Res, 1997 Apr, 69(1), 139 - 44
IL-6 production in human intestinal epithelial cells following stimulation with IL-1 beta is associated with activation of the transcription factor NF-kappa B; Parikh AA et al.; Recent studies suggest that interleukin-1 beta (IL-1 beta) stimulates interleukin-6 (IL-6) production in human intestinal epithelial cells, but the intracellular mechanisms of this response are not known . In other reports, the nuclear factor-kappa B (NF-kappa B) regulated IL-6 production in certain cell types . We tested the hypothesis that IL-6 production in the enterocyte is associated with activation of NF-kappa B . Caco-2 cells, a human intestinal epithelial cell line, were grown in tissue culture whereafter they were treated with IL-1 beta (0.5 ng/ml) . Cells were preincubated with pyrrolidine dithiocarbamate (PDTC; 10-500 microM), tosyl-lys-chloromethylketone (TLCK; 10-500 microM), or genistein (25-75 microM), all of which are known inhibitors of NF-kappa B . IL-6 levels in the culture media were measured after 24 hr by enzyme-linked immunosorbent assay (ELISA) and IL-6 messenger RNA (mRNA) levels were determined after 4 hr by competitive reverse-transcriptase polymerase chain reaction (RT-PCR) . NF-kappa B activity was determined by electrophoretic gel mobility shift assay (EMSA) . PDTC, TLCK, and genistein each inhibited IL-1 beta-induced IL-6 production by the Caco-2 cells in a dose-dependent fashion . These responses were also associated with a decrease in IL-6 mRNA levels . There was no NF-kappa B activity in untreated cells, but the addition of IL-1 beta resulted in the activation of NF-kappa B as determined by EMSA . The results suggest that IL-1 beta-induced IL-6 production in the enterocyte is associated with activation of NF-kappa B . The inhibition of IL-6 production by the NF-kappa B inhibitors indicates that the IL-6 production is regulated by NF-kappa B, although further experiments are needed to test that hypothesis.

Int J Dev Biol, 1997 Apr, 41(2), 267 - 73
The use of whole rat embryo cultures to identify and characterize causes of reproductive failure; Klein NW; The most important problem facing human teratology today is to identify the actual causes of this health problem . We have used cultures of whole rat embryos to address this problem using blood sera from individuals at risk as embryo culture media for this purpose . Through serum fractionations and nutrient supplementations to the serum we have studied drugs (dilantin, valproic acid), nutrient deficiencies (methionine) and an embryotoxic autoantibody to the protein laminin . In addition to identifying these factors it has been possible to address their mechanisms of action and to provide recommendations for treatment.

Hum Reprod, 1997 Apr, 12(4), 785 - 91
Optimization of a method for deactivation of platelet-activating factor:acetylhydrolase in serum for use in in-vitro fertilization culture media; Ammit AJ et al.; Embryos produced by in-vitro fertilization (IVF) may produce less platelet-activating factor (PAF) than is optimal for development . It was previously shown that supplementation of culture media with PAF results in a significant increase in pregnancy rate . Human embryos are often cultured in media supplemented with serum containing the enzyme PAF:acetylhydrolase (PAF:AH; EC 3.1.1.47), which hydrolyses PAF to its inactive form, lyso-PAF . Thus, effective supplementation of media with PAF requires inactivation of this enzyme . In this study we examine the efficacy of the methods of PAF:AH deactivation used for PAF supplementation of IVF culture medium . When the effectiveness of a commonly used acid treatment protocol (pH 3.0 at room temperature for 5 min) was examined, it was found that it was not completely effective for the majority of sera . When synthetic PAF was added to 18 serum samples which had been acid treated, five had 90-100% of the original PAF remaining after 24 h (showing that the acid treatment was effective), eight had from 10-90% of the original PAF remaining after 24 h, and five samples had 0-10% . The extent to which PAF:AH was susceptible to deactivation was not associated with the activity in the serum prior to treatment, the serum oestradiol concentration, or the cause of infertility . The period of acidification and the incubation temperature were assessed to develop a new acid-treatment protocol (20 min acid treatment at 37 degrees C) which was able to deactivate PAF:AH effectively in all sera (53/53) examined . A trial was performed to assess the effect of acid treatment of serum for 5 min at room temperature compared with the new protocol (20 min at 37 degrees C) on IVF outcome, following PAF supplementation of IVF culture medium . Oocyte recovery, fertilization and embryo development rates were equivalent for both groups and approximately equal numbers of embryos were transferred or cryopreserved . Pregnancy rates were not significantly different (14.6 versus 20.0%) for the two treatments, with a trend towards a higher pregnancy rate with the new acid-treatment protocol . The results show that this new procedure for acid treatment of serum in combination with PAF supplementation does not have detrimental effects on embryos and their pregnancy outcome and is therefore suitable for use in IVF.

In Vitro Cell Dev Biol Anim, 1997 Apr, 33(4), 282 - 8
Growth requirements and neoplastic transformation of two types of normal human breast epithelial cells derived from reduction mammoplasty; Kao CY et al.; A chemically defined culture medium was developed to support the growth of two distinctly different types of normal human breast epithelial cells (HBEC) derived from reduction mammoplasty . Type I cells expressed luminal epithelial cell markers and were deficient in gap junctional intercellular communication (GJIC), whereas Type II cells expressed basal epithelial cell markers and were efficient in GJIC . In this study, we examined and compared the growth factor and hormone requirements of these two types of cells and a series of cell lines that were obtained by sequential transfection with SV40 DNA (extended lifespan, nontumorigenic), treatment with 5-bromodeoxyuridine (BrdU)/black light (immortal and weakly tumorigenic), and infection of a virus carrying the neu oncogene (highly tumorigenic) . Growth of Type I cells was inhibited by withdrawing epidermal growth factor (EGF), hydrocortisone (HC), or insulin (INS) from the culture media, but was enhanced by fetal bovine serum (FBS) supplementation . Growth of Type II cells was inhibited by withdrawal of EGF, HC, or INS from the media, and was inhibited by FBS supplementation . Withdrawal of human transferrin (HT) or 17 beta-estradiol (E2) from the media did not alter the growth of Type I or Type II cells . SV40 transfected Type I cell lines still required EGF, HC, or INS for optimal growth . However, the highly tumorigenic cell line did not show a growth dependence on EGF, HC, or INS but did appear to require HT and 3,3',5-triiodo-D.L . thyronine (T3) for optimal growth . In addition, FBS stimulated the growth of these cell lines . Thus, this study shows that Type I HBEC are distinctly different from Type II HBEC in growth response to FBS and that neoplastically transformed Type I cells could become growth factor and hormone independent.

Curr Eye Res, 1997 Apr, 16(4), 387 - 95
Differential effects of transforming growth factors on localization of adhesion complex proteins following corneal epithelial cell wounding; Gassner HL et al.; PURPOSE: The differential effects of transforming growth factor (TGF) alpha, beta 1 and beta 2 on the de novo localization of heparan sulfate proteoglycan, collagen type VII and laminin-1 to the adhesion complex were analyzed using an in vitro model of corneal epithelial cell wound healing . METHODS: Bovine corneal explants were maintained in culture media containing either no growth factor or 1, 5, or 10 ng/ml TGF alpha, TGF beta 1 or TGF beta 2 . After 24 or 48 hours in culture, cryostat sections of explants were processed for immunofluorescence microscopy using antibodies directed against heparan sulfate proteoglycan, collagen type VII or laminin-1 . RESULTS: A comparison of antibody labeling patterns and relative fluorescence intensity of antibody labeling to controls suggested that TGF alpha inhibits the spatial polarization of proteins into the reforming adhesion complex during early stages of wound healing . Both TGF beta 1 and beta 2 enhanced the linear localization of the three proteins to the site of the reforming adhesion complex . However, in our model TGF beta isoforms did not have identical functions . TGF beta 2 accelerated the temporal localization of collagen type VII to the adhesion complex, an effect which was not observed with TGF beta 1 . CONCLUSIONS: TGF beta, but not TGF alpha, may play an important role in corneal epithelial cell wound healing by accelerating the reformation of the adhesion complex and subsequent epithelial cell-extracellular matrix adhesion.

Surgery, 1997 Apr, 121(4), 440 - 8
Measurement of interleukin-6, interleukin-10, and tumor necrosis factor-alpha levels in tissues and plasma after thermal injury in mice; Kawakami M et al.; BACKGROUND: Cytokines are important modulators of physiologic alterations after thermal injury . Indeed, an increase in the level of circulating cytokines has been documented after thermal injury . However, the mechanism of the increase has not been clarified . We determined cytokine levels in local tissue after thermal injury to identify the tissues responsible for the increase . METHODS: Female C57BL/6 mice each received a 20% full-thickness burn injury . Blood, burned skin, unburned skin, muscle underlying the burn, and muscle of the thigh, liver, spleen, and mesenteric lymph node were sampled at 1, 2, 4, 8, and 24 hours after injury . Uninjured control mice were treated similarly . The samples were cultured, and concentrations of tumor necrosis factor-alpha, interleukin-6 (IL-6), and IL-10 in the culture media were measured by using an enzyme-linked immunosorbent assay . RESULTS: IL-6 levels in unburned skin were significantly increased at 1 hour and decreased at 24 hours, compared with the control . IL-6 levels in muscle underlying the burn were significantly decreased at 8 hours . No elevation of plasma IL-6 levels was observed after injury . Neither tumor necrosis factor-alpha IL-10 was detected in any tissue . CONCLUSIONS: Results indicate that unburned skin may be a major source of IL-6 production after thermal injury and may contribute to the physiologic alterations occurring after such injury.

Proc Soc Exp Biol Med, 1997 Apr, 214(4), 367 - 73
Human lipoproteins as a vehicle for the delivery of beta-carotene and alpha-tocopherol to HepG2 cells; Martin KR et al.; Highly differentiated human cell lines represent a useful in vitro model for the study of carotenoid uptake, metabolism, and function . Carotenoids are usually introduced into tissue culture media either in organic solvents or as micelles, whereas carotenoids are localized in lipoproteins in vivo . Initially, the stability of beta-carotene and alpha-tocopherol in micelles and human lipoproteins under standard tissue culture conditions was compared . Recovery of beta-carotene and alpha-tocopherol was 27% +/- 2% and 73% +/- 2%, respectively, after overnight incubation of micellar beta-carotene and alpha-tocopherol in serum-free medium without cells . This marked loss of beta-carotene was attenuated by inclusion of alpha-tocopherol in micelles . In contrast, recovery of beta-carotene and alpha-tocopherol was 88%-95% when medium containing the total lipoprotein fraction isolated from beta-carotene supplemented individuals was incubated overnight without cells . Cellular accumulation of beta-carotene and alpha-tocopherol from medium containing total lipoproteins (1 mg/ml) was proportional to their concentrations in the lipoprotein fraction (r = 0.94 for beta-carotene and 0.74 for alpha-tocopherol) . Cells exhibited similar capability of acquiring beta-carotene and alpha-tocopherol from medium containing either low- or high-density lipoproteins . These data show that lipoproteins represent a stable vehicle for delivery of beta-carotene and alpha-tocopherol to HepG2 human liver cells.

Lab Invest, 1997 Apr, 76(4), 591 - 600
Basic fibroblast growth factor stimulates the release of preformed transforming growth factor beta 1 from human proximal tubular cells in the absence of de novo gene transcription or mRNA translation; Phillips AO et al.; Interstitial fibrosis is significantly correlated with the progression of renal impairment for most causes of renal insufficiency . Transforming growth factor beta 1 (TGF-beta 1) and basic fibroblast growth factor (bFGF) are two of a group of profibrotic cytokines that have been associated with the development of renal interstitial fibrosis . We have previously demonstrated that alterations in D-glucose concentrations modulate the synthesis of TGF-beta 1 by human renal proximal tubular cells (HPTC) in vitro . The aim of the present study was to examine the influence of bFGF on TGF-beta 1 synthesis by HPTC in culture and to examine any modulation of this response by changes in ambient glucose concentration . Incubation of growth-arrested HPTC (72 hours in serum-free medium) with bFGF resulted in a dose-dependent increase in latent TGF-beta 1 secretion . Maximal release of TGF-beta 1 was seen at a bFGF dose of 50 ng/ml in cells incubated in 5 mM D-glucose (7.48 +/- 2.5 ng/ml, mean +/- SEM; n = 3; p = 0.04) . This release of TGF-beta 1 in response to bFGF was unaffected by increasing the concentration of glucose in the culture media to 25 mM (7.76 +/- 1.3, mean +/- SEM; n = 3; p < 0.02) . It was also unaffected by pretreatment of cells with either actinomycin-D or cycloheximide . TGF-beta 1 secretion was, however, inhibited in a dose-dependent manner by the exposure of cells to the microtubule-disrupting agent vinblastine, indicating that the generation of TGF-beta 1 was dependent on the secretion of preformed, stored TGF-beta 1 . In a separate series of experiments, exposure of HPTC to TGF-beta 1 (10 ng/ml) led to the induction of bFGF mRNA, which was first apparent at 12 hours and reached maximal levels 24 hours after stimulation (normalized bFGF/alpha-actin mRNA ratio was 1.5 times that of the control) . This increase in bFGF mRNA was accompanied by a time-dependent increase in bFGF protein production, which was maximal after 24 hours (19.83 +/- 12.7 pg/ml versus 2.49 +/- 0.34 pg/ml, mean +/- SEM, stimulated versus control; n = 3; p = 0.03) . These findings demonstrate that bFGF stimulates the secretion of preformed, latent TGF-beta 1 by HPTC but does not induce de novo TGF-beta 1 gene transcription or TGF-beta 1 protein synthesis . We have also demonstrated a positive-feedback loop involving TGF-beta 1 and bFGF and postulate that this may be involved in the progressive nature of renal fibrosis in vivo.

Int J Food Microbiol, 1997 Apr 1, 35(2), 117 - 27
Enumeration of fungi in barley; Rabie CJ et al.; Estimation of fungal contamination of barley grain is important as certain fungi can proliferate during the malting process . The following factors which may affect the enumeration of fungi were evaluated: dilution versus direct plating, presoaked versus unsoaked grain, five culture media: potato dextrose agar (PDA), acidified Czapek-Dox agar (ACA), pentachloronitrobenzene agar; (PCNB) dichloran rose bengal chloramphenicol agar (DRBC) and malt salt agar; two disinfectants' ethanol/water (80:20 v/v) and sodium hypochlorite (3.5% w/v in H2O) . Two barley samples, one having a high incidence of storage fungi and one with a high incidence of field fungi were used and most fungi were identified to species level . Results showed that direct plating was superior to dilution plating for assessing the mycoflora of barley . Unsoaked grain gave significantly higher counts than presoaked grain in the case of Alternaria alternata, Rhizopus oryzae, Epicoccum nigrum and Mucor spp . Presoaked grain resulted in higher counts of Penicillium spp . Chlorine disinfection resulted in significantly higher counts of Aspergillus flavus, Eurotium spp . and Penicillium spp . Ethanol disinfection resulted in higher counts of Mucor spp., Phoma sorghina, Rhizopus oryzae and Aspergillus restrictus . PDA and ACA, in general gave some what better results than DRBC for both field and storage fungi . PCNB consistently gave the highest Fusarium counts . More than thiry fungal genera were found in the two samples.

Stroke, 1997 Apr, 28(4), 799 - 804
Gelatinase activity and the occurrence of cerebral aneurysms; Chyatte D et al.; BACKGROUND AND PURPOSE: Cerebral aneurysms are associated with decreased arterial collagen content; however, whether this deficiency results from impaired collagen synthesis or enhanced collagen degradation is unknown . This study tested the hypothesis that enhanced collagen degradation, not impaired collagen synthesis, is associated with the occurrence of cerebral aneurysms . METHODS: Cultured skin fibroblasts and serum samples were studied in patients with angiographic evidence of aneurysm (n = 31) and control subjects (n = 14) . Transcription of the type III collagen gene was assessed with the use of Northern blots prepared from RNA harvested from confluent cultured fibroblasts . Translation of type III collagen was assessed by Western blot analysis of proteins produced by cultured skin fibroblasts . Collagen metabolism was assessed by radioimmunoassay for type I (PICP) and type III (PIIINP) procollagen peptides in conditioned tissue culture media and serum . We assessed collagen degradation in serum and conditioned tissue culture media by evaluating gelatinase activity using quantitative zymography . RESULTS: Type III collagen synthesis was the same in aneurysm and control patients . Neither the molecular weight nor the relative amount of type III collagen mRNA differed between aneurysm and control patient fibroblasts . Western blot analysis revealed no difference in the relative amount or molecular weight of procollagen III synthesized by aneurysm and control cells . Aneurysm patients had a threefold increase in native serum gelatinase activity compared with control subjects (P = .004) . This increase occurred along with serum evidence of increased collagen metabolism . Serum levels of PICP (P = .03) and PIIINP (P = .02) were decreased in aneurysm patients . Elevated serum gelatinase activity and altered collagen metabolism could not be explained by enhanced secretion of gelatinase by cultured fibroblasts or altered net collagen synthesis by fibroblasts . High serum gelatinase activity was more common in men than in women (P = .04) . CONCLUSIONS: These findings are consistent with the hypothesis that accelerated enzymatic degradation of collagens and other structural proteins compromises the mechanical integrity of the cerebral vessel wall and leads to conditions that favor aneurysm formation.

Am J Pathol, 1997 Apr, 150(4), 1457 - 64
Differentiation-dependent p53 regulation of nucleotide excision repair in keratinocytes; Li G et al.; The role of the tumor suppressor p53 in repair of ultraviolet light (UV)-induced DNA damage was evaluated using a host-cell reactivation (HCR) assay . HCR determines a cell's ability to repair UV-damaged DNA through reactivation of a transfected CAT reported plasmid . Most UV damage is removed through nucleotide excision repair (NER) . Primary murine keratinocytes isolated from p53-deficient and wild-type p53 mice were used in the HCR assay . The NER was reduced in p53-/- keratinocytes as compared with p53+/+ keratinocytes . The reduced DNA repair in p53-/- mice was confirmed with a radioimmunoassay comparing cyclobutane dimers (CPDs) and (6-4) photoproducts in p53+/+ and p53-/- keratinocytes after the cells were exposed to UV irradiation . Our results demonstrate that wildtype p53 plays a significant role in regulating NER . Furthermore, as there is evidence that p53 protein levels decrease after keratinocytes become differentiated, we sought to determine whether p53 plays a role in NER in differentiated keratinocytes . Differentiation of the keratinocytes by increasing the Ca2+ concentration in the culture media resulted in a marked reduction in NER equally in both p53+/+ and p53-/- groups . This finding suggests that reduced DNA repair after differentiation is p53 independent . A similar reduction in HCR was confirmed in differentiated human keratinocytes . These data, taken together, indicate that p53 or p53-regulated proteins enhance NER in basal undifferentiated keratinocytes but not in differentiated cells . As nonmelanoma skin cancers originate from the basal keratinocytes, our findings suggest that loss of p53 may contribute to the pathogenesis of this common skin cancer.

Mol Reprod Dev, 1997 Apr, 46(4), 551 - 66
Culture conditions affect meiotic regulation in cumulus cell-enclosed mouse oocytes; Downs SM et al.; To test the hypothesis that culture conditions influence meiotic regulation in mouse oocytes, we have examined the effects of six culture media, four organic buffers, and pH on spontaneous maturation, the maintenance of meiotic arrest and ligand-induced maturation in cumulus cell-enclosed oocytes from hormonally primed immature mice . The media tested were Eagle's minimum essential medium (MEM), Ham's F-10 (F-10), M199, M16, Waymouth's MB 752/1 (MB 752/1), and Leibovitz's L-15 (L-15) . All six media supported > or = 94% spontaneous germinal vesicle breakdown (GVB) during a 17-18 hr incubation period, but polar body formation was lower in M199 and MB 752/1 than in the other media . The incidence of polar bodies could be increased in these two media by the addition of pyruvate . With the exception of M16 and MB 752/1, 4 mM hypoxanthine maintained a significant number of cumulus cell-enclosed oocytes in meiotic arrest . Inhibition could be restored by the addition of glutamine to M16 and pyruvate to MB 752/1 . Follicle-stimulating hormone (FSH) and epidermal growth factor (EGF) stimulated GVB in those media in which hypoxanthine was inhibitory . dbcAMP was able to maintain meiotic arrest in all of the media, but was least effective in M16 . FSH stimulated GVB in all dbcAMP-arrested groups except L-15, and FSH became stimulatory in L-15 when the pyruvate level was reduced to 0.23 mM and galactose was replaced with 5.5 mM glucose . When MEM was buffered principally with the organic buffers MOPS, HEPES, DIPSO, or PIPES (at 20 mM), high frequencies of GVB and polar body formation were observed in inhibitor-free medium . dbcAMP suppressed GVB in all groups; hypoxanthine also maintained meiotic arrest in all buffering conditions, although this effect was nominal in PIPES-buffered medium . FSH and EGF stimulated GVB in all dbcAMP- and hypoxanthine-treated groups . When the concentration of HEPES was increased from 20 mM to 25 mM, a more pronounced suppressive effect on maturation in both dbcAMP- and hypoxanthine-supplemented groups was observed in the absence of FSH . But whereas HEPES reduced the induction of maturation by FSH in dbcAMP-arrested oocytes, this buffer had no effect on FSH action in hypoxanthine-treated oocytes . When MEM was buffered with HEPES and the pH was adjusted to 6.8, 7.0, 7.2, or 7.4, a dramatic effect of pH on meiotic maturation was observed . pH had no significant effect on hypoxanthine salvage by oocyte-cumulus cell complexes, but FSH-induced de novo purine synthesis was significantly augmented by increased pH, in parallel with increased induction of GVB . The results of this study demonstrate that the use of different culture media, or minor changes in culture conditions, can lead to significant variation in (1) the spontaneous maturation of oocytes, (2) the ability of meiotic inhibitors to suppress GVB, or (3) the efficacy of meiosis-inducing ligands . Furthermore, such observations provide a unique opportunity to examine specific molecules and metabolic pathways that can account for this variation and thereby gain valuable insights into the mechanisms involved in meiotic regulation.

Fertil Steril, 1997 Apr, 67(4), 616 - 20
Unstimulated immature oocyte retrieval: early versus midfollicular endometrial priming; Russell JB et al.; OBJECTIVE: To assess the ability to retrieve transvaginally unstimulated immature oocytes from assisted reproductive technology (ART) patients and to determine their competency to mature, fertilize, and implant with early versus midfollicular exogenous estrogen endometrial priming . DESIGN: Prospective randomized study . SETTING: In vitro fertilization unit, private reproductive endocrinology and infertility practice . PATIENT(S): Fourteen patients, all had failed at least one IVF cycle . INTERVENTION(S): Early follicular endometrial priming was initiated on cycle day 3 with 2 mg 17 beta-E2 twice per day (group A) . Midfollicular endometrial priming was initiated with 1 to 2 mg/d of 17 beta-E2 between cycle days 5 and 7 and gradually increased by 1 to 2 mg/d until the oocyte retrieval (group B) . The oocytes were allowed to mature in 0.075 IU FSH or hMG, 0.5 IU of hCG, 1 microgram of 17 beta-E2 in Eagle's or Tissue Culture Media 199, fertilized, and transferred 72 hours later . MAIN OUTCOME MEASURE(S): Maturation, fertilization, and pregnancy rate . RESULT(S): Group A patients had 83 oocytes retrieved (11.8 +/- 6.1) versus 78 oocytes (11.1 +/- 2.7) from group B . The maturation rate in group A was 39.7% (34/83) versus 61.5% (48/78) in group B . The fertilization rate was 75.7% (25/34) in group A versus 75.0% (36/48) in group B . The cleavage arrest rate was significantly higher, 36.0% (9/25) in group A versus 8.3% (3/ 36) in group B . The number of embryos transferred was 1.8 embryos per retrieval in group A versus 4.0 embryos per retrieval in group B . One pregnancy was established in a patient with tubal disease in group B who delivered at 36 weeks gestation . CONCLUSION(S): Unstimulated immature oocyte retrieval can be performed successfully in ART patients . Midfollicular endometrial priming was able to achieve successful maturation (60%), fertilization (75%), and cleavage (92%), with the delivery of a successful pregnancy.

Arch Med Res, 1997 Spring, 28(1), 29 - 36
Biologically active steroid and thyroid hormones stimulate secretion of sex hormone-binding globulin by human term placenta in culture; Diaz L et al.; In this study, the influence of steroid and thyroid hormones and epidermal growth factor on the production of SHBG by placental tissue explants was investigated . Explants of trophoblastic tissue obtained from normal term placentas were cultured for 48 h in serum free culture medium, and then for an additional 24 h period in the presence or absence of various concentrations of either estradiol (0.25-5 nM), testosterone (0.5-500 nM), triiodothyronine (0.01-100 nM) or EGF (2-40 microM), respectively . Human SHBG concentration in culture media was estimated on each day by specific two-site time-resolved fluoroimmunometric assay and the results expressed as pmol/mg tissue protein . Binding characteristics and molecular structure of secreted SHBG were determined by {3H}5 alpha-DHT binding assays and Western blot analysis, respectively . Estradiol and triiodothyronine but not testosterone increased significantly (p < 0.05 vs . control) the secretion of SHBG into the culture media . Addition of EGF did not significantly change the production of SHBG at the various concentrations studied . {3H} 5 alpha-DHT binding assays and Western blot analysis of placental SHBG resulted in identical binding affinities (Kd 2.0 +/- 0.16 x 10(-9)M) and molecular structure to those obtained in serum from normal pregnant women . These findings support and extend previous observations by our laboratory indicating that SHBG gene is expressed in the placenta and provide further evidence on the hormonal regulatory characteristics of this steroid-binding protein in cultured placenta.

Biochim Biophys Acta, 1997 Mar 13, 1324(2), 171 - 81
Impermeant antitumor sulfonylurea conjugates that inhibit plasma membrane NADH oxidase and growth of HeLa cells in culture . Identification of binding proteins from sera of cancer patients; Kim C et al.; The antitumor sulfonylurea LY237868 (N-(4-aminophenyl-sulfonyl)-N'-(4-chlorophenyl)urea) was conjugated through the A ring to alpha-cyclodextrin or agarose bead material (Affigel 10) to prepare impermeant conjugates for activity measurements and affinity isolation of binding proteins from serum . When conjugated to alpha-cyclodextrin, the resulting LY237868 conjugate inhibited both NADH oxidase activity and growth of HeLa cells in culture . The conjugate was at least one order of magnitude more potent as an inhibitor than the parent compound . These findings confirm previous results that demonstrate an antitumor sulfonylurea-binding protein with NADH oxidase activity at the external plasma membrane surface of HeLa cells that is shed into culture media conditioned by growth of HeLa cells . A comparable activity, responsive to sulfonylurea, was present in sera of cancer patients . LY237868 conjugated to agarose beads as the affinity support bound a large number of serum proteins . However, compared to serum from normal patients, the affinity support bound two proteins of M(r) approx . 33.5 and 29.5 not found in sera of normal patients . The 33.5 kDa protein from human sera reacted with antisera to a 33.5 kDa protein from culture media conditioned by growth of HeLa cells that blocked and immunoprecipitated the sulfonylurea-responsive activity from HeLa cell plasma membranes . The results point to the 33.5 kDa protein from cancer patient sera that bound to the sulfonylurea affinity support as representing the circulating equivalent of the previously identified 34 kDa sulfonylurea-binding protein, with NADH oxidase activity at the external cell surface of cultured HeLa cells and a corresponding 33.5 kDa protein shed into culture media conditioned by growth of HeLa cells.

J Neurol Sci, 1997 Mar 10, 146(2), 97 - 102
Acidity is involved in the development of neuropathy caused by oxidized cellulose; Nagamatsu M et al.; Recently we demonstrated that oxidized cellulose (OC), a surgical topical hemostatic agent, induces subjacent nerve fiber degeneration by a diffusible chemical mechanism . Since OC is highly acidic, we examined the role of acidity in the development of neuropathy by OC in this study . Fifteen minutes' exposure to culture media containing OC (2 mg/ml, pH 3.47 or 10 mg/ml, pH 2.57) suppressed the subsequent neurite outgrowth of precultured rat DRG neurons in vitro . However, the neurotoxicity of OC disappeared when the pH of the media was restored to 7.42 . Topical application of 20 mg OC lowered the pH in the subperineurium of the adjacent rat sciatic nerve to around 3, and kept it below 4 for 2 h in vivo . Application of 0.1 ml neutralized physiological saline containing 40 mg OC did not produce pathological changes in the adjacent rat sciatic nerve in vivo, in contrast to the marked subperineurial nerve damage by direct application of 20 or 40 mg OC observed in our previous study . These results strongly indicate that local neurotoxicity of OC is due to its high acidity . Further care is needed to avoid direct application of large amounts of OC to peripheral nerve.

Hum Reprod Update, 1997 Mar-Apr, 3(2), 125 - 35
Polyvinyl alcohol and amino acids as substitutes for bovine serum albumin in culture media for mouse preimplantation embryos; Biggers JD et al.; The effect of replacing bovine serum albumin (BSA) in a simple defined medium (KSOM) with polyvinyl alcohol (PVA) and/or amino acids on the percentages of mouse zygotes that develop to at least the blastocyst stage and that hatch at least partially or completely is reported . Blastocysts could form when BSA was replaced with only PVA, but at a moderately reduced rate; however, partial hatching, and hence complete hatching, were severely impaired when BSA was replaced with only PVA . The substitution of BSA with amino acids alone resulted in a high rate of blastocyst formation and moderate impairment of hatching . The addition of PVA to BSA-free KSOM supplemented with amino acids had no extra effect . BSA had significant effects when added to BSA-free KSOM supplemented with amino acids . The BSA caused a significant increase in the rate of partial hatching, and may even have had a small effect on the rate of blastocyst formation . The results also showed that glucose, at a high concentration of 5.56 mM, does not inhibit the development of mouse zygotes to hatched blastocysts when cultured in KSOM supplemented with amino acids.

Coron Artery Dis, 1997 Mar-Apr, 8(3-4), 189 - 201
High-dose diltiazem prevents migration and proliferation of vascular smooth muscle cells in various in-vitro models of human coronary restenosis; Voisard R et al.; BACKGROUND: Restenosis after coronary angioplasty is considered to be caused mainly by increased migration and proliferation of smooth muscle cells (SMC) . The concept of local, site-specific delivery of pharmacologic therapies has opened the door for new, high-dose drug regimes . METHODS AND RESULTS: SMC were isolated by enzymatic disaggregation with collagenase/elastase from human coronary plaque tissue of 29 patients (pSMC) and post mortem from the coronary media of 33 corpses (mSMC) . Endothelial cells were isolated from human umbilical veins by enzymatic disaggregation with collagenase/dispase . By positive reaction with antibodies against smooth muscle alpha-actin and von Willebrand factor cells were identified as SMC or endothelial cells . In proliferation studies 5-150 micrograms/ml diltiazem was added to the culture media of pSMC, mSMC and endothelial cells . After 5 days there was a significant dose-dependent inhibition of cell proliferation (for pSMC with > 50 micrograms/ml, for mSMC with > 25 micrograms/ml, and for endothelial cells with > 5 micrograms/ml) . In migration studies the effect of 5-150 micrograms/ml diltiazem on the velocity of migration of pSMC was investigated over a period of 48 h . Administration of diltiazem at concentrations of 100 and 150 micrograms/ml caused a significant inhibition of the migration of pSMC . The cytoskeletal components smooth muscle alpha-actin, vimentin, and alpha-tubulin of pSMC and the expression of von Willebrand factor of endothelial cells were investigated after an incubation period of 5 days with 50 and 150 micrograms/ml diltiazem . In the transfilter coculture model the effect of 50 micrograms/ml diltiazem on mSMC was investigated after mechanical injury of cocultured endothelial cells . Administration of diltiazem at a concentration of 50 micrograms/ml inhibited the development of a neointimal proliferate in the transfilter coculture model significantly (P < 0.001) . CONCLUSIONS: A high dose of diltiazem inhibited the migratory and proliferative activities of coronary SMC significantly . In further experimental studies the effect of locally applied high doses of diltiazem on postangioplasty restenosis should be elucidated.

In Vivo, 1997 Mar-Apr, 11(2), 157 - 61
Interferons alpha, beta and gamma induce different patterns of gene expression in cultured human epidermal keratinocytes; Arany I et al.; Differences in the effects on confluent epidermal keratinocytes of treatment with interferons (IFNs) alpha, beta, and gamma were observed in their modulation of the mRNA levels of representative structural and functional cellular proteins . Comparisons of the responses in culture media with varying cellular maturation potential indicated the dependence of the modulation on the stage of differentiation . Differentiation was correlated with upregulation of all the genes by interferon gamma, but this effect was not seen with the other interferons . Even though IFNs alpha and beta share the same cell surface receptor, their effects on gene expression were clearly distinguishable and varied with the culture medium . These findings might have relevance in the treatment of skin lesions with varying degrees of differentiation.

J Reprod Fertil, 1997 Mar, 109(2), 187 - 91
Induction of early pregnancy factor activity in vitro by platelet-activating factor in mice; Lash GE et al.; The rosette inhibition test was used to determine early pregnancy factor activity in culture media from oestrous mouse ovaries and oviducts stimulated in vitro for early pregnancy factor production under different experimental conditions . Embryo conditioned media, platelet-activating factor and cortical granule release media could all stimulate the production of early pregnancy factor by oestrous mouse ovaries and oviducts . This stimulation was completely blocked by the presence of BN 52021, a platelet-activating factor receptor antagonist . This study indicates that platelet-activating factor is the 'ovum factor' released by the zygote on fertilization to initiate the synthesis of early pregnancy factor.

Immunotechnology, 1997 Mar, 3(1), 31 - 43
Cloning and expression of human V-genes derived from phage display libraries as fully assembled human anti-TNF alpha monoclonal antibodies; Mahler SM et al.; BACKGROUND: With the advent of phage antibody libraries, access to completely human antibody fragments is feasible, either by direct selection from human antibody libraries, or by guided selection . After selection, Fabs and scFvs may need to be expressed as complete antibodies in mammalian cells for further characterisation, or if effector functions are required . OBJECTIVES: To rebuild and express the human anti-TNF alpha antibody Fab-P3A2 (isolated as a Fab fragment from phage display libraries by guided selection) as a fully assembled, functional human antibody (gamma-1, lambda) in Sp2/0 myeloma cells, and to perform preliminary characterisation studies of the secreted IgG1 molecule . A further objective was to investigate the kinetics of human antibody production and the stability of antibody secretion in transfectomas cultured in various media formulations . STUDY DESIGN: A tripartite strategy was employed for cloning heavy chain gene (VH)-P3 and light chain gene V lambda-A2-C lambda into mammalian cell expression vectors p alpha Lys-30 and p alpha Lys-17 respectively . The cell line P3A2.B5 was isolated after co-transfection of Sp2/0 mouse myelomas with the constructs, expanded and weaned into a protein free medium . Fully assembled Ig-P3A2 antibody was purified by Protein A affinity chromatography and characterised with respect to size of antibody chains, and affinity for human TNF alpha . Stability of secretion was investigated by extended serial sub-culture and analysis of P3A2.B5 sub-clones . Strategies of media enrichment were tested for any effect on antibody productivity by selected P3A2.B5 sub-clones . RESULTS: The cell line P3A2.B5 secreted an assembled, human antibody Ig-P3A2, with heavy and light chains of molecular weight 55 and 28 KD respectively . Equilibrium capture studies showed Ig-P3A2 to have a dissociation constant of approximately 1.5 x 10(-8) M . The mean specific productivity of the cell line increased from 1.2 pg/cell/day to 7.8 pg/cell/day by a combination of medium enrichment and serum reduction . Prolonged serial sub-culture of P3A2.B5 showed the cell line to be unstable with respect to antibody secretion . CONCLUSIONS: We have outlined a method for expression of human V genes as assembled antibodies in Sp2/0 myeloma cells . A cloning strategy for the stable expression of scFv or Fab genes isolated from phage display libraries as assembled human antibodies of the IgGl subclass in Sp2/0 myeloma cells has been described . For maximising specific productivity of antibody-producing cell lines, supplementation of culture media with glucose, glutamine and amino acids increases antibody yield significantly compared to that in conventional media, indicating the latter is stoichiometrically limiting for production purposes.

J Mol Cell Cardiol, 1997 Mar, 29(3), 929 - 37
Downregulation of polo-like kinase correlates with loss of proliferative ability of cardiac myocytes; Georgescu SP et al.; Cardiac myocytes rapidly increase the cell number during the fetal and early neonatal period, but they lose their proliferative ability soon after birth . To understand the mechanism of how cardiac myocytes exit from the cell cycle, we examined the role of a newly identified serine/threonine kinase, polo-like kinase (Plk), in the process of proliferation of cardiac myocytes . Northern blot analysis revealed that Plk gene was abundantly expressed in cardiac myocytes and non-myocytes of fetal and neonatal rats but not in cardiocytes of adult rats . Western blot analysis showed that Plk protein was also detected only in fetal and neonatal hearts . During the early stage of cardiac differentiation . Plk expression was well correlated with the proliferative ability of cardiocytes . Plk mRNA was most abundant in undifferentiated embryonic stem (ES) cells and the mRNA levels decreased along with cardiac differentiation in the developing ES cell system . Once serum was deprived from the culture media, expression levels of Plk were markedly decreased and DNA was not synthesized in both cardiac myocytes and non-myocytes of neonatal rats . Re-addition of serum stimulated Plk gene expression and DNA synthesis in non-myocytes but not in cardiomyocytes . All these results taken together with the critical role of Plk in DNA synthesis in many cell types suggest that downregulation of Plk is important for the permanent withdrawal of cardiomyocytes from the cell cycle.

Cell Biol Int, 1997 Mar, 21(3), 133 - 44
Inhibition of malignant cell proliferation by culture media conditioned by cardiac or skeletal muscle; Zinman T et al.; The present work is an attempt to explain the high resistance of muscles to cancer development . We used primary cultures of rat skeletal and cardiac muscle, and examined the effect of the supernatant of these cultures (conditioned medium; CM) on proliferation of cancer cells . The results demonstrated that CM inhibited the proliferation of several types of malignant cells by more than 50%, without a significant inhibition on normal cells . Cell cycle analysis revealed that CM increased the number of cells in S and G2 phases, suggesting a cytostatic effect of CM . For defining the biological properties of the factor(s) which are present in the CM, skeletal muscle cultures were grown in chemically defined medium (serum free medium) . The concentrated sample was applied to a Sephadex G-50 column and three fractions were obtained . Only one fraction showed inhibitory activity . Four protein bands were observed in this fraction, as revealed by SDS-PAGE . We suggest that some, or all of these proteins are responsible for inhibition of tumor cell replication.

Med Sci Sports Exerc, 1997 Mar, 29(3), 326 - 32
The effect of ultrasound on collagen synthesis and fibroblast proliferation in vitro; Ramirez A et al.; Ultrasound has been applied therapeutically to accelerate connective tissue healing although there is little direct scientific evidence to support its use . This investigation was conducted to determine the effects of ultrasound on the rate of collagen synthesis and cell proliferation using cultured fibroblasts derived from Achilles tendons of neonatal rats . Ultrasound (intensity = 0.4 W.cm-2; frequency = 1 MHz) was applied to experimental cells growing as monolayers in culture flasks . Ultrasound had no effect on the rate of collagen synthesis by control fibroblasts over a period of 9 d . The addition of vitamin C to culture media stimulated collagen synthesis to the same extent in both control and ultrasound-treated cultures . Partial digestion of cell matrices with collagenase (used to simulate injury) resulted in an approximately 20% increase in the rate of collagen synthesis . Synthesis was further increased with ultrasound treatment (50-67%) . For example, after a single ultrasound treatment, the rate of collagen synthesis was 3.0 +/- 0.4 pg.micrograms-1 DNA.h-1 in cultures treated with collagenase, compared with 1.8 +/- 0.3 pg.micrograms-1 DNA.h-1 in collagenase-treated cultures not treated with ultrasound and 1.4 +/- 0.3 pg.micrograms-1 DNA.h-1 in controls . Ultrasound applied to preconfluent cultures resulted in significant increases in the rate of thymidine incorporation and DNA content . Three daily ultrasound treatments caused a 100% increase in the rate of thymidine incorporation and a 28% increase in DNA content . The results indicate that ultrasound stimulates collagen synthesis in tendon fibroblasts in response to an injury of the connective tissue matrix and that ultrasound stimulates cell division during periods of rapid cell proliferation.

Glycobiology, 1997 Mar, 7(2), 305 - 14
Sulfation and sialylation requirements for a glycoform of CD34, a major endothelial ligand for L-selectin in porcine peripheral lymph nodes; Shailubhai K et al.; Leukocyte recruitment from blood into peripheral lymph nodes is controlled in part by a specific interaction of lymphocyte-associated L-selectin with endothelial cell receptors known as peripheral addressins . In murine lymph nodes, two peripheral addressins have been identified, Gly-CAM-1, a 50 kDa molecule that also appears as a secreted form in plasma, and CD34, a 90 kDa membrane-associated sialomucin . A predominant 105 kDa CD34 mucin-like protein has also been identified in human tonsil as peripheral addressin . We have identified a 120 kDa sialomucin as the predominant peripheral addressin in porcine lymph nodes . Validation of the 120 kDa porcine molecule as a peripheral addressins was based on its ability to bind MECA-79, a monoclonal antibody previously used to isolate peripheral addressins from mouse and human tissues, and to bind an L-selectin-Fc chimera (LS-Fc) . The binding with LS-Fc was abolished in the presence of fucoidin, a sulfated polysaccharide known to inhibit L-selectin-receptor interactions . To address the possibility that the 120 kDa ligand may contain common recognition determinants for MECA-79 and L-selectin, the requirements for sialylation and sulfation were compared . Whereas desialylation of 120 kDa ligand drastically reduced its binding to LS-Fc, this treatment appeared to enhance the binding of 120 kDa ligand to MECA-79 . In contrast, the binding of both MECA-79 and LS-Fc to 120 kDa ligand was drastically reduced when de novo sulfation of this ligand was reduced by including chlorate, a metabolic inhibitor of sulfation, in the culture media . N-Terminal amino acid sequences of the porcine 120 kDa protein revealed homology with human CD34 . Taken together, these findings suggest that the porcine 120 kDa peripheral addressin is an L-selectin-binding glycoform of CD34.

Arterioscler Thromb Vasc Biol, 1997 Mar, 17(3), 483 - 9
Thrombin promotes activation of matrix metalloproteinase-2 produced by cultured vascular smooth muscle cells; Galis ZS et al.; Thrombin generated at sites of vascular injury not only participates in the coagulation cascade but can signal other events related to development and complication of atherosclerotic plaques . We investigated here a novel non-thrombotic action of thrombin: the possibility that this protease influences the expression or activation of matrix metalloproteinases (MMPs) produced by vascular smooth muscle cells (SMCs) . Matrix-degrading proteinases likely contribute to several aspects of vascular lesion development . Vascular SMCs constitutively elaborate the zymogen form of gelatinase A (MMP-2), found in cell supernatants complexed with its inhibitor, the tissue inhibitor of metalloproteinases (TIMP)-2 . When activated, MMP-2 digests collagens and elastin and may thus promote cell migration and vascular remodeling . Analysis of culture supernatants harvested from either human or rabbit vascular SMCs by gelatin zymography revealed that compared with supernatants of unstimulated SMCs, media conditioned by thrombin-stimulated cells contained increased amounts of proteolytically processed MMP-2, suggesting activation of this MMP . Further experiments tested whether thrombin directly activates MMP-2 . In cell-free experiments, when added to medium harvested from unstimulated SMCs, alpha-thrombin increased in a dose- and time-dependent manner the amount of proteolytically processed MMP-2, as shown by zymography and by Western blotting with specific antibodies . Thrombin cleaved pro-MMP-2 within 4 hours, even when the gelatinase was bound with its inhibitor, TIMP-2 . Thrombin treatment rendered culture media of unstimulated SMCs able to degrade collagen type IV, consistent with generation of active MMP-2 . Addition of inhibitors of either thrombin or MMPs decreased this type IV collagenolytic activity, but thrombin in the absence of SMC-conditioned medium containing pro-MMP-2 exhibited only minimal collagenolysis . Our results suggest that at sites of vascular injury, thrombin may activate locally produced MMP-2 and thereby facilitate cell migration and proliferation . In the case of complicated atherosclerotic plaques, episodes of intraplaque hemorrhage or plaque disruption with thrombosis may promote plaque instability by increasing local matrix-degrading activity.

Thorax, 1997 Mar, 52(3), 213 - 7
Epithelial barrier formation by airway basal cells; Erjefalt JS et al.; BACKGROUND: Epithelial shedding processes in airway inflammation and defence may produce damaged areas where basal cells are the main remaining epithelial cell type . The present study examines the capacity of basal cells to form an epithelial barrier structure after loss of columnar epithelial cells . METHODS: A technique was developed which allows selective removal of columnar epithelial cells from isolated airways . A drop of tissue adhesive glue was applied on the mucosal surface shortly after excision of guinea pig trachea and human bronchus . Gentle removal of the glue, together with attached columnar cells, left a single layer of cobbled, solitary basal cells . The tissue was kept in culture media . Morphological changes of the basal cells were monitored by immuno-histochemistry and scanning and transmission electron microscopy at several time points . RESULTS: After 20 minutes the basal cells had undergone extensive flattening and established contact with each other . The basement membrane thus became covered by a poorly differentiated epithelium in both guinea pig and human airways . Abundant interdigitating cytoplasmic protrusions were observed at cell borders . CONCLUSIONS: Basal cells promptly flatten out to cover the basement membrane at loss of neighbouring columnar cells . These data may explain why the epithelial barrier function may be uncompromised in desquamative airway diseases . Furthermore, they suggest the possibility that sacrificial release of columnar epithelial cells and prompt creation of a barrier structure constitute important roles of basal cells in airway defence against severe insults.

Placenta, 1997 Mar-Apr, 18(2-3), 99 - 108
Culture of syncytiotrophoblast for the study of human placental transfer . Part II: Production, culture and use of syncytiotrophoblast; Bloxam DL et al.; The conditions necessary for producing syncytical syncytiotrophoblast are examined . Tissue disaggregation conditions, culture media composition, different extracellular matrices and the influence of placental gestational age are all assessed . The importance of evaluating the biochemical and functional differentiational state of the cells is also stressed . Evidence is summarized that syncytiotrophoblast in culture is morphologically and ultrastructurally very similar to syncytiotrophoblast in vivo, and what is so far known biochemically is largely consistent with what is known in vivo . Studies published to date on microvillous membrane uptake and release and relationships with intracellular metabolism using syncytiotrophoblast in conventional culture are outlined from the point of view of the advantages and potential of this model . The present state of development of the two-sided model is assessed, mentioning factors to be considered such as the supporting membrane to be used, accounting for passive diffusion and paracellular leak components of transport and dealing with quantitative effects in kinetic studies of the presence of the supporting membrane . It is concluded that satisfactory methods are now in place for preparing pure villous syncytial syncytiotrophoblast in culture from cytotrophoblast derived from term (but not early) placentae, suitable for studying microvillous membrane transport and relationships with intracellular metabolism . Cytotrophoblast from early gestational age placenta may require different conditions to form true syncytiotrophoblast . A two-sided model for studies of overall transfer, basal transport and basal control mechanisms is now available and possibly with some development should be a good model for such investigations.

Trends Biotechnol, 1997 Mar, 15(3), 109 - 13
Integrated approaches to the design of media and feeding strategies for fed-batch cultures of animal cells; Xie L et al.; Animal cell culture has become an important approach for the production of biologically functional proteins for human therapy . The quantity and quality of protein production are influenced by the culture environment, which is subject to change over the course of cell cultivation . Therefore, it is vital to design an optimal culture environment and control it within an optimal region to maximize the productivity . This requires that the factors affecting the culture environment (nutrient concentrations, by-product accumulation, pH and osmolality) and cell growth be integrated into the design of culture media for fed-batch animal cell cultures.

FEMS Microbiol Lett, 1997 Mar 1, 148(1), 49 - 52
Magnesium ions are required for HEp-2 cell adhesion by enteroaggregative strains of Escherichia coli O126:H27 and O44:H18; Chart H et al.; Enteroaggregative strains of Escherichia coli, belonging to serotypes O44:H18 and O126:H27, were used to show that magnesium ions were essential for the adhesion of these enteroaggregative strains to HEp-2 cells . The removal of Mg2+ ions from culture media was correlated with the inability of strains to produce an outer membrane-associated protein of 18 kDa and a pellicle . It was concluded that magnesium ions were directly involved with the expression of an 18 kDa outer membrane-associated protein by strains of E . coli O126:H27 and O44:H18, and that the outer membrane-associated protein was involved in both HEp-2 adhesion and pellicle formation.

J Neuropathol Exp Neurol, 1997 Mar, 56(3), 263 - 72
Accumulation of Alzheimer amyloid-beta peptide in cultured myocytes is enhanced by serum and reduced by cerebrospinal fluid; Mazur-Kolecka B et al.; Smooth muscle cells cultured from leptomeningeal vessels from old dogs with amyloid-angiopathy accumulate intracellular deposits that are immunoreactive for amyloid-beta peptide (A beta) . We used this cellular model in the present study to examine the influence of sera and cerebrospinal fluid on intracellular accumulation of A beta-immunoreactive deposits and on secretion of soluble A beta into culture media . We found that sera from old dogs significantly increased the percentage of A beta-positive smooth muscle cells in culture . The enhanced accumulation of A beta was associated with (a) lower secretion of A beta into media, (b) altered maturation of amyloid-beta-precursor protein (A betaPP) into A betaPP751-770 with faster electrophoretic mobility, (c) increased accumulation of C-terminal fragments of A betaPP (12-15 kD, 10kD and less), and (d) increased secretion of A betaPP into culture media . These findings suggest that age- or disease-related serum factors increase accumulation of A beta by affecting production and processing of A betaPP In contrast, cerebrospinal fluids reduced accumulation of A beta . Involvement of A beta-carrier proteins-apolipoprotein E and transthyretin-in accumulation of A beta is demonstrated . Accumulation of A beta in cultured smooth muscle cells-a model of beta-amyloidosis-may be regulated by factors that alter production and processing of A betaPP as well as the fate of soluble A beta in extracellular space.

J Immunol Methods, 1997 Feb 28, 201(2), 233 - 41
Reduction of bovine immunoglobulin contamination from monoclonal antibodies by SOURCE 15PHE chromatography; Grunfeld H et al.; The presence of bovine immunoglobulin in cell culture media, and its nature as a polyclonal antibody, imposes increasing difficulties in resolution from the MAb intended for in vivo human applications . Particular difficulties are encountered when murine MAbs are the target antibody . This study presents model cases to simulate this problem, and suggests an efficient method for reduction of bovine IgG (BGG) from MAb preparations . Utilizing the new small particle size hydrophobic interaction chromatography resin, SOURCE 15PHE, up to complete resolution was achieved with a chimeric MAb . A 17-fold reduction of BGG was achieved with a murine MAb . In both cases, high recoveries of the MAb were obtained . The general applicability of the method is suggested.

J Immunol Methods, 1997 Feb 14, 201(1), 57 - 66
Quantitative analysis of the products of IgG chain recombination in hybrid hybridomas based on affinity chromatography and radioimmunoassay; Massino YS et al.; On the model of a hybrid hybridoma (quadroma) to alpha-endorphin (END) and horseradish peroxidase (HRP), we have elaborated a general approach to analyse H and L chain interactions in hybrid hybridomas and to evaluate their efficiency as producers of bispecific antibodies (bAbs) . This strategy is based on quantitative analysis of quadroma produced Abs by affinity chromatography and radioimmunoassay . First, Abs produced by quadroma cells in culture media (IgG pools from three quadroma clones) were fractioned with respect to specificity . Second, Ab concentrations in each fraction (bispecific, anti-END, anti-HRP and inactive) were measured by specific radioimmunoassays, using rabbit antiserum against mouse IgG and 125I-labelled affinity purified quadroma Abs . Then the experimentally obtained Ab distributions were compared with the predicted Ab distributions for different models of IgG chain recombination in quadroma cells (random H/L pairing, preferential homologous H/L association) . As follows from these models, in a random H/L recombination the yield of bAbs in quadroma produced IgG cannot exceed 12.5%, and the ratio of bAbs and inactive Abs cannot exceed 0.5 . In the analysed clones the yield of bAbs amounted to about 30% of total IgG, and the ratio of bAbs and inactive Abs was about 5-8, giving strong evidence for preferential homologous H/L association in these cells . The ratio of anti-HRP and anti-END Abs was about 10:1, suggesting unequal production of parental IgG chains in quadroma cells . The result of quantitative analysis of quadroma IgG was further supported by two-dimensional gel analysis of affinity-purified fractions of quadroma IgG and of two parental mAbs.

Lipids, 1997 Feb, 32(2), 219 - 26
An esterification protocol for cis-parinaric acid-determined lipid peroxidation in immune cells; McGuire SO et al.; Loss of fluorescence from cis-parinaric acid (cPnA) is a sensitive indicator of lipid peroxidation . The purpose of this study was to utilize cPnA to determine, at the level of the intact immune cell, whether enrichment of membranes with polyunsaturated fatty acids (PUFA) increased lipid peroxidation . P388D1 macrophages were labeled by addition of cPnA as an ethanolic solution . Within two minutes of addition, in the absence-of serum, cPnA rapidly intercalated into the plasma membrane . Lipid peroxidation was initiated by addition of Fe(2+)-EDTA resulting in a dose-dependent decrease in fluorescence with increased oxidant concentration . Cells previously enriched with PUFA and labeled by intercalation showed no differences in spontaneous or Fe(2+)-induced lipid peroxidation . In separate experiments, 20 microM cPnA in ethanolic solution was injected into cell culture media containing 0.1% essentially fatty acid free bovine serum albumin (BSA) . Cells were resuspended and incubated for 90 min at 37 degrees C . After washing with BSA to remove cPnA which had not incorporated, 0.5% (0.1 microM) of the added cPnA was found esterified within cellular lipids . This level of cPnA provided a 100-fold increase over basal autofluorescence levels . Cells labeled in this manner also lost fluorescence in a dose-dependent manner as levels of oxidant stress increased . Cells enriched with PUFA and labeled by esterification had significantly increased rates and total amounts of lipid peroxidation . Co-incubation with alpha-tocopherol and PUFA resulted in a decrease in lipid peroxidation which was not significantly different from control cells . In conclusion, esterification of cPnA into membrane phospholipids can sensitively detect changes in lipid peroxidation induced by alteration of membrane PUFA and/or vitamin E content.

J Endocrinol, 1997 Feb, 152(2), 265 - 74
Degradation of IGF-binding protein-3 by proteases in cultured FRTL-5 rat thyroid cells; Wang JF et al.; In this study, we have found that IGF-binding protein-3 (IGFBP-3) in calf serum added to tissue culture medium is degraded by cultured FRTL-5 cells and a major 31 kDa fragment of IGFBP-3 is produced . When FRTL-5 rat thyroid cells were cultured in 6H medium (modified F-12M medium containing TSH, insulin, hydrocortisone, somatostatin, transferrin, and glycyl-histidyl-lysine) containing 5% calf serum, both 44-46 and 31 kDa IGFBPs were found in conditioned medium by ligand blot analysis using 125I-labelled IGF-II . However, predominantly the 44-46 kDa IGFBP was detected in unconditioned 6H medium containing 5% calf serum . When calf serum in the media was replaced by human serum similar results were obtained, and the 44-46 kDa and 31 kDa IGFBPs were recognized using a human IGFBP-3 antibody following Western blot analysis . FRTL-5 cells secreted only small amounts of an endogenous 29 kDa IGFBP, thought to be IGFBP-5 . To separate the 31 kDa fragment of IGFBP-3 from the endogenous IGFBP-5, culture media were fractionated by concanavalin-A-Sepharose chromatography and aliquots of both flow-through and eluate from the column were analyzed by ligand blotting . A 31 kDa IGFBP was found in the eluate fractions from concanavalin-A-Sepharose chromatography following the separation of conditioned 6H medium supplemented with calf serum, suggesting that this species was an N-linked glycoprotein and could be derived from the degradation of serum IGFBP-3 by FRTL-5 cells . Using a modified zymographic assay, we examined whether the degradation of IGFBP-3 could depend on the cell membrane . Confluent FRTL-5 cells were washed with PBS and overlaid with liquid agarose solution . After the agarose had solidified, unconditioned 6H medium containing 5% calf serum was incubated with the cells at 37 degrees C for 16 h . Both 44-46 and 31 kDa IGFBP species were found in the overlying, conditioned medium by ligand blot . However, the 31 kDa IGFBP was not found in medium in the absence of FRTL-5 cells, and no IGFBP could be found in serum-free conditioned medium from agarose-covered FRTL-5 cells . This suggests that the 44-46 kDa IGFBP-3 in serum was degraded to yield a 31 kDa fragment, while any endogenous IGFBP-5 could not pass out of the agarose . The degradation of 44-46 kDa IGFBP-3 in the modified zymographic assay was inhibited by phenylmethylsulfonyl fluoride, EDTA, and aprotinin, but not by leupeptin . In summary, these results indicated that IGFBP-3 in calf serum added to culture medium could be degraded by FRTL-5 cells and that this may involve calcium-dependent serine proteases.

J Endocrinol, 1997 Feb, 152(2), 221 - 7
IGF-binding protein-6 is involved in growth inhibition in SH-SY5Y human neuroblastoma cells: its production is both IGF- and cell density-dependent; Babajko S et al.; The IGF system is involved in the growth and differentiation of neuroblastoma cells, but the precise roles played by the IGF-binding proteins (IGFBPs) remain unknown . We have examined the expression and functions of IGFBPs produced by the neuroblastoma cell line, SH-SY5Y, in the presence of: insulin, IGF-I, IGF-II, des(1-3)IGF-I (an IGF-I analogue with weak affinity for IGFBPs), acidic fibroblast growth factor, basic fibroblast growth factor, or nerve growth factor . Under basal conditions, SH-SY5Y cells in serum-free medium secreted IGF-II, and traces of IGF-I, IGFBP-2 and IGFBP-4 . After 24 h of culture, comparative mitogenic potencies were: des(1-3)IGF-I > IGF-I > IGF-II > insulin . After 48 h, when IGFBP-2 and IGFBP-4 concentrations in the culture media had increased, des(1-3)IGF-I remained the most active, but the activity of insulin now equalled or exceeded that of IGF-I and IGF-II . This suggests a negative feedback mechanism involving partial sequestration of IGF-I and IGF-II by IGFBP-2 and IGFBP-4 . At high cell density and with high concentrations of IGF-I, des(1-3)IGF-I (40 ng/ml) or IGF-II (80 ng/ml), the mitogenic activities of the IGFs diminished concomitantly with the appearance in the culture medium of an additional IGFBP identified as IGFBP-6, whose production depended on activation of the type 1 IGF receptor . These findings suggest that IGFBP-6 contributes as an autocrine inhibitor in the regulation of growth by the IGF system in these neuroblastoma cells.

J Endocrinol, 1997 Feb, 152(2), 201 - 9
Growth hormone as an amplifier of insulin-like growth factor-I action: potentiated oestradiol accumulation; Xu YP et al.; The interaction between GH and IGF-I in modulating oestradiol biosynthetic capacity was examined in cultured porcine granulosa cells . Granulosa cells (2 x 10(6) viable cells/culture) were initially cultured for 96 h (treatment period) in an androstenedione-free medium in the absence or presence of IGF-I (0 or 50 ng/ml), with or without GH at 100 ng/ml . At the conclusion of this period, culture media were discarded and the cells were washed twice with the androstenedione-free medium and reincubated for an additional 24 h (test interval) in an androstenedione (10(-7) M)-supplemented medium for oestradiol accumulation . GH alone induced no stimulation (P > 0.05) of basal oestradiol accumulation . In contrast, concurrent treatment with IGF-I produced a 4.3-fold increase (26 vs 112 ng/ 2 x 10(6) cells per 24 h, P < 0.001) in oestradiol accumulation . GH amplified IGF-I-induced oestradiol production in a dose (minimal dose requirement of 0.3 ng/ml)- and time (minimal time requirement of 24-48 h)-dependent manner . Studies on the site(s) of action indicated that GH exerts its amplifying effects on IGF-I-induced oestradiol production both proximal and distal to cAMP generation . As the specificity study and the inhibitory study indicated, GH amplification of IGF-I-induced oestradiol production is a process involving gene transcription and/or translation and the synergism is not solely specific to IGF-I as IGF-II-induced oestradiol production was also amplified in the presence of GH.

Curr Eye Res, 1997 Feb, 16(2), 131 - 43
Characterization of beta-glucuronidase in the retinal pigment epithelium; Ray J et al.; PURPOSE: To study the biochemical and molecular characteristics of the lysosomal enzyme beta-glucuronidase (GUSB) in the retinal pigment epithelium (RPE) and other tissues of different species . METHODS: Freshly isolated and cultured cells were harvested, and GUSB activity was measured fluorimetrically in cell homogenates or tissue culture media using the synthetic substrate 4-methyl-umbelliferyl beta-D-glucuronide (4-MUG) . The temperature and pH optima, and thermal stability of GUSB in the RPE and fibroblasts were established . Distribution of glycosaminoglycans (GAGs), the natural substrates of GUSB, in the RPE and fibroblast cell layer and media was examined by cellulose acetate electrophoresis following 72 h of metabolic labeling with Na2(35)SO4 . Total or poly A(+)-RNA isolated from cells or tissues of different species were examined in Northern blots to identify GUSB mRNA transcripts . RESULTS: Among all the species, the activity of GUSB and its mRNA level was found to be consistently high in RPE cells . In RPE cultures, the activity was detected in the cell layer and the media, and the activity decreased in both compartments with serial passage . While the temperature and pH optima for the enzyme activity was similar across the species, the thermal stability was remarkably different . The GAG profiles in RPE cells were different from fibroblasts . Supplementation of the cultured cells with selected GAGs moderately increased the GUSB activity . A GUSB transcript was detected in all the tissues examined . In man, mouse, dog, and cat the size of the transcript was 2.4 kb, while the rat GUSB transcript was 2.7 kb . CONCLUSIONS: The ubiquitous distribution of GUSB was evident from the biochemical and molecular studies . Presence of a high level of GUSB activity in the RPE makes it an ideal model for studies of this enzyme both in normal as well as in diseases resulting in GUSB deficiency.

Cell Growth Differ, 1997 Feb, 8(2), 243 - 50
Collagenase 3 (matrix metalloproteinase 13) gene expression by HaCaT keratinocytes is enhanced by tumor necrosis factor alpha and transforming growth factor beta; Johansson N et al.; Collagenase-3 (matrix metalloproteinase 13; MMP-13) is a novel matrix metalloproteinase, the expression of which to date has only been detected in human breast carcinoma tissue and osteoarthritic cartilage . Here, we show that MMP-13 transcripts are expressed by human HaCaT keratinocytes but not by primary human epidermal keratinocytes . The levels of MMP-13 mRNAs in HaCaT cells were enhanced up to 130- and 45-fold by tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta), respectively . The maximal induction of MMP-13 mRNAs by TNF-alpha was noted after a 6-h incubation, whereas with TGF-beta, the maximal stimulation was observed after 24 h . The up-regulation of MMP-13 mRNA abundance by TNF-alpha and TGF-beta was dependent on protein synthesis and was prevented partially by dexamethasone and retinoic acid . Nuclear run-on assays demonstrated activation of MMP-13 gene transcription by TNF-alpha maximally at the 2-h time point and by TGF-beta after 12 h of treatment . Incubation of HaCaT keratinocytes with TNF-alpha and TGF-beta also increased production of proMMP-13 into the culture media, as detected by Western blotting . Our data indicate that the MMP-13 gene is expressed by transformed epidermal keratinocytes, suggesting a role for MMP-13 in the invasive capacity of human epidermal malignancies.

J Neurosci Res, 1997 Feb 1, 47(3), 311 - 21
Kainate/AMPA receptors expressed on human fetal astrocytes in long-term culture; Cauley K et al.; Long-term cultivation of primary human fetal brain cells has yielded a homogeneous population of glial progenitors of extended life span . These human astrocyte precursor (HAP-1) cells have been in culture for greater than 1 year, are diploid, and do not form colonies in soft agar . The culture was established in 10% fetal calf serum (FCS), although cells greatly increase their proliferative rate when both basic fibroblast growth factor and FCS are present in the culture media . HAP-1 cells express the cytoskeletal proteins glial fibrillary acidic protein, vimentin, and nestin . HAP-1 cells express the AMPA/kainate receptor subunit genes GluRs 1, 3, and 4 and the kainate receptor subunit genes GluR6, KA1, and KA2 . Immunohistochemistry confirms the expression of GluR subunit proteins . HAP-1 cells demonstrate a kainate-responsive current found to be blockable by CNQX . HAP-1 cells will serve in the study of human glial cells and ligand-gated ion channels and in the identification of compounds which might act as agonists or antagonists at these receptor-ion channel complexes.

J Biomed Mater Res, 1997 Feb, 34(2), 189 - 99
Controlling cell interactions by micropatterning in co-cultures: hepatocytes and 3T3 fibroblasts; Bhatia SN et al.; The repair or replacement of damaged tissues using in vitro strategies has focused on manipulation of the cell environment by modulation of cell-extracellular matrix interactions, cell-cell interactions, or soluble stimuli . Many of these environmental influences are easily controlled using macroscopic techniques; however, in co-culture systems with two or more cell types, cell-cell interactions have been difficult to manipulate precisely using similar methods . Although microfabrication has been widely utilized for the spatial control of cells in culture, these methods have never been adapted to the simultaneous co-cultivation of more than one cell type . We have developed a versatile technique for micropatterning of two different cell types based on existing strategies for surface modification with aminosilanes linked to biomolecules and the manipulation of serum content of cell culture media . This co-culture technique allowed manipulation of the initial cellular microenvironment without variation of cell number . Specifically, we were able to control the level of homotypic interaction in cultures of a single cell type and the degree of heterotypic contact in co-cultures over a wide range . This methodology has potential applications in tissue engineering, implant biology, and developmental biology, both in the arena of basic science and optimization of function for technological applications.

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