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