Culture medium

Exp Hematol, 1989 Sep, 17(8), 865 - 71
A four-step procedure for the purification of thrombopoietin; McDonald TP et al.; The present work reports the preparation of a highly bioactive and stable thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) by a four-step purification procedure, i.e., Sephadex column chromatography, ethanol precipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and reverse phase-high performance liquid chromatography . The molecular weight (MW) of the purified product depended upon the method of purification, i.e., using denaturing buffers at 56 degrees C for 10 min, the MW was approximately 30,000 daltons; whereas, after preparing in denaturing buffers and heating to 100 degrees C for 10 min, the purified protein had an apparent MW of approximately 15 kd . Both moieties had significant biological activity . The data indicate that TSF may exist normally as a dimer (30 kd), but can disassociate to 15 kd without loss of bioactivity . The present work illustrates that the purified TSF has an isoelectric pH of 4.47 and exists in trace amounts in human embryonic kidney (HEK) cell culture media . The final product prepared in the presence of Tween-20 had a specific activity of approximately 21,000 U of TSF per mg of protein, representing a purification factor of approximately 164,000 . Using this four-step purification procedure, a homogeneous product was obtained as judged by SDS-PAGE and chromatofocusing . This purified material will be suitable for further studies, including amino acid sequencing.

Zhongguo Yao Li Xue Bao, 1989 Sep, 10(5), 453 - 7
Effects of Tremella polysaccharides on immune function in mice; Xia D et al.; It was found in vitro that Tremella polysaccharides (TP) (50, 100, 150 and 200 micrograms/ml) augmented lymphocyte proliferation induced by Con A and did not antagonize the suppressive effect of hydrocortisone on lymphocyte proliferation . In vivo TP promoted the plaque-forming cell (PFC) response to SRBC in mice . TP 50 and 100 mg/kg ip for 5 d produced 77.6% and 81.8% increases in PFC response respectively . At the doses of 150 and 200 micrograms/ml, TP decreased the interleukin 2 (IL-2) activities in the supernatant of culture media of mouse spleen cells . TP (50 micrograms/ml) enhanced the lymphocyte proliferation induced by Con A and increased the PFC response to SRBC by 47.1% in 14-month-old mice.

Am J Gastroenterol, 1989 Sep, 84(9), 1100 - 2
Eikenella corrodens isolated from a polymicrobial hepatic abscess; Massey BT; A 71-yr-old woman developed a hepatic abscess from which Eikenella corrodens was isolated as part of a polymicrobial flora . This facultatively anaerobic organism is now recognized as a true pathogen . Selective culture media may be required to isolate Eikenella corrodens, and it may be resistant to antibiotics commonly used for anaerobic infections . Special attempts to isolate this organism may be indicated when hepatic abscesses are likely to have resulted from spread of oral or abdominal infections.

J Neurochem, 1989 Sep, 53(3), 912 - 6
Sulfation of rat apolipoprotein E; Gebicke-Haerter PJ et al.; The synthesis of a 37-kilodalton (kDa) protein which has been shown recently to be identical with apolipoprotein E (apo-E) was increased after sciatic nerve injury of the rat . When regeneration of the nerve was allowed, its synthesis returned to control levels at about 8 weeks post injury . In this report it is shown that similar time-course studies of the protein in the rat optic nerve revealed a delayed increase of the protein but a comparably high level of synthesis at 3 weeks post injury . This level was maintained up to at least 18 weeks after crush . Furthermore, two-dimensional electrophoresis revealed that the characteristic "trailing" of the protein is due to its sialylation, because it was reduced after neuraminidase treatment . This treatment, however, detected a neuraminidase-resistant heterogeneous form in CNS tissue and a homogeneous form in peripheral nervous tissue . The trailing persisted up to 18 days of culture of optic nerve explants, of CNS glial cells, and of peritoneal macrophages, but disappeared during the first culture days of sciatic nerve explants and was not observed in Schwann cell culture media . Incorporation studies with 35SO4 revealed that apo-E was the major sulfated protein in culture media conditioned by CNS glial cells, whereas sulfation of the protein was undetectable in Schwann cell cultures . Because macrophages are likely to be the major source of apo-E in both peripheral and central glial cell cultures as well as in injured optic and sciatic nerves, it is hypothesized that resident cells of sciatic nerves secrete potent sulfatases . As a result, sialic acid residues may be more susceptible to degradation.(ABSTRACT TRUNCATED AT 250 WORDS)

Zhonghua Bing Li Xue Za Zhi, 1989 Sep, 18(3), 188 - 90
{The effect of prostaglandin E2 (PGE2) on growth, proliferation, morphology and protein synthesis of aortic smooth muscle cells (SMC) in vitro}; Lei ZZ et al.; SMC are the key element in the pathogenesis of atherosclerotic (AS) plaques . The effect of PGE2 on the growth, proliferation, morphology and protein synthesis of SMC were studied in vitro . SMC from tunica media of aorta of New Zealand white rabbits were used for cultivation . After 7 to 13 passages of subcultures, the cells were divided into control and experimental groups with a dosage of PGE2.5 micrograms,10 micrograms and 20 micrograms per milliliter of medium . After 3 days of successive culture, the cells were prepared for phase contrast microscopy, protein(P) and DNA(D) determination, and electron microscopy(TEM) . Additionally, similar groups of cells were grown on the cover glass for autoradiographic study of cell proliferation by adding 3H-thymidine in culture media . Under TEM, characteristic thin bundles of myofilaments and dense bodies were observed inside the cytoplasm . Mitochondria, Golgi complex,rER were also abundant in the control but not so in the experimental groups . Synchronously with the increase of PGE2 concentration, the P/D value which denotes protein synthesis was significantly decreased from 74.89 +/- 4.68 to 57.01 +/- 3.08, 45.81 +/- 4.61, 32.23 +/- 4.22 and the percentage of 3H-thymidine labelled cells from 37.60 +/- 5.30% to 15.60 +/- 4.20%, 10.18 +/- 3.00%, 3.75 +/- 0.80% respectively . Results showed that PGE2 may act as an inhibitor for growth, proliferation and protein synthesis of aortic SMC in vitro.

Rev Esp Fisiol, 1989 Sep, 45(3), 227 - 33
{Proliferation and liberation kinetics of the tissue polypeptide antigen of MCF-7 cells . Hormonal influence}; Lopez-Gonzalez JD et al.; The presence or absence of tissular polypeptide antigen (TPA) in the culture medium of hormone dependent breast cancer tumoral cells, the relationship between TPA concentration and cellular proliferative activity, have been investigated as well as whether TPA levels change in response to steroid preparations or the action of different antiestrogens . Results show that in MCF-7 cell line culture media, proteins antigenically related to TPA can be detected in concentrations which parallel the number of cells in culture . Consequently, TPA can be considered a cell proliferation marker . Hydroxy-tamoxifen at a concentration of 10(-7) M inhibited both cell proliferation and TPA antigen release, while the relative proportion between number of cells (valued by DNA quantification) and TPA concentration remained unchanged.

Mol Biol (Mosk), 1989 Sep-Oct, 23(5), 1364 - 72
{Properties and conditions of formation of the fibronectin-collagen complex in a culture of human embryo fibroblasts}; Belkin VM et al.; In our previous study the macromolecular complexes mostly consisting of fibronectin and procollagen were isolated from human fibroblast culture media using immobilized antibodies against fibronectin . At present an attempt was made to elucidate at what stage the formation of the fibronectin-collagen complex occurs--either in the course of incubation of the immobilized antibodies with labelled proteins secreted by fibroblasts, or in the extracellular space while labelling fibroblasts, or intracellularly . The results obtained show that the fibronectin-collagen complex: 1) pre-exists even before incubation with the immobilized antibodies and 2) it is of intracellular origin . Thus, the considerable amount of the fibroblast-secreted fibronectin (no less than 20%) is released from the cell not in the free form but in the complex with procollagen . It was suggested that the fibronectin-collagen complex presents a stage in the formation of the insoluble extracellular matrix.

Nippon Hifuka Gakkai Zasshi, 1989 Sep, 99(10), 1059 - 65
{Production of monoclonal antibodies to human T-cell lymphotropic virus type 1 (HTLV-1) and studies of their specificity}; Iwatsuki K et al.; Mouse monoclonal antibodies to HTLV-1 core proteins, p19 and p24, were obtained by hybridoma technique . The specificity was studied by indirect immunofluorescence, enzyme-linked immunosorbent assay (ELISA), dot blot, and Western blot studies . Polyclonal antibodies reactive with HTLV-1 viral proteins were raised by rabbits and guinea pigs . A sandwich ELISA and a dot blot method using these antibodies detected the soluble viral antigens in the ATL cell lysate and ATL culture media . No reaction was observed when IL-2-activated human T-cell lysate and the culture media were used as controls . These data showed that our sandwich ELISA and dot blot systems are available for quantitative analysis of the soluble HTLV-1 viral antigens and contribute to understanding the ability of viral replication by ATL cells.

Mol Cell Endocrinol, 1989 Sep, 66(1), 83 - 91
18-Hydroxylase activity in the Y1 adrenal cell line; Ramirez LC et al.; 18-Hydroxylase activity, reported here for the first time in the mouse adrenal tumor cell line (Y1), was expressed in the metabolism of 11-deoxycorticosterone (DOC) and corticosterone (B) . Detected after 24 h of incubation, it was more evident after 48 h and produced mostly 18-hydroxy-20 alpha-DHB from these exogenous substrates . However, 18-hydroxylation was quantitatively less significant than the metabolism of 20 alpha-reduction and 11 beta-hydroxylation (of DOC) . The latter is also the predominant metabolism of progesterone in this cell line, during the conversion of cholesterol from the serum-supplemented culture media . The cytochrome P-450 11 beta activity of the Y1 cells is similar to that of the mouse in vivo which catalyzes the production of an 11 beta 18-dihydroxylated metabolite as the principal 18-hydroxylated steroid . It is different from that of other species, such as the rat and the bovine, both in terms of the ratio of 11 beta- to 18-hydroxylated metabolites and of the structure of these metabolites.

Vopr Med Khim, 1989 Sep-Oct, 35(5), 103 - 8
{Changes in the cAMP levels and acid phosphatase activity in a monolayer primary culture of hepatocytes from newborn rats during anoxia and substrate deprivation}; Nikulina SE et al.; Acid phosphatase activity and cAMP level were studied in primary hepatocyte culture of new born rats under conditions of anoxia and substrate deprivation (incubation of the cells in Hanks salt solution) . Incubation of hepatocytes in Hanks salt solution within one hour under conditions of anoxia caused a significant increase in free (cytosolic) enzyme activity . Substitution of Hanks salt solution by normal tissue culture medium and reoxygenation after 1 hr anoxia resulted in a decrease of free acid phosphatase activity, whereas activity of the enzyme in lysosomal fraction was increased . Content of cAMP was decreased distinctly after 15 min incubation of hepatocyte culture under conditions of anoxia and substrate deprivation and was increased above control values within 5 min of reoxygenation and substitution of Hanks salt solution by normal tissue culture media . Addition of cAMP-containing liposomes to hepatocyte culture under these experimental conditions led to a decrease in free acid phosphatase activity and to an increase of the enzyme activity in lysosomal fraction . cAMP appears to modulate the lability of hepatocyte lysosomal membrane . The mechanisms involved in these processes are discussed.

Br J Cancer, 1989 Sep, 60(3), 343 - 50
Partial protection of oncogene, anti-sense oligodeoxynucleotides against serum nuclease degradation using terminal methylphosphonate groups; Tidd DM et al.; Under certain circumstances sequence-specific inhibition of gene expression may be achieved in intact cells using exogenous anti-sense oligodeoxynucleotides . The efficacy of this approach to investigating gene function is limited in part by the rapid serum nuclease mediated degradation of oligodeoxynucleotides in culture media . In order to determine the relative contributions of 3'-exonuclease, 5'-exonuclease and endonuclease activity in fetal calf serum to oligodeoxynucleotide destruction, we have tested chimeric N-ras anti-sense sequence molecules protected against exonuclease attack with terminal methylphosphonate diester linkages . An 18-mer with two methylphosphonate diester linkages at the 3'-terminus, a 20-mer with two methylphosphonate diester groups at both ends, and the 16-mer 3'-methylphosphonate monoester components of their respective piperidine hydrolysates were totally resistant to venom phosphodiesterase, whereas the 16-mer 3'-hydroxyl components of the hydrolysates were rapidly degraded . Both the chimeric oligodeoxynucleotides and 3'-methylphosphonate monoesters were considerably more stable than normal 3'-hydroxyl oligodeoxynucleotides at 37 degrees C in McCoy's 5A medium containing 15% heat inactivated fetal calf serum . Typically 20-30% of the former (initial concentration 10-100 microM) remained intact at 20 h as compared to the latter which were 88-100% degraded in 4 h and undetectable at 20 h . We conclude that a 3'-phosphodiesterase activity is a predominant nuclease responsible for oligodeoxynucleotide degradation by fetal calf serum, and that for cell culture studies, significant protection of oligodeoxynucleotides may be achieved by incorporating 3'-terminal methylphosphonate diester or even monoester end groups.

J Clin Invest, 1989 Sep, 84(3), 863 - 75
Inhibition of tumor cell growth by interferon-gamma is mediated by two distinct mechanisms dependent upon oxygen tension: induction of tryptophan degradation and depletion of intracellular nicotinamide adenine dinucleotide; Aune TM et al.; Growth of a variety of human tumor cell lines is inhibited by interferon-gamma (IFN-gamma) in vitro . This mechanism is not well understood . The present experiments identify two separate mechanisms which account for the growth inhibitory activity of IFN-gamma . Cell lines most sensitive to IFN-gamma (inhibited by 10-30 U/ml IFN-gamma in 3 d) were stimulated by IFN-gamma to oxidize tryptophan in media to kynurenine and completely eliminated tryptophan from the culture media after 48-72 h . Addition of L-tryptophan, but not other aromatic amino acids, other essential amino acids, or D-tryptophan, prevented inhibition of cell growth by IFN-gamma . The amount of IFN-gamma required to yield 50% inhibition of cell growth was directly related to the concentration of L-tryptophan in culture media and increased from approximately 3 to 600 U/ml as the concentration of tryptophan in the media was increased from 25 to 1,000 microM . By contrast, inhibition of growth of the cell lines, BT20 and HT29, was not prevented by addition of tryptophan . Inhibition by IFN-gamma (100-300 U/ml after 5-6 d) was, however, completely prevented by addition of two inhibitors of adenosine diphosphate-ribosyl transferase (ADP-RT), 3-aminobenzamide or nicotinamide . Activity of ADP-RT was increased in these cell lines after addition of IFN-gamma . ADP-RT catalyzes the incorporation of the ADP moiety of nicotinamide adenine dinucleotide (NAD) into proteins and causes depletion of intracellular NAD . All tumor cell lines tested had reduced levels of intracellular NAD after treatment with IFN-gamma and loss of NAD preceded inhibition of cell growth by 12-24 h . Inhibitors of IFN-gamma-mediated inhibition of cell growth prevented loss of levels of intracellular NAD . Generation of reactive oxygen species lead to DNA strand breaks which result in activation of ADP-RT . Increased DNA strand breaks were induced in BT20 and HT29 cells but not ME180 and A549 cells after culture with IFN-gamma . The two enzymes known to catalyze the decyclization of tryptophan to kynurenine require superoxide anion for activity . Increased amounts of superoxide anion were released from ME180 and A549 cells after culture with IFN-gamma . Reduced oxygen concentration decreased the ability of IFN-gamma to inhibit tumor cell growth in vitro . Intracellular glutathione has been shown to protect cells against oxidative damage by various agents . Elevation or reduction of intracellular glutathione concentrations lowered or raised sensitivity of cell lines to IFN-gamma, respectively . These data indicate that at least two distinct mechanisms can account for IFN-gamma-madiated inhibition of tumor cell growth . Both mechanisms appear to be sensitive to oxygen tension and to changes in intracellular glutathione concentrations, and both mechanisms lead to loss of intracellular NAD.

J Biol Chem, 1989 Aug 5, 264(22), 13150 - 6
The structure of avian type XII collagen . Alpha 1 (XII) chains contain 190-kDa non-triple helical amino-terminal domains and form homotrimeric molecules; Dublet B et al.; The monoclonal antibody 75d7, specific for type XII collagen (Sugrue, S.P., Gordon, M.K., Seyer, J., Dublet, B., van der Rest, M., and Olsen, B . R . (1989) J . Cell Biol., in press), was used to characterize the intact form of type XII collagen from chick embryo leg tendons . On an immunoblot of a 6% polyacrylamide gel of tendon extracts, one sharp band is recognized by the antibody at Mr = 220,000, while two fuzzy and poorly resolved bands are seen at Mr = 270,000 and Mr = 290,000 . By immunoprecipitation of radiolabeled tendon culture media and electrophoresis of the precipitated material, bands with the same mobilities are observed, indicating that type XII collagen is not proteolytically processed in the extracellular space . Type XII collagen was extracted from tendons with 1 M NaCl in a Tris-HCl buffer and partially purified by concanavalin A-Sepharose and gel permeation chromatographies, using dot immunoblots to monitor the purification . Fractions highly enriched in bacterial collagenase-sensitive proteins with the same electrophoretic properties as type XII collagen were obtained . These fractions did not stain with Alcian blue and neither they nor the immunostained type XII collagen were affected by chondroitinase ABC digestion, indicating that type XII collagen is not a proteoglycan . A disulfide-bonded trimeric CNBr peptide was isolated by affinity chromatography on an antibody column and further purified by gel electrophoresis . Its NH2-terminal amino acid sequence was shown to be unique, demonstrating that type XII collagen is a homotrimer {alpha 1 (XII)}3 . After bacterial collagenase digestion, both the immunopurified radiolabeled preparation and the purified tendon extract fraction showed by gel electrophoresis the presence of a large disulfide-bonded, 3 x 190-kDa, collagenase-resistant domain . Rotary shadowing and electron microscopy of the purified type XII fraction demonstrated that the molecule has the structure of a cross consisting of a 75 nm collagenase-sensitive tail, a central globule, and three 60 nm arms each ending in a small globule . After heat denaturation and renaturation, only a very large globule can be seen, attached to the triple helical tail . These results show that type XII collagen has a unique structure and is different from the other matrix constituents described so far.

Biol Reprod, 1989 Aug, 41(2), 347 - 54
Immunological characterization and immunocytochemical localization of a progesterone-dependent cat endometrial secretory protein; Verhage HG et al.; The major objective of this study was to make a polyclonal antibody to a previously described group of progesterone (P)-dependent low molecular weight secretory proteins of the cat uterus . Proteins present in uterine flushings obtained from a P-treated cat were partially purified using Sephadex G-75, separated on two-dimensional polyacrylamide gels, and transferred to nitrocellulose membranes . The region containing the polypeptides were cut out, solubilized in dimethyl sulfoxide, mixed with Freund's adjuvant, and injected at 2-wk intervals into a male rabbit . The antiserum used in this study was obtained 8 wk after the initial injection and crossreacted with antigens on Western blots of uterine flushings and uterine culture medium obtained from ovariectomized estradiol (E2)-primed cats treated with P, and from pregnant preimplantation animals . Polypeptides in three molecular weight regions crossreacted with the antisera (Mr approximately equal to 28,000, pI 5.5 6.0; Mr approximately equal to 36,000, pI 6.0 6.5; Mr approximately equal to 41,000, pI 5.5 6.0), and each region consisted of several isoelectric variants . The Mr approximately equal to 28,000 proteins were the dominant form observed in culture media, and the Mr approximately equal to 36,000 proteins were the major form present in uterine flushings . The antigens were not detected in uterine flushings or culture medium obtained from ovariectomized, E2-treated, and estrous animals . The antigens were also absent in serum and other reproductive and nonreproductive tract tissues . Immunocytochemical analysis demonstrated that antigen staining was limited to the epithelial cells of the deeper uterine glands of the P-dominated animal . Immunoperoxidase staining was diffuse throughout the cytoplasm of these epithelial cells . Thus, the epithelial cells of the uterine glands in the P-dominated cat synthesize and secrete a complex group of P-dependent, uterine-specific proteins that may have potential functional significance during early blastocyst development and implantation.

Liver, 1989 Aug, 9(4), 242 - 9
Human hepatic sinusoidal endothelial cells in culture produce von Willebrand factor and contain Weibel-Palade bodies; Harrison RL et al.; Human hepatic sinusoidal endothelial cells were derived from cadaveric human livers . Cells were grown in culture for several weeks to produce small patches of confluent endothelial cells . The ultrastructure of sinusoidal endothelial cells was examined, cell monolayers were stained immunocytochemically for von Willebrand factor antigen, and antigen in cell culture media was measured by enzyme-linked immunosorbent assay . Human hepatic sinusoidal endothelial cells contained von Willebrand factor antigen and Weibel-Palade bodies, were fenestrated, and released von Willebrand factor antigen into media in a time-dependent manner . Although in some respects human hepatic endothelial cells were different from vascular cells, there was no evidence that there were qualitative differences in their capacity to produce von Willebrand factor.

Neurosurgery, 1989 Aug, 25(2), 196 - 201
Cyst fluids of malignant human brain tumors contain substances that stimulate the growth of cultured human gliomas of various histological type; Westphal M et al.; The contents of 14 cysts that were located within human intracranial tumors were obtained at surgery by needle aspiration . These tumor cyst fluids (TCFs) were mostly derived from glial tumors (10 cases) . TCFs from one metastasis from a mammary carcinoma, one cystic meningioma, one hemangioblastoma, and a cystic acoustic neurinoma were also included . These TCFs were added to primary cultures of human gliomas, established human glioma cell lines, and normal human arachnoid cells in culture . The presence of proliferation-promoting factors in all cyst fluids could be demonstrated . On the basis of the response patterns of the cultures, it was possible to distinguish different levels of growth autonomy and growth factor sensitivity among these cultures and to speculate about varying degrees of cellular autocrine activation . The TCFs appear to contain factors that are not normally present in fetal calf serum, which is a regular constituent of most cell culture media . Some primary cultured cells as well as cell lines react in an oversensitive manner to the addition of TCFs.

J Clin Endocrinol Metab, 1989 Aug, 69(2), 259 - 66
Effects of human and salmon calcitonin on human articular chondrocytes cultivated in clusters; Franchimont P et al.; The effects of different pharmacological concentrations (0, 5, 10, 100, and 1000 ng/mL) of synthetic human calcitonin (hCT) and salmon calcitonin (sCT) on the incorporation of {3H}thymidine and production of proteoglycans (PG) and type II collagen (coll II) by human articular chondrocytes during a 20-day period were studied in a tridimensional chondrocyte culture model . {3H}Thymidine uptake was measured in chondrocyte clusters, and specific PG and coll II RIAs were performed every 4 days on the culture medium and cell aggregates; total PG and coll II production were also assessed at different culture durations by adding the amounts found in culture media and their corresponding clusters . Incubation with hCT or sCT did not affect {3H}thymidine uptake regardless of the dose . For each culture period, PG and coll II release into culture medium, cluster content, and total production increased significantly in a dose-dependent manner . Cumulative curves for these parameters showed a progressive significant increase with culture duration at hCT and sCT doses of 0, 5, and 10 ng/mL . Cumulative curves obtained with 10, 100, and 1000 ng/mL were seldom significantly different from one another . No differences emerged between the use of hCT or sCT . Thus, CT exerted no proliferative effect on human articular chondrocytes in tridimensional culture, but displayed a dose-dependent and prolonged stimulatory effect on PG and coll II production . CT may possess chondroprotective properties in addition to its other known effects.

J In Vitro Fert Embryo Transf, 1989 Aug, 6(4), 213 - 7
Human amniotic fluid for fertilization and culture of human embryos: results of clinical trials in human in vitro fertilization (IVF) programs; Gianaroli L et al.; We report the outcome of clinical trials carried out in two IVF programs, comparing the use of human amniotic fluid (HAF) as a complete medium to Whittingham's T6 medium containing human serum (T6 + 10% HS) for egg incubation, insemination, embryo culture, and embryo transfer . There were no significant differences in the clinical trials between HAF used alone as a complete medium and T6 + 10% HS in fertilization rates of eggs, cleavage rates of embryos up to 48 hours in culture, pregnancy success rates after embryo replacement or the outcome of pregnancies . There was no advantage in using T6 + 10% HS for fertilization of eggs and HAF as a complete medium for embryo culture and transfer in any of the parameters examined . We conclude that HAF does not meet the complete requirements of human eggs and embryos in vitro and further developments of culture media are required to obtain embryo development equivalent to that in vivo.

Mol Endocrinol, 1989 Aug, 3(8), 1197 - 206
Differential effects of estrogen on luteinizing hormone-releasing hormone gene expression in slice explant cultures prepared from specific rat forebrain regions; Wray S et al.; Five serially sectioned tissue slices (400 microns) from the preoptic area/hypothalamus of postnatal day 4 rats were cultured using a slice explant roller culture technique . After 18 days in culture, these slices thinned sufficiently to allow immunocytochemical and in situ hybridization histochemical assays for LHRH peptide and LHRH mRNA, respectively . Large numbers of neurons containing mRNA encoding LHRH were detected in these slices using in situ hybridization histochemistry (ISHH) . These 35S-labeled cells were distributed in the cultured slices in a pattern similar to that found with LHRH immunocytochemistry and ISHH in vivo, indicating that LHRH neurons were maintained in these cultures in an organotypic manner . Densitometric single cell analyses after ISHH of the culture slices were performed using a Loats image analysis system, so as to provide a density value per cell (density/cell) . Comparisons of these density values from the slice explants cultured in presence or absence of 10(-7) M estradiol found that: 1) under basal (control) culture conditions there were no consistent differences in the frequency distributions of the density/cell values between all the five slices derived from either male or female rats, 2) mean density/culture values under control conditions did not differ significantly between slices and sexes, 3) the presence of estradiol in the culture media resulted in an overall decrease in density/cell values, with the most significant decrease occurring in slice 3 which is comparable to the level of the organum vasculosum lamina terminalis/rostral preoptic area (OVLT/rPOA) in vivo, and 4) this decrease in density/cell values in slice 3 due to estradiol treatment, was greater in cultures derived from female vs . male tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Jpn J Pharmacol, 1989 Aug, 50(4), 495 - 8
Effects of short chain fatty acids on the production of heat-labile enterotoxin from enterotoxigenic Escherichia coli; Takashi K et al.; Each addition of some short chain fatty acids (SCFAs) into casamino acids-yeast extract culture media at a concentration of 2 mg/ml reduced the production of heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli in proportion to the elongation of carbon chain from C-2 to C-7 . The LT-production was inversely recovered by the addition of longer chain fatty acids . The reduction of LT-production by SCFAs seems to depend on the disturbance of the biosynthesis of LT itself, since LT was not detected in the cells treated with n-heptyric acid at 2 mg/ml, which abolished the LT-production.

Cutis, 1989 Aug, 44(2), 113 - 4
Recent developments in sexually transmitted diseases: chancroid--epidemiology, diagnosis, and treatment; Felman YM; The most important recent developments concerning chancroid have been in its epidemiology (a tremendous increase in the incidence of the disease in the United States over the past several years), diagnosis (better culture media), and treatment (several newer and older drugs that have been shown to be effective against chancroid in recent studies).

Arch Biochem Biophys, 1989 Aug 1, 272(2), 386 - 92
Augmentation of inorganic pyrophosphate elaboration in cartilage by serum factors; Rosenthal AK et al.; The disordered production of inorganic pyrophosphate (PPi) by articular cartilage is thought to have an important role in the pathogenesis of calcium pyrophosphate dihydrate deposition disease and perhaps osteoarthritis . We have previously shown that fetal calf serum added to the culture media of porcine articular cartilage explants increases the elaboration of PPi into the ambient media . We have examined this PPi stimulatory activity by studying the effects of adult human serum (HS), serum derived from adult human plasma (HP), and an acid-alcohol extract of human platelets (PE) on PPi production in cartilage organ culture . Ten percent HS produces a 1.4-fold increase in PPi production after 48 h of culture, while cartilage incubated in media containing 10% HP produces no more PPi than that incubated in media alone . PE stimulates a mean 2-fold increase in PPi production at 48 h in the presence of low concentrations of HP, and has no effect alone . It does not appear to up-regulate the activity of the ectoenzyme nucleoside triphosphate pyrophosphohydrolase (NTPPPH), nor does it promote the release of enzyme substrate into the extracellular space . Cartilage exposed to 0.5% HP and PE has 1.51 +/- 0.36 units of NTPPPH activity whereas cartilage exposed to 0.5% HP alone has 1.52 +/- 0.41 units of enzyme activity . PE does not increase the release of {14C}adenine-labeled compounds into the media . Approximately 13% of soluble 14C counts was found in the media of chondrocytes treated with PE while 18% of counts was released in the presence of HP alone . We have demonstrated a factor or factors present in FCS, HS, and an acid-ethanol extract of human platelets which represent(s) the first known physiologic modulators of PPi production in articular cartilage and may increase PPi production without affecting NTPPPH activity.

Shika Kiso Igakkai Zasshi, 1989 Aug, 31(4), 372 - 8
{In vitro cultivation of human pulpal fibroblast strains--permanent and deciduous teeth}; Tsukamoto Y et al.; We succeeded in separating and the cultivating stable monolayer cultures of dental pulp fibroblast strains derived from permanent and deciduous human teeth . Human permanent (n = 67) and deciduous teeth (n = 26) were extracted under acupuncture anaesthesia for the correction of malocclusion . After splitting the teeth, the pulp tissues were carefully removed, placed in tissue culture flasks, and grown in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum (FCS) . The human pulpal fibroblasts (HPF) of permanent teeth and deciduous teeth (DHPF) were subcultured . Both the HPF and DHPF appeared to migrate from adherent tissues within 24 to 48 hr after explanation . They proliferated in the pulp explants, and lined up in parallel rows of cells closest to the explant tissue within 7 to 10 days in all of the experimental cases . The outgrowing cells were subcultured at 1.3 x 10(4) cells/cm2 in tissue culture flasks every 4-11 days . They showed vigorous proliferation . The average number of cells in the 6-7 day cultures of HPF were 5.6 x 10(4) cells/cm2 from 3 to 16 passages . It was 4.7 x 10(4) cells/cm2 from 3 to 10 passages with DHPF . However, no difference was observed between HPF and DHPF in the amount of synthesized protein in culture flasks . Furthermore, the growth rate of DHPF was more sensitive than that of HPF to the FCS percentages of the culture media.

Nippon Sanka Fujinka Gakkai Zasshi, 1989 Aug, 41(8), 971 - 80; discussion 1000-7
{Fundamental and clinical studies on biochemical properties of endometriosis in comparison with endometrium}; Taketani Y; 1 . Regulatory mechanism of cell growth of endometriosis in comparison with endometrium . Estradiol alone has no growth-promoting effect on both endometriotic and endometrial cells . Epidermal growth factor (EGF) stimulates cell growth of both cell types . Endometrial cells but not endometriotic cells produce and release EGF into culture media so that stimulatory effect of exogenous addition of EGF is blunted in endometrial cells . Estradiol exerts its mitogenic action by enhancing the mitogenic effect of EGF in endometrium . By contrast, the effect of estradiol is minimal in endometriotic cells, showing less dependency on estradiol for their proliferation . Progesterone inhibits cell growth of the both cell types in the same manner . 2 . A biological role of EGF in endometriosis . Endometriotic cells possess EGF receptors . The affinity of the receptor is the same as that of endometrial cells . However, the number of receptor per cell is about half of that for endometrium . Estradiol increases the number of EGF receptors in endometrial cells which may explain the mitogenic effect of estradiol in the face of EGF . However, stimulatory effect of estradiol for EGF receptors is less pronounced in endometriotic cells . Mitogenic action of EGF is suggested to be mediated by phosphorylation of tyrosine residues of 170 kd protein in the tissues . EGF increases the production of tissue plasminogen activator (t-PA) and activates the aromatase activity of the both cell types . However, the stimulatory action of EGF on progestin receptor is observed only in endometrial cells . 3 . Biochemical characterization of endometriotic cells in comparison with endometrial cells . Endometriotic tissues accumulate less amount of glycogen and XIII factor of blood coagulation as compared to endometrial tissues . The ability of endometriotic cells to release prostaglandin is also weaker, suggesting suppressed differentiated function of endometriotic cell . Endometriotic cells produce the same amount of CA125 as endometrial cells . Danazol and EGF inhibit the release of CA125 into culture media when standardized per cell . Therefore, normalization of CA125 levels during the treatment dose not always mean the reduction of the lesions but reflect the suppressed function of the endometriotic tissues . 4 . Altered microenvironment of endometriotic tissues . An analysis of peritoneal fluid . The amount of peritoneal fluid (PF) with endometriosis increased throughout the menstrual cycle . A number of macrophage is reported to increase in PF with endometriosis.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochem Biophys Res Commun, 1989 Jul 14, 162(1), 217 - 23
Concomitant secretion of big endothelin and its C-terminal fragment from human and bovine endothelial cells; Emori T et al.; A specific radioimmunoassay (RIA) for the carboxyl-terminal fragment (CTF) of big porcine endothelin (pET), an intermediate form of pET, was established to characterize big ET-like and its CTF-like immunoreactivity (LI) secreted from cultured bovine and human endothelial cells (EC) . The antibody used crossreacted equally with big pET(1-39) and its CTF(22-39), but not with pET(1-21) . Serial dilution curves of the culture media from bovine and human EC were parallel to that of standard CTF . Reverse-phase HPLC coupled with RIAs for big ET and ET of the culture media from bovine and human EC revealed essentially the same elution profiles: two major CTF-LI components, one corresponding to big pET(1-39) and the other to its CTF(22-39), in addition to one major ET-LI component corresponding to pET(1-21) . The amounts of CTF-LI were almost equal to that of ET-LI on a molar basis . These data suggest that big ET is processed by a putative ET converting enzyme to yield its CTF and the mature ET(1-21) in EC.

Vet Clin North Am Food Anim Pract, 1989 Jul, 5(2), 291 - 300
Tremorgenic syndromes in livestock; Nicholson SS; Grasses that are essential components of livestock grazing programs sometimes are the source of tremorgenic toxicants to the animals consuming them . Morbidity can be high but mortality need not be if management closely observes the cattle daily and removes them at first sign of trouble . Specific treatment generally is not available nor needed . Survivors recover completely within a few days or weeks, except in chronic phalaris poisoning, where sheep and cattle may die after prolonged illness--or at least not make an economical recovery . Certain poisonous plants are responsible for tremorgenic signs in livestock and horses . White snakeroot and rayless goldenrod pose a public health risk to individuals who might drink milk from a goat or cow grazing toxic amounts of these weeds . Poisonous weeds and trees often are a local or regional problem, and often are seasonal . A veterinarian new to the area who has a food animal practice should seek out information relative to poisonous plants, nutritional deficiencies, and diseases endemic to the practice area . The ability of certain fungi to produce toxic metabolites in feed-stuffs creates the potential for tremorgenic or other types of toxicosis in most classes of livestock . Wet grain byproducts from ethanol production and other processes can provide the right culture media for fungi.

Am J Respir Cell Mol Biol, 1989 Jul, 1(1), 13 - 20
Bronchial epithelial cells produce lung fibroblast chemotactic factor: fibronectin; Shoji S et al.; The interaction between the epithelial cells and the subjacent mesenchymal cells in the airway is thought to play a major role during tissue repair after airway injury and lung morphogenesis . To evaluate this interaction, we cultured human lung fibroblasts, and bovine and human bronchial epithelial cells, and determined that bronchial epithelial cell-conditioned medium has a chemotactic activity for lung fibroblasts . This activity had the characteristics of protein: it was nondialyzable, heat-labile, pepsin-labile, acid-stable, and lipid-inextractable . Molecular sieve chromatography on Sephadex G-150 and affinity chromatography on gelatin-Sepharose revealed that there was one peak of chemotactic activity in high molecular weight range, which bound to gelatin, thus suggesting that the chemotactic factor might be fibronectin . Production and secretion of fibronectin into the culture media were demonstrated by biosynthetic incorporation of radioactive amino acid into fibronectin followed by immunoprecipitation on SDS-PAGE and autoradiography . Release into the culture medium was confirmed by ELISA . The identity of fibronectin as the chemotactic activity was confirmed by the addition of antifibronectin antibody to the conditioned medium, which inhibited chemotaxis in dose-dependent manner . Thus, bronchial epithelial cells produce fibronectin which can function as a chemotactic factor for lung fibroblasts . This production of fibronectin by bronchial epithelial cells may play an important role in regulating interaction between the bronchial epithelial cells that line the lumenal surface of the bronchial epithelial wall and the mesenchymal fibroblasts that underlie the bronchial epithelial basement membrane.

Diagn Microbiol Infect Dis, 1989 Jul-Aug, 12(4), 303 - 8
Evaluation of four mycobacterial blood culture media: BACTEC 13A, Isolator/BACTEC 12B, Isolator/Middlebrook agar, and a biphasic medium; Agy MB et al.; Four commercially available mycobacterial blood culture systems were compared for sensitivity and time to detection of growth . A 5-ml volume of SPS-anticoagulated blood was cultured in a BACTEC 13A vial and a modified M7H11/BHI biphasic medium . In addition, two aliquots of Isolator concentrates, each derived from 5 ml of blood, were inoculated into a BACTEC 12B vial and onto a pair of Middlebrook 7H11 agar plates (M7H11) . Mycobacteria were recovered from 32 of 180 cultured specimens (17.8%) . Growth was detected in 30 (93.7%) of the 13A vials, 27 (84.4%) of the M7H11 agar plates, 26 (81.2%) of the 12B vials, and 14 (43.8%) of the biphasic bottles . The mean times to growth detection in the 13A vial (14.2 days) and the 12B vial (13.7 days) were shorter than in either the M7H11 plates (20.8 days) or the biphasic medium (24.1 days) . When the Isolator/12B vial-and-M7H11 plates were evaluated as a single system, 29 cultures (90.6%) had a mean time to growth detection of 13.5 days . Colony-forming units per ml were inversely associated with time to growth detection . Delay in transport (greater than 24 h) appeared to reduce viability . The direct inoculation feature makes the 13A vial very suitable for mycobacterial blood cultures.

J Neurochem, 1989 Jul, 53(1), 297 - 9
Direct evidence that excitotoxicity in cultured neurons is mediated via N-methyl-D-aspartate (NMDA) as well as non-NMDA receptors; Frandsen A et al.; Cultured GABAergic cerebral cortex neurons were exposed to the excitatory amino acid (EAA) L-glutamate, kainate (KA), N-methyl-D-aspartate (NMDA), or RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolopropionate (AMPA) . To ensure a constant glutamate concentration in the culture media during the exposure periods, the glutamate uptake inhibitor L-aspartic acid beta-hydroxamate was added at 500 microM to the cultures that were exposed to glutamate . Each of these EAAs was able to induce neurotoxicity . It was not possible to reduce or prevent glutamate-induced cytotoxicity by blocking only one of the glutamate receptor subtypes with either the NMDA receptor antagonist D-(-)-2-amino-5-phosphonopentanoate (APV) or with one of the specific non-NMDA antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX) . However, if the cultures were exposed simultaneously to glutamate and the antagonists in combination, i.e., APV plus CNQX or APV plus DNQX, the toxicity was completely prevented . Furthermore, CNQX and DNQX were shown to be selective blockers of cytotoxic phenomena induced by non-NMDA glutamate agonists with no effect on NMDA-induced cell death . Likewise, APV prevented NMDA-induced cell death without affecting the KA- or AMPA-induced neurotoxicity . It is concluded that EAA-dependent neurotoxicity is induced by NMDA as well as non-NMDA receptors.

J Endocrinol Invest, 1989 Jul-Aug, 12(7), 443 - 48
Effects of growth hormone-releasing hormone (GHRH) on densely granulated somatotroph adenomas and sparsely granulated somatotroph adenomas in vitro: a morphological and functional investigation; Kawakita S et al.; The effects of growth hormone-releasing hormone (GHRH) were studied on densely granulated somatotroph adenoma cells and sparsely granulated somatotroph adenoma cells in culture by measuring release of growth hormone (GH) as well as ultrastructural morphometrical parameters and comparing them with those of control adenoma cells . Both types of adenoma cells cultured with GHRH showed similar increases of GH release into culture media and exhibited similar increases in cytoplasmic volume densities (CVD) of endoplasmic reticulum and Golgi apparatus and decreases in CVD of secretory granules and secretory granule diameter . These results indicate that (1) both types of somatotroph adenoma cells react similarly to GHRH stimulation, despite their morphologic differences, and (2) GHRH stimulates GH synthesis as well as GH release by somatotroph adenoma cells.

Rev Soc Bras Med Trop, 1989 Jul-Sep, 22(3), 119 - 24
Usefulness of serology for the evaluation of Trypanosoma cruzi transmission in endemic areas of Chagas' disease; Chuit R et al.; Thirteen communities from 7 Argentinian provinces were selected for the evaluation of serology as an indicator of transmission of Chagas disease . Of the communities appraised, 6 did not have a history of previous treatment with insecticides and 7 had received sporadic or continuous insecticide treatment . The inhabitants of 20% of the houses of each locality were studied by serology . The samples were obtained by finger pricking and 50 microliters of blood were mixed with 15 microliter of 50% glycerine solution in tissue culture media to be assayed by Indirect Hemagglutination and Indirect Immunofluorescence tests . In untreated areas, the prevalence of infection in infants 0-4 years old was 17.5%, reaching to over 22% for the 5-9 year old group, and to 33.3% in 10-14 year old individuals . The prevalence in treated and surveyed areas was 2.6% in 0-4 year old children, 5.4% in 5-9 year old and 6.2% in 10-14 year old youngsters . The differences between both areas were statistically significant (p less than 0.005) . This study favors serology as a valid indicator for the evaluation of transmission of Chagas disease in rural areas.

J Invest Dermatol, 1989 Jul, 93(1), 10 - 7
Murine keratinocyte cultures grown at the air/medium interface synthesize stratum corneum lipids and "recycle" linoleate during differentiation; Madison KC et al.; In a recent investigation we showed that murine keratinocyte cultures grown at the air/medium interface in the presence of dermis exhibit morphologic differentiation comparable to that seen in vivo, including the formation of lamellar granules and stratum corneum intercellular lipid lamellae . In the present study, lifted cultures were found to more closely reproduce the lipid composition of the parent epidermal tissue than submerged cultures grown on plastic . In addition, the specific fatty acid profile of individual lipid classes in lifted cultures was, in general, remarkably well maintained in vitro . Acylceramides, which are highly enriched in linoleic acid in vivo, remained enriched in vitro; however, the linoleic acid content of the cultures was substantially lower than that in vivo, confirming previous reports of the relative essential fatty acid deficiency of standard culture media . As the lifted cultures differentiated over time, the lipid composition changed to reflect the formation of a stratum corneum with its different complement of lipids . Label from {U-14C}linoleic acid was specifically incorporated into linoleate-containing lipids during short pulses in both submerged and lifted cultures . Changes in label distribution over a long chase period in lifted cultures indicated that linoleate was transferred from phospholipids to ceramides, providing evidence for the "recycling" of essential fatty acids in epidermis.

Brain Res, 1989 Jun 19, 490(1), 14 - 25
Endogenous opioid systems regulate growth of neural tumor cells in culture; Zagon IS et al.; Endogenous opioid systems (i.e., opioids and opioid receptors) play a role in neural cancer . Using a tissue culture system of S20Y murine neuroblastoma to assess the effects of opioids on growth, {Met5}-enkephalin was the most potent compound to influence cell replication . With a median effective concentration of 10(-10) M, this peptide inhibited cell proliferation in a stereospecific and naloxone-reversible manner . {Met5}-Enkephalin depressed both DNA synthesis and mitosis . {Met5}-Enkephalin was detected in neuroblastoma cells by radioimmunoassay, and was found to increase in concentration in culture media over time, suggesting that these cells produced the peptide . Immunocytochemistry showed {Met5}-enkephalin-like activity in the cortical cytoplasm, but not the cell nucleus, of neuroblastoma cells . Binding of {3H}-{Met5}-enkephalin specific and saturable, and Scatchard analysis yielded a Kd of 1.2 +/- 0.1 nM and a binding capacity of 50.2 +/- 4.3 fmol/mg protein . {Met5}-Enkephalin also depressed the growth of N115 murine neuroblastoma, SK-N-MC human neuroblastoma, and HT-1080 human fibrosarcoma . These results indicate that {Met5}-enkephalin, a naturally occurring pentapeptide that is derived from proenkephalin A, is a potent inhibitor of cell growth . Since cancer cells produce {Met5}-enkephalin, and contain a binding site to this ligand, endogenous opioid systems appear to control cell proliferation by an autocrine mechanism.

FEBS Lett, 1989 Jun 19, 250(1), 85 - 90
A new family of growth factor-like peptides . 'Trefoil' disulphide loop structures as a common feature in breast cancer associated peptide (pS2), pancreatic spasmolytic polypeptide (PSP), and frog skin peptides (spasmolysins); Thim L; Four peptides present in completely different biological sources have been shown to exhibit a large degree of structural similarity . The peptides include: (i) a 60 amino acid residue breast cancer associated pS2 peptide isolated from human gastric juice and the culture media of the human breast cancer cell line MCF-7; (ii) a 106 amino acid residue pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas and pancreatic juice; and (iii) a 49 and 50 amino acid residue peptide predicted from a cDNA isolated from the skin of the frog, Xenopus laevis . These peptides are characterized by having one (pS2 and the frog peptides) or two (PSP) domains of a highly conserved 38-39 amino acid residue consensus sequence not found in any other known peptides or proteins . The domain sequences contain 6 cysteine residues in nearly the same positions and it is suggested that these 6 residues are linked by 3 disulphide bonds to form a characteristic 'trefoil' disulphide loop structure common in all four peptides . From the sources of which the peptides have been isolated and from experiments showing that PSP has a growth factor stimulatory effect on MCF-7 cells, it is further suggested that these peptides may represent members of a new family of growth factors.

Thromb Res, 1989 Jun 15, 54(6), 655 - 75
Biosynthesis of factor V by normal adult rat hepatocytes; Mazzorana M et al.; The synthesis of coagulation factor V was investigated in isolated rat hepatocytes maintained in long-term primary culture . Two culture conditions were compared . A clotting assay and an immunoprecipitation experiment with rabbit anti rat factor V IgG were used to demonstrate not only the presence of factor V in the cells but also active secretion into the culture medium . Both the inhibition of the clotting reaction in presence of the antibody and absence of thrombin in culture media confirmed the specificity of the clotting assays . Electron microscopic examination located factor V in the endoplasmic reticulum and Golgi apparatus of hepatocytes in common with other liver specific plasma proteins . Examination of liver tissue sections confirmed the production of factor V in hepatocytes but not in hepatic endothelial cells although it did not exclude a transit pathway of factor V through these cells . Addition of Russell viper venom factor V activating enzyme to the culture medium had no effect on the factor V activity . In contrast, treatment of cell extracts did increase the coagulant activity . This suggests that hepatocytes contained principally an unactivated form or procofactor, whereas factor V present into the culture medium was mainly in an activated form . These data provide evidence for synthesis and secretion of an hepatocytic factor V.

J Immunol, 1989 Jun 15, 142(12), 4372 - 7
Expression of a binding structure for sialic acid-containing glycoconjugates on rat bone marrow-derived macrophages and its modulation by IFN, TNF-alpha, and dexamethasone; Gessl A et al.; Rat macrophages express a binding structure for sialic acid-containing glycoconjugates (sialic acid-binding receptor, SAR) which can be detected by a rosette assay utilizing SRBC coated with bovine brain gangliosides (E-G) . Freshly isolated rat bone marrow cells (BMC) contain about 5% SAR-positive cells . Rat BMC cultured for 1 wk with tissue culture media containing CSF-1 differentiate into a virtually pure population of bone marrow-derived macrophages (BMDM phi) . All BMDM phi bound E-G coated with an optimal concentration of gangliosides (100 micrograms/ml) . When BMC were cultured for 1 wk with murine recombinant granulocyte-macrophage CSF, irrespective of the dose of GM-CSF, approximately 90% of the cells were identified as rat macrophages, and practically all expressed SAR . Only about 50% of BMDM phi bound SRBC coated with a suboptimal concentration of gangliosides (20 micrograms/ml) . However, this percentage increased markedly after 8 to 72 h incubation with 1 to 10,000 U/ml purified murine IFN-alpha or IFN-beta, whereas murine or rat rIFN-gamma at doses above 10 U/ml led to a decrease of E-G binding . Human and murine rTNF-alpha enhanced rosette formation in a dose-dependent manner . These effects could be blocked by the respective anti-cytokine antibodies . Treatment of BMDM phi with dexamethasone also augmented E-G rosetting . The enhancement of E-G binding was abolished by pretreatment of BMDM phi with cycloheximide and actinomycin D but not with mitomycin C, suggesting that de novo synthesis of protein and RNA, but not DNA, is required . Our results demonstrate that all rat BMDM phi constitutively bear SAR, and that murine IFN-alpha, IFN-beta, and TNF-alpha, as well as dexamethasone, may augment SAR expression.

Mol Biochem Parasitol, 1989 Jun 1, 35(1), 73 - 8
Trichomonas vaginalis and Tritrichomonas foetus secrete neuraminidase into the culture medium; Costa e Silva Filho F et al.; Supernatants taken from axenic cultures of Trichomonas vaginalis and Tritrichomonas foetus contain a neuraminidase activity, the detection of which is augmented when the trichomonad culture media are supplemented with 30% supernatant of confluent epithelial cultures . The enzyme was active against human erythrocytes, which became highly reactive to peanut agglutinin lectin . The specificity of the enzyme was checked by using a substrate specific to neuraminidase: 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuramic acid.

Blood, 1989 Jun, 73(8), 2074 - 6
Multimeric composition of endothelial cell-derived von Willebrand factor; Tsai HM et al.; The multimeric composition of human endothelial cell (EC)-derived von Willebrand factor (vWF) was studied using SDS-agarose gel electrophoresis and autoradiography . Two multimers were found in lysates prepared from confluent cultures of human umbilical vein endothelial cells . The smaller multimer had a molecular weight (mol wt) of approximately 950 Kd, while the second was larger than those seen in plasma . When electrophoresis was performed using the discontinuous buffer system of Ruggeri and Zimmerman, the small multimer consisted of a single band migrating with the slowest-moving component of the corresponding plasma triplet . The large EC-vWF multimer was detected in culture media conditioned with EC monolayers for ten minutes . It remained the only multimer in media conditioned for up to three days . Calcium ionophore A23187 increased the amount of the large vWF multimer released into the culture media, but did not change its multimeric composition . The small multimer was never detected in the EC-conditioned media . These findings suggest that (1) a large, fully polymerized multimer of vWF is released from the ECs, while the small multimer probably represents a major intermediate component in the process of multimerization, and (2) plasma vWF multimers are probably generated from the large endothelial vWF after it is released into the circulation.

J Endocrinol, 1989 Jun, 121(3), 513 - 9
Indomethacin inhibits the effects of oestrogen in the anterior pituitary gland of the rat; Rosental DG et al.; Two inhibitors of prostaglandin synthesis, indomethacin and aspirin, blocked the increase of oestrogen-binding sites in the nuclear subcellular fraction, an increase which occurs after the administration of oestradiol . Consequently the biological effects of oestrogens in the anterior pituitary gland of the rat (prolactin synthesis, concentration of progesterone-binding sites and cell proliferation) are diminished . The anterior pituitary gland synthesized prostaglandin F2 alpha (PGF2 alpha), PGE2 and PGD2 from arachidonic acid . This synthesis was blocked when indomethacin was added to the culture media . Oestrogen increased the concentration of PGE2: an increase that was partially prevented by indomethacin . Prostaglandins may have an important role on the effects of oestrogen in the anterior pituitary gland of the rat.

Int J Dev Biol, 1989 Jun, 33(2), 267 - 75
Production of fibronectin and collagen types I and III by chick embryo dermal cells cultured on extracellular matrix substrates; Robert J et al.; Dermal cells isolated from the back skin of 7-day chick embryos were cultured on homogeneous two-dimensional substrates consisting of one or two extracellular matrix components (type I, III, or IV collagen, fibronectin and several glycosaminoglycans (GAGs): hyaluronate, chondroitin-4, chondroitin-6, dermatan and heparan sulfates) . The effect of these substrates on the production of fibronectin, of types I, III and IV collagen by cells was compared with that of culture dish polystyrene . Using immunofluorescent labeling of cultured cells, it was observed that, on all substrates, in 1-day and 7-day cultures, 85 to 95% of cells contain type I collagen in the perinuclear cytoplasm; label was absent from cell processes . Type I collagen was also detected in extracellular fibers extending between neighboring cells . By contrast, on all substrates, only 5 to 20% of cells produced type III collagen . Otherwise distribution of type III collagen was similar to that of type I collagen . With anti-type IV collagen antibody no staining of either cell content or extracellular spaces was detected . Staining with anti-fibronectin antibody revealed two types of distribution patterns . On polystyrene and on all but type I collagen substrates, labeling revealed clusters of short thick strands and patches of fibronectin-rich material in extracellular spaces . On type I collagen substrate, however, immunostaining revealed a delicate network of regularly spaced parallel fibrils of fibronectin extending between and along cells . Using quantitative radioimmunoassay of the culture media, it was shown that, after 7 days of culture, cells secreted more type I than type III collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Tissue Res, 1989 Jun, 256(3), 487 - 94
The effect of low- and high-density lipoprotein cholesterol on steroid hormone production and ACTH-induced differentiation of rat adrenocortical cells in primary culture; Heikkila P et al.; We studied the effects of lipoprotein-derived cholesterol on the ACTH-induced differentiation of cultured fetal rat adrenocortical cells . For this purpose human plasma high-density lipoprotein3 (HDL3) or low-density lipoprotein (LDL) was added to culture media devoid of cholesterol, and thereafter the morphological changes in cells were monitored and the amounts of steroids synthesized were measured . It could be demonstrated that, ultrastructurally, upon ACTH-stimulation the adrenocortical cells differentiated into fasciculata-like cells even in the absence of lipoproteins in the culture medium . The addition of either HDL3 or LDL caused an increase in the number and size of cytoplasmic lipid droplets suggesting uptake and deposition of lipoprotein-derived cholesterol into the differentiating cells . The amount of steroids secreted from cells differentiating in media devoid of cholesterol was only half that observed in cells differentiating in serum-supplemented medium . Addition of either HDL3 or LDL increased the ACTH-stimulated steroid synthesis to the levels observed in serum-supplemented medium . This study demonstrates that both HDL3 and LDL are able to provide cholesterol for steroid synthesis accompanying the ACTH-induced differentiation of fetal rat adrenocortical cells.

Yan Ke Xue Bao, 1989 Jun, 5(1-2), 32 - 5
Study in cytotoxicity of gentamycin to corneal epithelium and endothelium in tissue culture; Lin N et al.; A study in cytotoxicity of gentamycin to tissue-cultured bovine corneal endothelial cells and rabbit corneal epithelial cells is reported . When the cultured cells reached confluence, they were exposed to tissue culture media containing gentamycin for 6 hours . We found that 0.5% gentamycin caused significant damage to corneal epithelial cells--diffuse plasmolysis, with scattered cell necrosis and 5% loss . While corneal endothelial cells were exposed to 1.6 mg/ml gentamycin, extensive cell loss (approximately 15%) was observed . The damaged cells recovered their normal morphology after 24 hours . When the concentration of gentamycin increased twice, serious damage to cells occurred . The area of cell loss reached 40%, and the recovery of cellular morphology was much slower . This study demonstrates that gentamycin potential cytotoxicity to corneal epithelium and endothelium, suggesting that gentamycin should be rationally used in the treatment of ocular diseases.

Toxicology, 1989 May 31, 56(1), 107 - 17
Comparative toxicity of allylamine and acrolein in cultured myocytes and fibroblasts from neonatal rat heart; Toraason M et al.; Allylamine is toxic to the cardiovascular system causing aortic, valvular and myocardial lesions . Acute toxicity is believed to involve metabolism of allylamine to highly reactive acrolein . Comparative toxicity of allylamine and acrolein was evaluated in cardiac fibroblasts and myocytes, which were obtained from neonatal rat hearts by a differential plating technique . Allylamine and acrolein were added directly to serum supplemented culture media (M199) . Toxicity was assessed by measuring lactate dehydrogenase (LDH) release as an indicator of cell lysis . Spontaneous beating activity of myocytes, and adenosine 5' triphosphate (ATP) levels of myocytes and fibroblasts were also assessed . Cell lysis occurred 4 h after treatment of myocytes with 0.5 mM allylamine, whereas 20 mM allylamine was required to lyse fibroblasts . Acrolein, at a concentration of 0.05 mM, was equally toxic to fibroblasts and myocytes . Semicarbazide, a benzylamine oxidase inhibitor, protected myocytes from allylamine toxicity, but clorgyline, a monoamine oxidase inhibitor, was ineffective . Semicarbazide was ineffective against acrolein toxicity . Beating activity of myocytes was arrested by 0.05 mM acrolein and 0.5 mM allylamine, although 0.05-0.1 mM allylamine reduced beating activity . Myocyte ATP levels were reduced 4 h after exposure to 0.01 mM acrolein . Allylamine at 0.05 mM reduced ATP in myocytes, but 10 mM allylamine was required to reduce ATP in fibroblasts . ATP levels remained normal in myocytes exposed to 1 mM allylamine in the presence of 0.1 mM semicarbazide . The findings support the hypothesis that the toxicity of allylamine in cultured myocytes is dependent on its metabolism to acrolein, and that cytotoxicity may result from interference with energy production.

Eur J Biochem, 1989 May 15, 181(3), 721 - 6
Thrombospondin is synthesized and secreted by human osteoblasts and osteosarcoma cells . A model to study the different effects of thrombospondin in cell adhesion; Clezardin P et al.; In this study we have shown by both immunofluorescence and immunoprecipitation techniques that human osteoblasts and osteosarcoma cells synthesize and secrete thrombospondin, a 450-kDa glycoprotein initially found in platelets . Immunofluorescence with a mouse monoclonal antibody to human platelet thrombospondin yielded specific granular staining within the cytoplasm of human osteoblasts . SDS/polyacrylamide gel electrophoresis analysis of immunoprecipitates obtained with polyclonal and monoclonal anti-thrombospondin antibodies allows the identification of thrombospondin in the cellular lysates and the culture media of biosynthetically labelled osteoblasts and osteosarcoma cells . Kinetic and dose/response studies of osteoblasts and of two osteosarcoma cell lines (MG-63, SaOs-2) were performed to assess the ability of these cells to adhere to thrombospondin and type-I collagen . Thrombospondin promoted the attachment of human osteoblasts whereas it inhibited the adhesion of MG-63 and SaOs-2 cells, both when it was directly adsorbed to plastic and when it was bound to type-I collagen . Therefore osteoblasts and osteosarcoma cells may be valuable tools to study the role of thrombospondin in cell adhesion.

Int J Cancer, 1989 May 15, 43(5), 949 - 55
Presence of nerve growth factor-like immunoreactivity in carcinoid tumour cells and induction of a neuronal phenotype in long-term culture; Ahlman H et al.; Mid-gut carcinoid tumour cells expressed a neuronal phenotype, observed and characterized immunocytochemically in long-term culture . Initially the culture contained a main population of spherical tumour cells with granules immunopositive for serotonin (5-HT) and tachykinins (TK) . Production and secretion of these substances into media was verified biochemically . Cytoplasmic granules with 5-HT-like immunoreactivity (5-HT-LI) were markedly reduced during culture, while granules with TK-LI were unchanged in number, corresponding to the biochemical findings . After a few days in culture, tumour cells were flattened and fine neurite-like processes extended . After 2-3 weeks many endocrine tumour cells had converted to neuron-like cells with slender cell processes containing granules with TK-LI . Varicose enlargements and apparent growth cones were observed . When neurites were extended, 50-80% of the neuron-like cells were positive with antisera against the neurofilament triplet . Cells of both endocrine and neuronal phenotypes were positive with antisera against tetanustoxin, Thy 1-antigen, neuron-specific enolase, synapsin and a synaptic vesicle protein (p 38) supporting the concept of these tumour cells as para-neurons . Intermediate filaments, studied with monoclonal anti-vimentin, were found in all cells . Filaments were also observed ultrastructurally . Initially, nerve growth factor (NGF)-LI was found in granules of all spherical tumour cells . When neuritic processes were extended, the cells appeared to lose these granules . After 40 days in culture, NGF-LI was absent or very sparse . The studies indicate autocrine secretion of a growth factor, reacting with the NGF antiserum, by cultured mid-gut carcinoid tumour cells inducing a neuronal phenotype with enhanced NF and TK synthesis and suppressed 5-HT synthesis . In bioassay systems the culture media caused a delayed neurite reaction on PC12 cells, but no reaction on chick ciliary ganglion cells, indicating that the factor is not authentic NGF.

Biochem Biophys Res Commun, 1989 May 15, 160(3), 1415 - 20
Effect of two different glucose concentrations on insulin receptor mRNA levels in human hepatoma HepG2 cells; Briata P et al.; Glucose is known to affect mRNA levels of several genes . In order to investigate possible effects of glucose on insulin receptor mRNA levels, we cultured human hepatoma cells (HepG2) in two different culture media: DMEM containing 100 mg/dl glucose and DMEM containing 450 mg/dl glucose . Insulin receptor mRNA levels and insulin binding activity were reduced in HepG2 cultured at lower glucose concentrations . These data suggest that glucose affects insulin receptor gene expression.

Cancer Res, 1989 May 15, 49(10), 2584 - 7
Inhibition of carcinogen-inducible DNA amplification in a simian virus 40-transformed hamster cell line by ethacridine or ethanol; Burkle A; DNA amplification as a mechanism to increase gene expression has been established as a cause of cytotoxic drug resistance and appears to play a role in tumor cell progression . In order to investigate factors which control the process of DNA amplification we have been using a simian virus 40 (SV40)-transformed Chinese hamster cell line (CO60) as a model system . This cell line can be induced to amplify integrated viral DNA with a variety of agents . In this report the following is shown . (a) Addition of ethacridine, an intercalative compound, or ethanol to the culture media inhibits amplification induced by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine or by gamma-irradiation in a dose-dependent fashion . In the case of N-methyl-N'-nitro-N-nitrosoguanidine induction (50 microM), the highest concentrations of ethacridine (40 microM) or ethanol (2% v/v) tested reduced SV40 amplification from about 20-fold to less than 2-fold . (b) Neither substance induces significant amplification when applied alone over a wide range of concentrations (0.01-20 microM ethacridine; 0.001-2% ethanol) . (c) Significant inhibition of amplification is achieved with nearly nontoxic concentrations of both substances (10 microM; 1%), (d) Without direct interference with the inducer . It is concluded that ethacridine or ethanol treatment uncouples the toxic effects of an alkylating agent or ionizing radiation from their ability to induce amplification in CO60 cells.

Anal Biochem, 1989 May 15, 179(1), 24 - 7
Bidimensional reversed-phase high-pressure liquid chromatography analysis of cultured cell neuropeptides: application to atrial natriuretic factor; Nguyen TT et al.; We report here a one-step procedure for extraction and analysis of neuropeptides in chromaffin cell culture media and acid extracts using reversed-phase HPLC . The bidimensional HPLC system consists of a precolumn connected to a six-port switching valve which is on-line with an analytical column . The direct injection of the biological samples onto the precolumn previously equilibrated with 15% acetonitrile allows the elimination of interfering substances . The samples purified on the precolumn can then be eluted onto the analytical column via the switching valve for neuropeptide separation . This trace-enrichment system allows a minimum of sample handling, both saving time and reducing possibilities of loss and contamination . This method has been applied to monitor the precursor and mature forms of atrial natriuretic factor from chromaffin cell secretion media and cell content extracts . The recovery of atrial natriuretic factor is in the range of 80-100% . This procedure could be applied to the study of the precursor-product relationship of any neuropeptide, e.g., from radiolabeled extracts of pulse-chase experiments performed on cultured chromaffin cells.

J Immunol, 1989 May 15, 142(10), 3361 - 8
Regulation of antigen presentation . II . Anti-Ig and IL-2 induce IL-1 production by murine splenic B cells; Hawrylowicz CM et al.; Murine splenic B cells did not constitutively express IL-1 activity . After culture with anti-Ig and T cell-conditioned media and then fixation, B cells expressed membrane IL-1 and were able to stimulate growth of the IL-1-dependent T cell clone D10 . Expression of membrane IL-1 required stimulation of B cells for 2 days before fixation . Significant IL-1 activity was detectable in freeze-thaw lysates of identical B cell preparations by 12 h . B cells also released IL-1 into the culture media . In situ hybridization studies by using probes to murine IL-1 alpha and IL-1 beta genes supported these observations . Thus, messenger RNA for IL-1 alpha and IL-1 beta rose in parallel, were detected between 6 and 24 h of culture, and declined to low levels by 30 h . Despite the presence of mRNA for IL-1 alpha and IL-1 beta, only IL-1 alpha had functional activity as determined by the use of a mAb to IL-1 alpha . IL-2 was found to be an essential component of the T cell-derived supernatant . Although IL-4 or TNF did not induce significant B cell IL-1 expression, they both caused a modest, but reproducible enhancement when added in combination with IL-2 . IFN-gamma, by contrast, partially inhibited IL-1 induction.

In Vitro Cell Dev Biol, 1989 May, 25(5), 424 - 34
Modulation of actin mRNAs in cultured vascular cells by matrix components and TGF-beta 1; Kocher O et al.; Alpha-smooth muscle actin is currently considered a marker of smooth muscle cell differentiation . However, during various physiologic and pathologic conditions, it can be expressed, sometimes only transiently, in a variety of other cell types, such as cardiac and skeletal muscle cells, as well as in nonmuscle cells . In this report, the expression of actin mRNAs in cultured rat capillary endothelial cells (RFCs) and aortic smooth muscle cells (SMCs) has been studied by Northern hybridization in two-dimensional cultures seeded on individual extracellular matrix proteins and in three-dimensional type I collagen gels . In two-dimensional cultures, in addition to cytoplasmic actin mRNAs which are normally found in endothelial cell populations, RFCs expressed alpha-smooth muscle (SM) actin mRNA at low levels . alpha-SM actin mRNA expression is dramatically enhanced by TGF-beta 1 . In addition, double immunofluorescence staining with anti-vWF and anti-alpha-SM-1 (a monoclonal antibody to alpha-SM actin) shows that RFCs co-express the two proteins . In three dimensional cultures, RFCs still expressed vWF, but lost staining for alpha-SM actin, whereas alpha-SM actin mRNA became barely detectable . In contrast to two-dimensional cultures, the addition of TGF-beta 1 to the culture media did not enhance alpha-SM actin mRNA in three-dimensional cultures, whereas it induced rapid capillary tube formation . Actin mRNA expression was modulated in SMCs by extracellular matrix components and TGF-beta 1 with a pattern very different from that of RFCs . Namely, the comparison of RFCs with other cell types such as bovine aortic endothelial cells shows that co-expression of endothelial and smooth muscle cell markers is very unique to RFCs and occurs only in particular culture conditions . This could be related to the capacity of these microvascular endothelial cells to modulate their phenotype in physiologic and pathologic conditions, particularly during angiogenesis, and could reflect different embryologic origins for endothelial cell populations.

Endocrinology, 1989 May, 124(5), 2464 - 72
Immunological characterization and immunocytochemical localization of oviduct-specific glycoproteins in the baboon (Papio anubis); Verhage HG et al.; Oviducts obtained from estradiol-treated ovariectomized baboons synthesize and release a family of high mol wt (100,000-130,000) glycoproteins during short term explant culture . The objective of this study was to make a polyclonal antibody to these glycoproteins and then use the antibody to determine the presence of the glycoproteins in oviduct flushings, tissue culture media, and tissues obtained from cycling and steroid-treated baboons . Oviduct culture medium proteins from estradiol-treated baboons were separated on one-dimensional polyacrylamide gels and transferred to nitrocellulose membranes . The region containing the glycoproteins was cut out, solubilized in dimethylsulfoxide, mixed with Freund's adjuvant, and injected at 2-week intervals into a male rabbit . The anti-serum used in this study was obtained 6 weeks after the initial injection and cross-reacted with antigens on Western blots of oviduct flushings and oviduct culture media obtained from follicular stage and estradiol-treated baboons . The antigens were absent in oviduct flushings obtained from luteal stage, ovariectomized and estradiol-primed baboons treated with estradiol and progesterone or progesterone alone . The antigens were not detected on Western blots of other reproductive and nonreproductive tract culture media or in serum obtained from follicular stage or estradiol-treated baboons . Immunoperoxidase staining was limited to discrete granules in the apical cytoplasm of secretory cells in oviducts obtained from follicular stage and estradiol-treated baboons . Thus, the secretory cells of the baboon oviduct synthesize and secrete a family of estradiol-dependent oviduct-specific glycoproteins that may have potential functional significance during fertilization and embryo development.

Kidney Int, 1989 May, 35(5), 1119 - 25
Micromolar aluminum levels reduce 3H-thymidine incorporation by cell line UMR 106-01; Blair HC et al.; Aluminum-induced osteomalacia is a frequent complication observed in patients on maintenance hemodialysis . However, it is not known whether there are direct effects of aluminum on osteoblasts, or alternatively, whether the observed changes are due to changes in PTH or other factors . We sought to determine the effect of micromolar levels of aluminum on osteoblasts using a well-defined cell line derived from a 32P induced osteosarcoma of rat, UMR 106-01, which is alkaline-phosphatase positive, responds to PTH, and synthesizes type I collagen . Aluminum exposure was controlled using tissue culture media with {Al } less than 1 microgram/liter (40 nM), produced by precipitation of aluminum salts at pH 8.5 . The effect of defined {Al }, from 20 to 800 micrograms/liter (0.7 to 30 microM), was then determined by adding back aluminum while measuring DNA and protein synthesis . We found that aluminum depressed DNA synthesis, as determined by 3H-thymidine incorporation, by 60%, with half maximal effect at 20 micrograms/liter (740 nM) in cells at a density of 20,000/cm2 . Alternatively, protein synthesis, as determined by 3H-leucine incorporation, did not decline, and in some cases increased . However, qualitative analysis of matrix proteins produced with and without 800 micrograms/liter (30 mM) {Al } showed no differences . Direct measurements of cell number and protein synthesis confirmed these findings . Al does not alter the PTH-induced cAMP response of these cells . Thus, aluminum has a direct effect on cell division, and probably on protein synthesis, in this osteoblast-like cell line . These effects occur at levels of aluminum below those commonly contaminating tissue culture media, and thus are seen reproducibly only in media of defined {Al }.(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1989 May, 55(5), 1209 - 13
Factors relevant in bacterial pyrroloquinoline quinone production; van Kleef MA et al.; Quinoprotein content and levels of external pyrroloquinoline quinone (PQQ) were determined for several bacteria under a variety of growth conditions . From these data and those from the literature, a number of factors can be indicated which are relevant for PQQ production . Synthesis of PQQ is only started if synthesis of a quinoprotein occurs, but quinoprotein synthesis does not depend on PQQ synthesis . The presence of quinoprotein substrates is not necessary for quinoprotein and PQQ syntheses . Although the extent of PQQ production was determined by the type of organism and quinoprotein produced, coordination between quinoprotein and PQQ syntheses is loose, since underproduction and overproduction of PQQ with respect to quinoprotein were observed . The results can be interpreted to indicate that quinoprotein synthesis depends on the growth rate whereas PQQ synthesis does not . In that view, the highest PQQ production can be achieved under limiting growth conditions, as was shown indeed by the much higher levels of PQQ produced in fed-batch cultures compared with those produced in batch experiments . The presence of nucleophiles, especially amino acids, in culture media may cause losses of PQQ due to transformation into biologically inactive compounds . Some organisms continued to synthesize PQQ de novo when this cofactor was administered exogenously . Most probably PQQ cannot be taken up by either passive diffusion or active transport mechanisms and is therefore not able to exert feedback regulation on its biosynthesis in these organisms.

J Neurochem, 1989 May, 52(5), 1576 - 81
Role of intracellular pH in the axolemma- and myelin-induced proliferation of Schwann cells; Saunders RD et al.; In order to provide additional information on the biochemical events that interact to cause Schwann cells to proliferate, we have monitored the intracellular pH of Schwann cells that have been stimulated to divide with myelin-enriched fractions (MEF) or axolemma-enriched fractions (AEF) . The intracellular pH of Schwann cells was monitored using 2',7'-bis(carboxymethyl)-5(6)-carboxyfluorescein (BCECF), which displays an increase in fluorescence upon alkalinization . Both AEF and MEF caused dose-dependent increases in the intracellular fluorescence of the Schwann cell cultures . At their maximum doses, AEF and MEF stimulation resulted in a 260 and 300% increase in intracellular fluorescence, respectively . The increase in intracellular fluorescence was abolished when cells were stimulated in Na+-free media, suggesting a role for the Na+/H+ exchanger . Mitotic stimulation required integrity of the Na+/H+ exchanger, as inhibition of the Na+/H+ exchanger for periods up to 1 h after addition of mitogen caused a significant inhibition of subsequent mitosis . Phorbol esters, which can potentiate AEF- and MEF-induced Schwann cell proliferation, increased intracellular fluorescence fivefold, an effect which was also dependent upon the presence of Na+ in the culture media . The specificity of the increase in intracellular pH for AEF and MEF was tested by incubating Schwann cells with liver microsomes and a biologically inactive phorbol alcohol, neither of which is significantly mitogenic for Schwann cells . Neither liver microsomes nor phorbol alcohol had a significant effect on intracellular pH . The implications of the increase in intracellular pH in Schwann cells with respect to inositol phospholipid metabolism, protein kinase C activation, and cellular proliferation are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Am J Vet Res, 1989 May, 50(5), 747 - 50
Proadifen-induced production of prostacyclin by equine peritoneal macrophages; Morris DD et al.; A study was performed to determine the effect of proadifen hydrochloride on prostacyclin (prostaglandin I2 {PGI2}) and thromboxane A2 (TxA2) synthesis by equine peritoneal macrophages and the effect of proadifen on endotoxin-induced synthesis of PGI2 and TxA2 by equine macrophages . Peritoneal macrophages (2.5 x 10(6)/ml) were incubated for 6 hours in tissue culture media containing 1) nothing (nontreated control), 2) proadifen hydrochloride (20, 100, 250, and 500 mumol/L, 3) endotoxin (5 ng/ml), or 4) the calcium ionophore A23187 (0.95 mumol/L) . In a second series of experiments, peritoneal macrophages were incubated with endotoxin (5 ng/ml) and proadifen (250 umol/L), for 6 hours . Concentrations of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and thromboxane B2, the stable metabolites of PGI2 and TxA2, were determined in the incubation media by radioimmunoassay . Proadifen caused increased synthesis of PGI2 by equine macrophages, without affecting TxA2 production . The increased PGI2 production was similar to that induced by endotoxin and calcium ionophore; however, the latter 2 agents significantly stimulated TxA2 production as well (P less than 0.05) . There were no significant differences among mean concentrations of 6-keto-PGF1 alpha in media from macrophages treated with 100, 250, or 500 mumol/L proadifen, but there was a significant curvilinear regression between their concentrations . The ratio of thromboxane B2 to 6-keto-PGF1 alpha was significantly lower than baseline in incubation media from macrophages exposed to proadifen, endotoxin, and calcium ionophore . Proadifen hydrochloride did not significantly change equine peritoneal macrophage production of PGI2 or TxA2 in response to endotoxin.

Endocrinology, 1989 May, 124(5), 2321 - 9
An insulin-like growth factor-binding protein in the baboon (Papio anubis) endometrium: synthesis, immunocytochemical localization, and hormonal regulation; Fazleabas AT et al.; The major secretory product of the baboon and human decidua during pregnancy is an insulin-like growth factor-binding protein (IGF-BP) . This study was designed to determine the site and regulation of synthesis of this protein in the nonpregnant baboon based on our previous findings that this molecule is biochemically and immunologically similar in the two species during pregnancy . Endometrial tissue was obtained from cycling baboons and steroid-treated ovariectomized animals . Portions of the tissue were fixed for immunocytochemical analysis, cultured in the presence or absence of cyclohexamide and actinomycin-D, or snap-frozen in liquid nitrogen for RNA isolation . Immunostaining using monoclonal antibodies to IGF-BP indicated that the protein was localized predominantly in the epithelium of the deep glands and was most intense during the late luteal stage of the cycle . Immunoreactive product was also present in tissue from estrogen-primed progesterone-treated ovariectomized animals . However, the staining pattern was more variable and less intense than that in intact animals . Western blots of explant culture media showed the presence of an immunoreactive product only in those tissues that also demonstrated immunocytochemical localization . The absence of an immunoreactive band in medium obtained from tissue incubated in the presence of cyclohexamide suggested that this protein was synthesized de novo . The mRNA coding for IGF-BP appeared to be stable, as synthesis in explant cultures continued in the presence of actinomycin-D . The cDNA probe hybridized to a single message transcript of 1.65 kilobases . The presence of mRNA in tissues coding for this protein correlates with the immunochemical data relating to the site and hormonal regulation of its synthesis . The presence of this protein in the glandular epithelium of the baboon endometrium may have implications in the autocrine and/or paracrine regulation of trophoblast growth and penetration during implantation.

Endocrinology, 1989 Apr, 124(4), 1595 - 601
Glucocorticoids stimulate plasminogen activator production by rat granulosa cells; Wang C et al.; We studied the direct effects of glucocorticoids on plasminogen activator (PA) production by rat granulosa cells . PA production was assayed by culturing rat granulosa cells on {125I}fibrin plates and determining the extent of fibrinolysis after addition of the specific substrate plasminogen . In granulosa cells from preantral follicles of immature rats, treatment with FSH caused dose-dependent increases in PA production whereas glucocorticoids by itself was without effect . Increasing concentrations (10(-10) to 10(-6) M) of both natural and synthetic glucocorticoids potentiated the stimulating effect of FSH on PA production by 120 to 170% . The stimulatory potencies of the natural corticosteroids correlated with the glucocorticoid potencies (cortisol/corticosterone greater than aldosterone/11-deoxycorticosterone) . In granulosa cells from Graafian follicles of mature rats, glucocorticoids on its own had direct stimulating effect on PA production . The stimulatory action of glucocorticoids on FSH-dependent PA production was completely blocked by simultaneous treatment with antiglucocorticoid RU 486 . Antiserum directed against tissue-type PA (tPA) neutralized the increased fibrinolytic actions of glucocorticoids . Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fibrin autography techniques, we showed that the increase in fibrinolytic activity in response to glucocorticoids resulted from increased production of tPA rather than urokinase-like PA . The effect of glucocorticoids on the production of PA inhibitors (PAI) was examined by 1) neutralization of urokinase activity by increasing amounts of culture media from granulosa cells treated with glucocorticoids, 2) reverse fibrin autography, and 3) Western blot analysis with a specific PAI antiserum . All these methods failed to detect a stimulatory action of glucocorticoids, with or without FSH, on PAI production by rat granulosa cells in the culture media . Our data showed that glucocorticoids have a direct stimulating effect on tPA production, but unlike its action on other in vitro systems, they have no significant effect on PAI production by rat granulosa cells in vitro.

Prenat Diagn, 1989 Apr, 9(4), 227 - 42
Differentiation in human chorionic villus cultures: hCG and HLA expression; Phillips CN et al.; The present report describes methods to separate, culture, and study syncytio-cytotrophoblast and mesenchymal core of the first-trimester human chorionic villus . The cultured outer layer cells (syncytio-cytotrophoblast) are multinucleated, pleomorphic, and active in the formation of human chorionic gonadotrophin (hCG) . The mesenchymal core cells are more fibroblast-like in appearance, do not show multinucleation, and have less hCG in their culture media . Both cultured cell types express HLA (ABC) Class I histocompatibility antigens but not HLA (DR) Class II antigens . These and previous studies from this laboratory postulate different embryonic origins: (1) Syncytio-cytotrophoblast cultures of chorionic villus derive from differentiated trophoblast and preserve multinucleation as well as hCG hormone function . (2) Cells cultured from the chorionic villus core originate from extraembryonic mesenchyme . (3) Amniocytes (AF cells) cultured from amniotic fluid resemble the multipotential and early-stage trophoblast, retaining pleomorphism, multinucleation, and lacunae formation as well as production of hCG, progesterone, oestrogen, basement membrane glycoprotein, and Type IV collagen . These cell types cultured from the chorionic villus and amniotic fluid provide a means for in vitro study of specific embryonic cell lineages.

Hum Reprod, 1989 Apr, 4(3), 327 - 30
Platelet aggregating activity in human embryo culture media free of PAF-acether; Amiel ML et al.; A factor activating human platelets and liberated by the embryo was sought in the culture media of human embryos using two bioassays . The first bioassay demonstrated the existence of a thrombocytopaenic factor after the i.p . injection of culture media into splenectomized mice . This factor was found more frequently in media which contained an embryo compared to those which contained a non-fertilized oocyte . PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was searched for with a specific bioassay, using washed rabbit platelets . This remained negative for all the media studied, including those which had contained an embryo giving rise to a pregnancy . In these experiments it was not possible to relate embryo-derived platelet activating factor (EDPAF) to PAF-acether . Neither were we able to use the detection of EDPAF to test embryonic viability, or attempt to identify those embryos which were susceptible to lead to a pregnancy after intrauterine transfer from among all the embryos transferred.

In Vitro Cell Dev Biol, 1989 Apr, 25(4), 358 - 64
Influence of branched-chain amino acid composition of culture media on the synthesis of plasma proteins by serum-free cultured rat hepatocytes; Montoya A et al.; Supplementation of Ham's F12 culture medium with essential amino acids (EAA) up to the rat plasma levels increased the rates of synthesis of albumin and transferrin by cultured rat hepatocytes by 1.3 and 1.7, respectively . Fifty percent of this increase could be attributed to three of the EAA: the branched-chain amino acids (BCAA: Leu Ile and Val) . Non-branched-chain essential amino acids (non-BC-EAA) stimulated only 25% of the increase produced by the whole EAA mixture . When each EAA was tested individually, none of them caused an appreciable increase in albumin and transferrin in culture medium . When the concentrations of all EAA were raised to rat postprandial portal levels, albumin and transferrin synthesis rates reached a maximum, increasing by 3.2 and 3.5, respectively . Supplementation with BCAA at postprandial portal concentrations increased albumin and transferrin synthesis rates by 2.2 and 2.0, respectively, and had no noteworthy effect on the synthesis of cellular proteins . Non-BC-EAA at their postprandial portal concentrations increased albumin and transferrin synthesis rates by 1.7 and 1.9, respectively . Supplementation with alanine to reach a nitrogen content equal to that of the modified EAA-enriched medium had no stimulatory effect . Our results show that EAA have a specific effect on the synthesis of plasma proteins by cultured hepatocytes, and that BCAA at physiologic concentrations account for the major part of this stimulatory effect . Consequently, EAA and particularly BCAA concentration should be elevated in serum-free nutrient media to sustain maximum plasma protein synthesis.

Ann Vasc Surg, 1989 Apr, 3(2), 167 - 9
Pericardial cells for graft seeding: isolation, culture and identification; Sugimoto JT et al.; The purpose of this study was to determine the feasibility of using the pericardium as a source of endothelial cells . Nineteen pieces of fresh pericardium were obtained from nine mongrel dogs . Cells were prepared by collagenase digestion of the pericardium for 24 minutes followed by centrifugation . The cells were divided into three groups: The supernatant subjected to no further steps, Group I (N = 6); filtration through a 15 micron porous mesh, Group II (N = 6); and Percoll gradient separation with medium 199, Group III (N = 7) . The cells obtained were cultured for seven days in tissue culture media . Yield (cells x 10(5)/gram fresh tissue) was determined with Methods I, II, and III, producing 32.4 +/- 25.9 (SD), 0.96 +/- 0.6 and 0.57 +/- 0.5, respectively (I vs II or III, p less than 0.01) . Fibroblast contamination determined by phase contrast light microscopy was demonstrated in 6/6 cultures with Method I, 3/6 with II and 1/7 for III (I vs III, p less than 0.01) . An assay for endothelial cells (Factor VIII) was positive in 2/6 cultures with Method I, 5/6 with II and 7/7 for III (I vs III, p less than 0.01) . The pericardium is a suitable organ for procurement of endothelial cells . Though reducing yield, filtration and Percoll gradient separation allows for isolation of a relatively pure culture of endothelial cells.

J Anim Sci, 1989 Apr, 67(4), 928 - 33
Acute and long-term lipogenic response to insulin and clenbuterol in bovine intramuscular and subcutaneous adipose tissues; Miller MF et al.; The present study was conducted to determine whether insulin and clenbuterol affected either short-term (2-h) incubations or long-term (48-h) tissue cultures of i.m . and s.c . adipose tissue explants . Samples were taken from control steers and steers fed 7 mg.head-1.d-1 clenbuterol for 50 d, after which time the drug was withdrawn from the diet for 90 d prior to slaughter . Neither short-term incubations nor long-term explant cultures contained bovine serum albumin (BSA) . Insulin (6.67 x 10(-9) M) had no effect (P greater than .05) on lipogenesis in s.c . and i.m . adipose tissue in 2-h tissue incubations of fresh adipose tissue . There was a substantial decrease in activity during the culture period, which was ameliorated somewhat in s.c . adipose tissue by the presence of insulin in the culture media . Clenbuterol exposure for 48 h in vitro decreased the production of lipids from acetate in both adipose tissue depots but had no effect in short-term adipose tissue incubations . Results from the present study confirm that omitting BSA from incubation media does not enhance the responsiveness of bovine s.c . adipose tissue or the less mature i.m . adipose tissue to insulin . Insulin may maintain greater cell viability in 48-h explant cultures.

Nippon Hifuka Gakkai Zasshi, 1989 Apr, 99(5), 529 - 36
{Effects of dihydrotestosterone on cultured hair papilla cells and localization of its receptors}; Katsuoka K et al.; We have reported that cultured papilla cells (PC) grown by isolation and cultivation of human hair papillae show some biological characteristics . In the present study, localization of androgen binding proteins in PC and effects of dihydrotestosterone (DHT) on PC in vitro were examined . Cytochemical staining of PC using DHT-peroxidase conjugate gave positive reactions in the nucleo of PC originating from scalp- and axilla-hair papillae . The cultivation of PC with added DHT in media for two weeks showed increases 3H-thymidine uptake and 14C-proline uptake over that of dermal fibroblasts . These results suggest that androgen receptors exist in PC and that DHT in culture media induces an accelerating effect upon the DNA synthesis and protein synthesis.

Cardiovasc Res, 1989 Apr, 23(4), 279 - 85
An in vitro system for study of effects of angiotensin I on cultured endothelial cells; Bagby SP et al.; Since certain non-vascular angiotensin II (AII) receptors may be activated by angiotensin I (AI), and since sustained increase in AI levels accompanies chronic treatment with converting enzyme inhibitors (CEI) which block conversion of AI to AII, the question of whether AI has significant biological effects is of clinical relevance . We therefore sought to develop an in vitro culture system in which effects of angiotensin I, independently of its conversion to AII, could be studied in cloned aortic vascular endothelial cells (VEC) . This was complicated by peptide degradation during the period of observation, both by angiotensin converting enzyme (ACE) on the surface of VEC and by angiotensinases in either the serum component of culture media or associated with the cell monolayer . Accordingly, we examined the half life of AI under relevant cell culture conditions, with and without confluent fetal bovine aortic endothelial cells (FBAEC) . Factors assessed included (1) fetal calf serum: commercial source, concentration in culture media, effects of converting enzyme inhibitor (CEI: MK422) and/or heat inactivation (superimposed on the commercially performed process); and (2) effect of FBAEC in monolayer culture, with and without CEI . Results showed that (1) in the absence of cells, loss of AI in culture media, when present, was solely due to the presence of fetal calf serum (FCS) and showed a dose dependent response; (2) FCS from differing sources may vary dramatically in capacity for AI breakdown; and (3) serum related AI disappearance included a heat resistant ACE like component (inhibitable by CEI) and a heat sensitive/CEI resistant component dominant at concentrations of FCS exceeding 5%.(ABSTRACT TRUNCATED AT 250 WORDS)

J Virol Methods, 1989 Apr-May, 24(1-2), 91 - 101
Concentration and purification of feline leukaemia virus (FeLV) and its outer envelope protein gp70 by aqueous two-phase systems; Hammar L et al.; The major protective antigens of retroviruses are considered to be their glycosylated envelope proteins . However, the methods commonly employed to enrich and purify virus from culture media such as pelleting and density-gradient centrifugation result in a low recovery of the viral external glycoproteins . This is an obvious drawback when the virus is intended for use in a vaccine . In search for alternative methods to concentrate and purify FeLV, we have attempted extraction in two-phase systems based on water-soluble polymers (Albertsson PA., Biochem Biophys Acta 1958; 27: 378-395) . A variety of polymer systems was tested . Some of them seem attractive for a large-scale concentration of the virus and/or its glycoprotein . The distribution between the phases of two FeLV proteins, the outer envelope protein, gp70, and the gag protein, p27, was determined . With a system composed of dextran sulfate and polyvinyl alcohol both the glycoprotein and the gag protein were almost completely recovered in the lower phase which constitutes about 3% of the total system in weight . The two proteins were more than 40-fold purified as calculated on protein basis . The proteins can be extracted readily.

Acta Med Okayama, 1989 Apr, 43(2), 97 - 103
Composition of culture media for steroid hormone secretion by murine adrenal tumor cells, Y-1 clone; Ichikawa Y; Murine adrenal tumor cells (Y-1 clone) were stimulated by adrenocorticotropic hormone (ACTH) and cyclic adenosine 3',5'-monophosphate (cyclic AMP) to produce steroid hormone (delta 4, 3-keto steroids) . The steroids were secreted into the medium immediately after synthesis . The optimum concentrations of ACTH and cyclic AMP for stimulation of steroid production were 10(-2) U/ml and 1.0 mM, respectively . In serum-free medium, ACTH and cyclic AMP stimulated steroidogenesis in Y-1 cells, but the amount of steroid hormone in the culture medium was low . However, a high level of steroid production was maintained with medium containing 10 mg/ml bovine serum albumin (BSA) . In culture medium containing a higher concentration of BSA, Y-1 cells did not become spherical as is usually the case when steroid production is stimulated by ACTH or cyclic AMP . The morphological changes did not always correlate with steroid secretion by Y-1 cells.

Arch Invest Med (Mex), 1989 Apr-Jun, 20(2), 113 - 22
Steroid conjugate formed by human endometrium; Garzon P et al.; Progesterone -6,7-3H (P*) was incubated in minces of human secretory and proliferative endometrium in absence as well as in presence of 10 and 100 micrograms/ml of unlabelled progesterone (P), in Eagle's Culture Medium throughout 72h . The following metabolites of P* were found in culture media: 1 . C-21 derivatives of P* reduced at C-5, and C-20 positions . Also, a 3 beta-hydroxy-5 alpha pregnane-20-One conjugated to a glucuronic acid moiety was identified . 2 . Concentrations of water-soluble derivative accounted for 21% and 29% of the recovered radioactivity in proliferative and secretory endometria, respectively . 3 . After beta-glucuronidase cleavage, 80% to 95% of the water soluble derivatives were released as free steroids . 4 . Approximately 60% corresponded to 3 beta -hydroxy- 5 alpha pregnane-20-one of the pooled water extracts . 5 . Alson, 17% and 13% as well as 9% and 8% recoveries under 10 and 100 micrograms/P were observed in proliferative and secretory endometria, respectively . Glucuronidation seems to be a compensatory route to metabolize P and P* in human endometrium as might also occur in other species.

Inflammation, 1989 Apr, 13(2), 233 - 44
Stimulus-specific production of cyclooxygenase and lipoxygenase metabolites of arachidonic acid by bovine alveolar macrophages; Laegreid WW et al.; Alveolar macrophages (AMs) are capable of producing a variety of inflammatory mediators including those derived from arachidonic acid, the prostaglandins (PGs), leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) . Inflammation associated with release of arachidonate-derived mediators is a result of the combined actions of all of these mediators . Thus, it is critical to determine the entire spectrum of arachidonate-derived metabolites that AMs are capable of producing . In this study bovine AMs were prelabeled with {3H}arachidonic acid prior to stimulation with serum-treated zymosan, phorbol myristate acetate (PMA), or the calcium ionophore A23187 . The total release of arachidonate metabolites into the culture media was measured by reverse-phase HPLC with on-line radiometric detection . All stimuli used induced production of metabolites of the cyclooxygenase pathway with thromboxane B2 and HHT being the major metabolites . Lesser amounts of PGF2 alpha, PGE2, and PGD2 were produced . Only stimulation with A23187 resulted in production of LTB4 and 5-HETE, products of the 5-lipoxygenase pathway . This latter result indicates that the two major pathways of arachidonate metabolism in AMs may be selectively stimulated . Such an effect could have important consequences in the development of pulmonary inflammation . Furthermore, the spectrum of arachidonic acid metabolites produced by bovine AMs closely resembles that of human AMs, in contrast to rodent AMs.

Thromb Res, 1989 Apr 1, 54(1), 41 - 52
Interleukin-1, endotoxin or tumor necrosis factor/cachectin enhance the level of plasminogen activator inhibitor messenger RNA in bovine aortic endothelial cells; Medina R et al.; It is known that either endotoxin (LPS) or interleukin-1 (IL-1) increase the activity of plasminogen activator inhibitor (PAI) in the culture media of human and bovine endothelial cells . We have confirmed these results in bovine aortic endothelial cells (BAEC) . To determine if this effect was mediated by increases in the level of PAI messenger RNA (mRNA) we examined the effects of these cytokines on PAI mRNA levels in BAEC, using RNA blot analyses . Treatment of BAEC with either IL-1, LPS, or human recombinant tumor necrosis factor/cachectin (TNF) dramatically increased the level of PAI mRNA . Since elevated levels of PAI will decrease fibrinolytic potential, this mechanism is in concert with the known increase in in vivo procoagulant potential induced by these agents and could contribute to thromboembolic phenomena.

Biol Reprod, 1989 Apr, 40(4), 873 - 85
Characterization of an insulin-like growth factor binding protein, analogous to human pregnancy-associated secreted endometrial alpha 1-globulin, in decidua of the baboon (Papio anubis) placenta; Fazleabas AT et al.; The major secreted protein of the human decidua (pregnancy-associated endometrial alpha 1-globulin {alpha 1-PEG}), is an insulin-like growth factor-binding protein (IGF-BP) that is immunologically and biochemically similar to placental protein 12 (PP12) extracted from term human placenta . Since previous studies have demonstrated that the baboon and human endometrium synthesize and release a number of biochemically and immunologically related polypeptides in culture, this study was undertaken to further characterize a related IGF-BP in baboon placental tissues . Decidua, chorio-amniotic membranes with adhering decidua (CAM-D), and placental villi were obtained from pregnant baboons between Days 134 and 160 of gestation by Cesarean sections . Portions of tissue were either cultured in the presence of 35S-methionine, fixed for immunocytochemistry, or frozen in liquid nitrogen for cytosol extraction . Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of tissue culture media (TCM) revealed that the major secretory product of the decidua and CAM-D was an acidic polypeptide (Mr 33,000) . Western blot analysis and immunoprecipitation of TCM with murine monoclonal antibody (B2H10) against human alpha 1-PEG demonstrated that this molecule, secreted by the baboon decidua and CAM-D, but not the placental villi, was immunologically identical to the human IGF-BP . Immunocytochemical localization of IGF-BP was intense in the cytoplasm of stromal cells in decidua and CAM-D and absent in the placenta . Gel filtration of TCM and cytosol followed by screening of eluates for 125I-IGF-I binding resolved two peaks (Mr greater than 100,000 and 35,000) of specific IGF-BP in decidua and CAM-D . The 35,000 peak had 100-200 times the binding capacity of the Mr greater than 100,000 peak and a Kd of 1.14-1.83 nM . The eluates contained in the Mr 35,000 peak were also immunoreactive to alpha 1-PEG, as accessed by a polyclonal radioimmunoassay . Affinity cross-linking with 125I-IGF-I followed by sodium dodecyl sulfate-PAGE revealed an immunoreactive complex of Mr 36,000, confirming that the baboon protein represents a high affinity IGF-BP . These studies indicate that the hypertrophied stromal cells of the baboon decidua and CAM-D synthesize and release an IGF-BP as their major secretory product, analogous to the situation in humans . The results of this study suggest that this protein may play a role in the regulation of IGF action during pregnancy.

FEBS Lett, 1989 Mar 27, 246(1-2), 25 - 9
Characterization of vesicles, containing an acylated oligopeptide, released by human colon adenocarcinoma cells . NMR and biochemical studies; Masella R et al.; RNA-containing vesicles, recovered from the supernatant of high-density cell samples of human colon carcinoma, produce a high-resolution 1H NMR spectrum of lipids characterized by isotropic tumbling; these vesicles contain large amounts of triglycerides and cholesterol esters . Both findings have strict analogies to what is displayed by the proteolipid complexes isolated from the sera of tumor-bearing patients {(1985) Proc . Natl . Acad . Sci . USA 82, 3455-3459; (1986) FEBS Lett . 203, 164-168} . Lipid analysis and enzymatic tests indicate that these vesicles are selected micromaps of plasma membranes, analogous to those that can be recovered from culture media in which tumor cells are grown {(1985) Dev . Biol . 3, 33-57} . Peculiar lipids, an acylated oligopeptide and a modified phospholipid, are also present in the vesicles.

Int J Cancer, 1989 Mar 15, 43(3), 478 - 86
Immunodetection of cathepsins B and L present in and secreted from human pre-malignant and malignant colorectal tumour cell lines; Maciewicz RA et al.; Pre-malignant and malignant human colorectal tumour epithelial cell lines both secreted precursor forms of the 2 cysteine proteinases, cathepsins B and L . The amount of proteinases secreted by these cell lines varied according to the cell density . Comparison at similar cell densities showed that the pre-malignant, adenoma-derived cell line (PC/AA) secreted as much, or more, of both cathepsin B and L precursors as did the malignant, carcinoma-derived cell line (PC/JW/FI) . However, mature forms of cathepsins B and L were detected in the culture media of only the carcinoma-derived cell line, thus indicating that the invasive potential of a tumour may be related to its ability to process extracellularly the secreted precursor enzyme to a mature and consequently active enzyme, rather than to the amount of proteinase synthesized and/or secreted . Similar results were obtained using 2 other epithelium-derived tumour cell lines, HT/29 (carcinoma) and SP/AN (adenoma) . Immunolocation studies showed that cathepsin B was lysosomal while cathepsin L appeared to have a distribution more consistent with a plasma membrane association . Purified human cathepsins B and L (mature form) were capable of solubilizing an isolated basement membrane matrix (bovine anterior lens capsule) in vitro, thus indicating that the secreted mature enzymes and the membrane-associated cathepsin L could potentially degrade basal laminae or sub-endothelial basement membranes in vivo.

J Rheumatol, 1989 Mar, 16(3), 355 - 62
Proteoglycan metabolism in tissue cultured human articular cartilage . Influence of piroxicam; Verbruggen G et al.; Proteoglycan metabolism was investigated in longterm tissue cultured human cartilage . Visually intact cartilage from adult donors showed improving accumulation rates for 35sulfate labelled proteoglycans over a 6-week period . The loss of newly synthesized molecules in the nutrient culture media was low and constant . Fibrillated cartilage from a 17-year-old male showed higher basal 35S incorporation rates and the proportions of 35S proteoglycan aggregates were higher than in normal tissue . These observations may reflect the immature status of the tissue or an attempt at repair . However samples lost increasing amounts of 35S proteoglycans in the incubation media . This material appeared to be monomeric proteoglycan . The amount of 35S activity retained in the fibrillated tissue matrix fell during culture as did the proportion of proteoglycan aggregates . Thus catabolic events were postulated in these fibrillated cartilage samples . When piroxicam was added to the incubation media more newly synthesized proteoglycans were retained in the intercellular matrix of the fibrillated samples . Increased accumulation of 35S activity was seen in some of the batches of visually intact cartilage.

J Helminthol, 1989 Mar, 63(1), 72 - 4
In vitro effect of ivermectin on Pseudoterranova decipiens survival; Manley KM et al.; Third larval stages (L3) removed from fish fillets, fourth larval stages (L4) raised in in vitro culture, and adults of Pseudoterranova decipiens, collected from grey seal (Halichoerus grypus) stomachs, were exposed to the broad spectrum anthelmintic, ivermectin . L3 and L4 parasites were exposed, in vitro, to 500, 100, 50, 20, 5 and 1 micrograms/ml concentrations of the drug, in culture media . Adult P . decipiens were exposed in vitro to a concentration of 500 micrograms/ml ivermectin, only . Controls consisted of parasites placed in culture media alone or culture media plus drug vehicle . These three developmental stages of P . decipiens were all found to be susceptible to the effects of ivermectin.

J Androl, 1989 Mar-Apr, 10(2), 145 - 51
Polyamine profiles in rat testis, germ cells and Sertoli cells during testicular maturation; Shubhada S et al.; Polyamine cellular concentrations (putrescine, spermidine and spermine) in the rat testis and testicular cell types were determined by fluorescence spectroscopy of their dansyl derivatives . A method is described to separate dansylated polyamines by high performance liquid chromatography in less than 12 minutes . In rat Sertoli cells, polyamine concentrations (per mg DNA) were greater than those in germ cells and the testis . The concentrations of all three polyamines increased with age . Concentrations of spermidine and spermine in germ cells also increased with age and leveled off after 27 to 35 days . On the other hand, higher putrescine levels were found in the testis of young rats (13 to 22 days) while the greatest spermidine and spermine contents were observed in the testis from rats of 31 to 35 days old . Of great interest, Sertoli cells from all age groups studied released a relatively large quantity of putrescine and a smaller amount of spermidine, but no spermine, into culture media . The amount of polyamine released by Sertoli cells varied with the age of the animal . Sertoli cells from 27-day-old rats released the greatest quantity of putrescine on a per mg DNA basis . The release of putrescine increased after hypotonic treatment that removed contaminating germ cells from the remaining Sertoli cells . It is concluded that cellular polyamine levels in the rat testis, germ cells and cultured Sertoli cells and the amount of polyamines released by Sertoli cells were age-dependent during the first wave of spermatogenesis.

Matrix, 1989 Mar, 9(2), 116 - 26
The degradation of collagen in pig synovium in vitro and the effect of colchicine; Fell HB et al.; Colchicine induced a rapid destruction of the collagenous matrix of pig synovial explants in culture in the presence of serum . The most efficacious doses were 0.01-0.1 micrograms/ml (2.5 x 10(-8) M - 2.5 x 10(-7) M) . The histological progression of the tissue breakdown induced by colchicine was very similar, although faster, to that described for other agents (Fell et al., 1986), with cells having basophilic nuclei accumulating in areas of fibril degradation . The loss of collagen correlated with an increase in collagenase production and at the peak of resorption (6 to 8 days) active collagenase was present in the culture media . Immunocytochemical methods demonstrated active collagenase bound to collagen fibrils after only 4 days in culture, before significant collagen degradation could be observed histologically . Collagen breakdown was completely inhibited by cortisol, and partially inhibited by indomethacin: only the inhibition by indomethacin could be reversed by exogenous prostaglandin E2 . Vinblastine at a higher dose was as effective as colchicine but the lumicolchicines, which do not disrupt microtubules, were ineffective . Although the precise mechanism of action of colchicine is unknown, this culture system provides a useful in vitro model for increasing our understanding of the cellular mechanisms of tissue breakdown and for elucidating the roles of active collagenase and related metalloproteinases.

J Exp Med, 1989 Mar 1, 169(3), 1021 - 9
Regulation of macrophage functions by L-arginine; Albina JE et al.; Sites of inflammation with prominent macrophage infiltration, such as wounds and certain tumors, are uniquely deficient in free arginine . The effects of arginine availability on macrophage physiology were investigated . When cultured in media containing less than 0.1 mM L-arginine, rat resident peritoneal macrophages exhibited enhanced spreading, tumor cytotoxicity, superoxide production, phagocytosis, and protein synthesis . Thus, arginine concentrations similar to those found in sites of inflammation can augment macrophage functions, while those found in plasma (approximately 0.1 mM) and in commonly used culture media (0.4 to 1.2 mM) are inhibitory . Culture in homoarginine, but not D-arginine, ornithine, citrulline, urea, histidine, or lysine also inhibited macrophage tumor cytotoxicity, indicating the specificity of the effect . In contrast to resident macrophages, the tumor cytotoxicity of peritoneal macrophages obtained after C . parvum injection was suppressed by culture in arginine-deficient media . However, L-arginine-deficient media enhanced all other activation-associated functions in C . parvum-elicited macrophages as in resident cells . Arginine-free wound fluid promoted resident macrophage tumoricidal activity when compared with rat serum, and again, the addition of L-arginine was inhibitory . The marked effects of L-arginine availability on macrophage functions, together with the knowledge that these cells modify the extracellular arginine concentration in sites of inflammation through arginase, provide evidence for an autoregulatory mechanism of macrophage activation.

Tsitologiia, 1989 Mar, 31(3), 267 - 72
{Ultrafine organization of Leptomonas peterhoffi flagellates cultured in broth and solid nutrient media}; Malysheva MN et al.; The morphology of in vitro grown lower trypanosomatids L . peterhoffi was studied by means of electron microscopy . The flagellates from both liquid and solid culture media are represented by uninucleate cells of two structural types . Type I flagellates are characterized by dense cytoplasm enriched with numerous ribosomes . Type II flagellates are most abundant in the cultures; they display a less dense cytoplasm and fewer ribosomes . The flagella of L . peterhoffi of both types form enlargements, which are most expressed at the outlet of the flagellar pocket . The nuclei of some cells contain twisted threads about 10 nm in diameter . L . peterhoffi from the liquid media usually possess long, narrow and curved flagellar pockets . On the solid medium, amoeboid and hemispherical colonies composed of both uninucleate and giant multinucleate cells are formed . In these cells the flagellar pockets are usually short and straight . Outside the flagellar pocket, the axoneme often becomes looped in the flagellar enlargements of the colonial uninucleate cells.

Biochim Biophys Acta, 1989 Feb 28, 973(2), 111 - 7
Purification of an acidic plastocyanin from Microcystis aeruginosa; Tan S et al.; Plastocyanin and cytochrome c-553 are two functionally equivalent electron carriers in the photosynthetic chain of cyanobacteria . Microcystis aeruginosa, a unicellular cyanobacterium which grows well at a high pH (8.6) and which was not known to possess plastocyanin, has been studied for its ability to synthesize plastocyanin in culture media with and without Cu . In the absence of Cu, an acidic cytochrome c-553 alone was isolated . With the inclusion of 2 microM Cu, cytochrome c-553 synthesis was partially suppressed and an acidic plastocyanin was isolated . A newly developed procedure, using high concentrations of ammonium sulfate to fractionate water-soluble proteins on Sephacryl S-200 was successfully used to isolate and concentrate the plastocyanin, thus allowing it to be further purified to homogeneity . This protein has an isoelectric point of 4.8 which is similar to the pI value reported for other acidic plastocyanins from higher plants and green algae . Its N-terminal sequence of the first 15 amino acids has been determined; 9 of these amino acids are identical to those in the sequence of the basic plastocyanin from Anabaena variabilis.

Brain Res, 1989 Feb 27, 481(1), 175 - 80
Monoclonal antibody directed against glutaraldehyde conjugated glutamate and immunocytochemical applications in the rat brain; Chagnaud JL et al.; Like other small-sized neurotransmitter molecules, glutamate (Glu) was conjugated to carrier proteins via glutaraldehyde (G) . Human serum albumin (HSA) and thyroglobulin (TH) conjugates were alternately injected into mice . When a relevant immune response was obtained for antibody affinity and specificity, hybridization of spleen activated lymphocytes with SP2/O/Ag myeloma cells was performed . Supernatant culture media of hybridomas were tested for the presence of anti-conjugated Glu antibodies with our ELISA method . Selected hybridomas giving good antibody affinity and specificity were then cloned by the limiting dilution technique . Using DEAE-chromatographed ascites fluid, Glu reactivity was observed on the cortex and the hippocampus . Staining obtained with this monoclonal antibody was in agreement with that observed with previous polyclonal antisera directed against conjugated Glu or monoclonal anti-gamma-glutamyl-Glu antibody.

FEBS Lett, 1989 Feb 27, 244(2), 328 - 32
Production of carrier proteins for insulin-like growth factors (IGFs) by rat osteoblastic cells . Regulation by IGF I and cortisol; Schmid C et al.; A bone-derived rat cell line, PyMS, releases IGF I and IGF carrier proteins which are similar to those found in rat serum . Western blot analysis of culture media conditioned by hormone-treated cells shows that growth hormone and IGF I stimulate and cortisol inhibits production of IGF carrier proteins in vitro . A glycosylated carrier protein species of 49-42 kDa is closely related to the subunits of the growth hormone-dependent carrier protein complex found in rat serum . In addition, rhIGF I rapidly induces a 32 kDa, non-glycosylated IGF-binding protein whose accumulation is markedly increased by cortisol.

J Biol Chem, 1989 Feb 25, 264(6), 3078 - 88
Biosynthesis and deposition of a noncovalent laminin-heparan sulfate proteoglycan complex and other basal lamina components by a human malignant cell line; Frenette GP et al.; The basal lamina components laminin, heparan sulfate proteoglycan (HSPG), and type IV collagen were synthesized and codeposited in the extracellular matrix (ECM) by a cultured human cell line from gestational choriocarcinoma (JAR) . Laminin and HSPG formed a noncovalent complex detected by the coimmunoprecipitation of HSPG with laminin from cell lysates and culture media . The complex was stable in the cell lysis buffer that contained detergents (1% Triton X-100, 0.5% deoxycholate, and 0.1% sodium dodecyl sulfate) and sodium chloride (from 0.15 to 1.0 M), but was dissociated by adding 8 M urea to the detergent lysates . Even though JAR cells produced roughly equal amounts of HSPG and chondroitin sulfate proteoglycan, only HSPG complexed with laminin, suggesting a specific interaction between these basal lamina components . The laminin-HSPG complex was deposited and retained in the ECM . This was shown biochemically by isolating an enriched fraction of ECM from JAR cells cultured on native type I collagen gels . At steady state, more than half (52%) of the laminin-HSPG in the culture was recovered in the ECM fraction, in contrast to 16% of the total laminin and 29% of the total type IV collagen, which were secreted to a greater extent than laminin-HSPG into the culture medium . The retention of the laminin-HSPG complex in the ECM suggests that it may participate in the assembly of the basal lamina-like extracellular matrix deposited by JAR cultures . Omission of ascorbate from the culture medium abolished the ECM deposition of type IV collagen but had little effect on the deposition of laminin or laminin-HSPG . This demonstrates that the stable deposition of laminin-HSPG and laminin in the collagen-based choriocarcinoma cultures is not dependent on an assembled network of type IV collagen.

Biochem Biophys Res Commun, 1989 Feb 15, 158(3), 660 - 6
Production of interleukin 6 and its relation to the macrophage differentiation of mouse myeloid leukemia cells (M1) treated with differentiation-inducing factor and 1 alpha,25-dihydroxyvitamin D3; Miyaura C et al.; We have studied the production of interleukin 6 (IL-6) and its relation to the macrophage differentiation in murine myeloid leukemia cells (M1) . As has been reported, differentiation-inducing factor (D-factor), 1 alpha, 25-dihydroxyvitamin D3 {1 alpha, 25(OH)2D3}, and recombinant IL-6 similarly induced differentiation of M1 cells into macrophages . The three compounds also induced mRNA expression of IL-6 in M1 cells . M1 cells treated with D-factor or 1 alpha, 25(OH)2D3 produced biologically active IL-6, but the amounts of IL-6 secreted into culture media did not appear to be enough to induce differentiation of M1 cells . Furthermore, simultaneous addition of anti-IL-6 antibody did not suppress the differentiation of M1 cells induced by D-factor or 1 alpha, 25(OH)2D3 . These results show that IL-6 production is an essential property associated with the macrophage differentiation of M1 cells, but it may not be responsible for the D-factor- and 1 alpha, 25(OH)2D3-induced differentiation.

Anal Biochem, 1989 Feb 15, 177(1), 156 - 60
A colorimetric assay for the measurement of D-glucose consumption by cultured cells; Blake DA et al.; A colorimetric method is described for measuring glucose consumption by tissue culture cells . This procedure, which utilizes the coupled activities of glucose oxidase and horseradish peroxidase, is insensitive to the spectral interferences caused by the phenol red and sera present in most tissue culture media . The spectral properties (absorbance maxima and apparent absorption coefficients) and stability of a large number of chromogenic horseradish peroxidase substrates were surveyed for their ability to perform in an assay for glucose in the presence of phenol red and sera components . One of these chromophores, the product of an oxidative couple between 4-aminoantipyrine and N-ethyl-N-sulfopropyl-m-toluidine, was subsequently used to develop a fixed time assay for glucose in media samples . The assay required only 10 microliters of media in a 1-ml assay volume; reproducibility studies showed variabilities of less than 5% in the assay of a single sample, and values obtained in glucose analyses correlated well with those obtained using commercially available test kits . The assay was used to study the rate of glucose consumption in two different cell types, bovine corneal endothelial cells and human diploid fibroblasts.

Cancer Res, 1989 Feb 15, 49(4), 983 - 90
Clonal variation in the production of tumor-associated alpha 2-macroglobulin in a malignant human melanoma and association with growth stimulation; Bizik J et al.; alpha 2-Macroglobulin (alpha 2-M) is known as a wide-spectrum proteinase inhibitor and to bind covalently certain growth factors . We have previously characterized tumor-associated alpha 2-M synthesized and secreted by human tumor cell lines . Of the cell lines studied, the melanoma cell line HMB-2 produced the largest amount of this glycoprotein . Immunofluorescence staining of cultured HMB-2 cells suggested that the cell population is heterogeneous with respect to alpha 2-M production . We have now isolated clones from the parental HMB-2 cells and characterized eight representative ones in detail . They varied considerably in the quantity of alpha 2-M secreted, from 4.2 to 46.5% of total protein . No relationship between the production of alpha 2-M by these clones and their pigmentation or tumorigenicity in nude mice was found . However, statistically there was a strong correlation between the modal chromosome number and population doubling time (r2 = 0.88, P less than 0.001) and also between the modal chromosome number and alpha 2-M production (r2 = 0.73, P less than 0.01) . The growth rate of the clones correlated with the level of alpha 2-M in culture medium (r2 = 0.69, P less than 0.01) . Clones with lower alpha 2-M production had a proportionally shorter population doubling time than the clones with intermediate or high production . Northern hybridization indicated quantitative variation in the alpha 2-M mRNA expressed by the parental cells and the clones, that was comparable but not identical with the quantity of alpha 2-M in the respective culture media; both parental cells and clones expressed platelet-derived growth factor A-chain mRNAs with little difference in levels . Serum-free medium from low alpha 2-M producer clones stimulated normal stationary fibroblasts significantly more than clones producing intermediate or high amounts of alpha 2-M . alpha 2-M decreased and anti-alpha 2-M IgG increased the stimulation . These results suggest that production of tumor-associated alpha 2-M is related to both autocrine and paracrine growth-stimulating activity of the tumor cells.

J Immunol Methods, 1989 Feb 8, 117(1), 131 - 6
Purification of monoclonal antibodies raised against prostate-specific acid phosphatase for use in vivo in radioimaging of prostatic cancer; Hakalahti L et al.; In developing diagnostic reagents for the radioimaging of prostatic cancer, methods were optimized for the purification of two mouse IgG1 monoclonal antibodies raised against prostate-specific acid phosphatase and produced in cell culture . Two different two-step methods were selected . One method consisted of two successive ion exchange chromatographic steps on Mono S and Mono Q; in the other method, Mono S chromatography was followed by hydrophobic interaction (Alkyl Superose) chromatography . In both cases, fast protein liquid chromatography (FPLC) instrumentation was used . The antibodies were purified from cell culture media containing fetal calf serum (1-5%) . Highly pure (greater than 95%) IgG1 antibodies, free of contaminating serum-derived proteins or column materials, were obtained in good yield (greater than 90% recovery) . The purified antibodies completely retained their immunological reactivity towards prostate-specific acid phosphatase and were sterile and pyrogen-free . Since the monoclonal antibodies produced were intended for applications in vivo, an essential feature of the methods selected was the availability of in situ cleaning procedures for sterilization of the gel materials and for the inactivation of viruses and pyrogens in the gels . The methods developed could be readily scaled up for preparative purposes.

Hybridoma, 1989 Feb, 8(1), 117 - 26
The use of continuous culture to enhance monoclonal antibody production; MacMichael GJ; Hybridoma 14-4-4S, ATCC HB32, was grown in batch and continuous culture in an attempt to increase the production of monoclonal antibody (MAb), and to determine the adequacy of each culturing method for optimizing culture media . Various concentrations of glucose, fetal bovine serum (FBS) and serum type were studied to determine their effects on the specific growth rate (mu), the specific monoclonal antibody production rate (MPR), the maximum population density and the final MAb concentration . Attempts to optimize the culture medium using the batch method resulted in ambiguous results . However, by growing the hybridoma in a cytostat at a standardized population density of 1.0 X 10(6) cells/ml, the growth rate and MPR were found to decrease below feed concentrations of glucose and FBS of 22.5 mM and 10% (v/v), respectively . In addition, the use of the cytostat demonstrated that FBS was superior to both new born bovine serum (NBS) and Serum PlusTM (SPS) . Batch cultures indicated that the production of MAb appeared to be related to the metabolic activity of the cells, and continuous culture demonstrated a direct relationship between mu and MPR.

Scand J Immunol, 1989 Feb, 29(2), 239 - 46
Liver sinusoidal blood containing natural killer-like cells; Lukomska B et al.; Rat liver sinusoidal washout cells were examined . These cells, which are marginated in sinusoids, could be washed out by simple flushing of the vasculature with culture media without enzymes and under physiological portal pressure . They revealed, in comparison to peripheral blood mononuclear cells, high cytotoxic activity commonly attributed to the natural killer (NK) and natural cytotoxic (NC) cells, and were found to be anti-asialo-GM1-negative . Liver sinusoidal cytotoxic cell (LSCC) activity has been found to be associated with the large granular lymphocytes in low-density cells in OX8-positive as well as in OX8-negative populations . The mononuclear cells washed out from the liver microvasculature could be stimulated with NK-sensitive targets to release soluble factors which selectively lyse YAC-1 tumour cells and inhibit growth of normal haematopoietic granulocyte-macrophage colony-forming cells in vitro . The cytotoxic cell population in the liver turned out to be blood-borne in origin and not resident . Our findings suggest that liver sinusoidal cytotoxic cells represent an NK population with a predilection for marginating in the liver and may be important in eliminating tumour or virus infected cells passing through the liver from the circulation . The mechanism of their accumulation in liver sinusoids remains unclear.

Nippon Ketsueki Gakkai Zasshi, 1989 Feb, 52(1), 80 - 5
T-cell colony formation in homosexual men with anti-human immunodeficiency virus; Wakabayashi Y et al.; In vitro differentiation and proliferation of precursor T-cells was examined in four homosexual men positive for anti-human immunodeficiency virus (HIV), one of whom had acquired immunodeficiency syndrome (AIDS) . Peripheral mononuclear cells from the men were cultured with phytohemagglutinin-P in semi-solid culture media containing methylcellulose for 7 days . Colony formation was significantly reduced in all subjects, even in the presence of IL-2 . Moreover, numerous colony component cells lacked T-cell specific surface markers . These results suggest that the impaired T-cell differentiation and proliferation occurs at the precursor T-cell level in HIV infection.

J Invest Dermatol, 1989 Feb, 92(2), 272 - 7
Surface tension in the developing mammal: anisotropic tension generation in the dorsal skin of the perinatal rat; Hoath SB et al.; It is common knowledge that skin biopsies retract following excision . The direction and the magnitude of this retraction is thought to be related to the field of tension previously experienced by the skin sections in vivo . We utilized this phenomenon to investigate the ontogeny, directionality, and regulation of skin tension in the perinatal rat from gestational day 20 to postnatal day 6 . In this study, geometrically-precise biopsies (circles and rectangles) were excised from the dorsal skin of perinatal rats and placed in tissue culture media . The excised skin sections rapidly exhibited changes in shape (to ellipses and helices) . These resulting conformations were characterized with regard to selected spatial attributes; i.e., the ratio of major to minor axes for elliptical figures and the "winding number" for helices . This simple methodological approach allowed the following conclusions: 1) skin retraction in the rat is anisotropic and is maximal in the rostral-caudal direction; 2) skin retraction is inversely related to postnatal age and to ambient temperature; 3) retraction is inhibited by in vivo treatment with epidermal growth factor (EGF) as well as by metabolic poisons such as sodium azide; 4) retraction is augmented in vitro by calcium chloride . Overall, these data support the hypothesis of a metabolically active, temperature-dependent, anisotropically organized retractile mechanism in the dorsal skin of the perinatal rat.

Endocrinology, 1989 Feb, 124(2), 612 - 7
Transcriptional regulation of osteocalcin production by transforming growth factor-beta in rat osteoblast-like cells; Noda M; Osteocalcin (OC) is one of the abundant non-collagenous bone matrix proteins produced exclusively by osteoblasts, and its serum level is used as an indicator of bone metabolism in patients . Transforming growth factor-beta (TGF beta) is abundant in bones and platelets, promotes wound healing in vivo, and is a potent stimulator of the production of extracellular matrix proteins in fibroblasts and osteoblasts . The effects of TGF beta on OC gene expression were examined in rat osteoblast-like cells, ROS17/2.8 . TGF beta 1 decreased OC levels in the culture media 2- to 3-fold . TGF beta 1 also decreased the level of osteocalcin mRNA about 3-fold in a dose-dependent manner . TGF beta 2 and TGF beta 1,2 a heterodimeric form, showed similar effects on OC mRNA levels as TGF beta 1 . The suppression of the OC message level was detectable at 24 h and lasted for up to 72 h . This effect on OC mRNA was blocked by cycloheximide . The stability of OC mRNA was not changed by TGF beta 1 . On the other hand, the rate of OC gene transcription was reduced 4- to 5-fold, as estimated by in vitro nuclear transcription (run-on) assay . TGF beta 1 blocked the increase in the OC mRNA level induced by PTH or 1,25-dihydroxyvitamin D3 . These results indicate that TGF beta inhibits osteocalcin gene expression at least in part through transcriptional control.

Nippon Sanka Fujinka Gakkai Zasshi, 1989 Feb, 41(2), 161 - 6
{Establishment and characterization of human ovarian endometrioid carcinoma cell line}; Fujii T; A new cell line, designated as HOC-I, was established from a recurrent region of ovarian endometrioid carcinoma . HOC-I was subcultivated more than 55 times . 1) The monolayer culture cells showed a pavement like arrangement with polygonal cells and had a tendency to pile up . PAS positive substance could be seen in the cytoplasm . Desmosomes, microvilli and well developed cell organelles could be found by electron microscopy . 2) The population doubling time was about 75 hours . The chromosomal number showed pseudodiploidy of which the mode was 46 . 3) By heterotransplantation to the nude athymic mouse, the tumor easily developed . 4) The effects of estradiol and progesterone on the cellular growth were assessed by the 3H-TdR uptake test . Estradiol increased 3H-TdR uptake of HOC-I but progesterone decreased it . These data suggested that HOC-I had sex-steroid hormone dependency . 5) Estrogen receptor was not detected in HOC-I by the ER-EIA method or the ER-ICA method . 6) The HOC-I cells produced CA125 and TPA in culture media.

J In Vitro Fert Embryo Transf, 1989 Feb, 6(1), 15 - 21
Lack of correlation of immunosuppressive activity secreted by human in vitro fertilized (IVF) ova with successful pregnancy; Armstrong DT et al.; The high rate of implantation failure in humans following in vitro fertilization (IVF) has been attributed to a lack of production of immunosuppressive factors by cleaved embryos, rendering them vulnerable to maternal immune attack just before or around implantation . Systemic as well as blastocyst-secreted suppressor factors have been described and claimed to be responsible for successful pregnancy . Experimentally, we have screened in a double-blind fashion the suppressive activity of human embryo culture media (B2 Menezo system, France) in which zygotes after decoronization were individually cultured during 24 hr on lymphocyte proliferation as well as natural killer (NK) activity . Suppressive activity in media from cleaved and uncleaved ova did not differ significantly, and activity in media from transferred embryos was not correlated significantly with successful pregnancy . The implications of these data are discussed.

J Dermatol, 1989 Feb, 16(1), 42 - 6
Glycosaminoglycan content in the media of cultured dermal fibroblasts derived from burn scar and normal skin; Nogami R et al.; The glycosaminoglycan (GAG) content of the extracellular matrix of burn scar in humans has been reported to differ from that of normal skin . In order to investigate whether the GAG content altered as a result of functional changes in fibroblasts, the GAG content was determined in culture media of fibroblasts derived from growing burn scar, mature scar, and normal skin tissue . No statistical differences were observed in the population doubling-times of scar and normal skin . Mature scar showed significantly higher values for all the concentrations of uronic acid, hexosamine, and sulfate measured in the glycosaminoglycan, as compared with normal skin values, and the concentrations from growing scar were slightly higher than those for normal skin . The above results may suggest an increase in glycosaminoglycan sulfate synthesis following the hyperplasia of the matrix in burn scar tissue.

Tohoku J Exp Med, 1989 Feb, 157(2), 153 - 62
Erythropoietic activity in culture media conditioned by rat mesangial cells; Fukushima Y et al.; Using a tissue culture technique we examined erythropoietin (EPO) producing cells in rat glomeruli . In 5 out of 6 independent glomerular cell cultures, EPO activity was found in the mesangial-cell proliferating phase, but not in the epithelial-cell proliferating phase . Therefore, mesangial cells seemed to be EPO producing cells.

Rev Fr Gynecol Obstet, 1989 Feb, 84(2), 101 - 5
{Evaluation of the probability of the occurrence of pregnancy during oligo-astheno-teratospermia}; Barriere P et al.; Analysis of several sperm counts makes possible the positive diagnosis of oligo-astheno-teratospermia . Attempts to determine their etiology and consequently their treatment, remain negative, in most cases . The physician involved must then try to answer the couple's concern about the prognosis . The prognosis depends on the alterations seen on the sperm count parameters; but there is no threshold figure under which no pregnancy is observed (except for zero), concerning the count, the gradual mobility or the morphology of the spermatozoids . Motility studies, survival tests in cervical secretions, in the female genital apparatus and in culture media or fertilization in vitro tests, improve the prognosis . However, an answer can only be provided if the fertility takes into account the age of the female partner and a study of her fertility as well as the duration of the infertility . Management will consist of a choice between discontinuation of the treatment, test of fertilization in vitro and temporizing, including trials of male treatment and treatment of possibly associated female factors.

Nippon Shokakibyo Gakkai Zasshi, 1989 Feb, 86(2), 227 - 36
{In vivo and in vitro studies on PGE2 production and function in human hepatocellular carcinoma}; Itakura M et al.; In order to investigate the production of PGE2 and its' function in human hepatocellular carcinoma, the effects of indomethacin and PGE2 on tumor growth were examined using in vivo and in vitro techniques . HH2-6 cells produced PGE2 in the culture media, and the inverse relationship was observed in between the cell proliferation and the culture supernatant PGE2 levels . While in vivo, plasma and tumor tissue PGE2 levels of tumor bearing nude mice were significantly increased for 1 or 2 weeks after tumor inoculation . In the case of which indomethacin was injected daily into the abdominal cavity (4 mg/kg body weight), the elevation of plasma and tissue PGE2 levels was remarkably suppressed, and the latent time of tumor growth was also prolonged . On the other hand, another case of which PGE2 was injected (10 micrograms or 0.1 microgram i.p.) at first 10 days revealed shortened latent time . These results indicate the intimate relation between PGE2 and latent time on tumor growth . Furthermore, histological findings suggest that tumor derived PGE2 might play an important role in tumor angiogenesis.

Horm Metab Res, 1989 Feb, 21(2), 92 - 5
Alpha-human atrial natriuretic polypeptide inhibits 19-hydroxy-androstenedione secretion by human adrenal cells; Higuchi K et al.; We investigated the effect of ACTH, angiotensin II (AII), and alpha-human atrial natriuretic polypeptide (alpha-hANP) which plays an important role of water-electrolytes balance, on 19-hydroxyandrostenedione (19-hydroxyandrost-4-ene-3,17-dione, 19-OH-A-dione) secretion by cultured human adrenal cells . 19-OH-A-dione in culture media was measured using a specific RIA . Basal 19-OH-A-dione secretion by adrenal cells was 0.69 +/- 0.08 ng/3h/10(6) cells and significantly rose to 1.17 +/- 0.14 ng/3h/10(6) cells in the presence of ACTH, but not in the presence of A II . These results demonstrate that 196-OH-A-dione is directly secreted from adrenal cells . alpha-hANP significantly inhibited both basal and ACTH-stimulated 19-OH-A-dione secretions, as well as aldosterone . These results demonstrate that alpha-hANP inhibits aldosterone activity by means of the inhibition of both aldosterone and 19-OH-A-dione (an aldosterone amplifier) secretion by adrenal cells.

Br J Oral Maxillofac Surg, 1989 Feb, 27(1), 1 - 11
Interleukin 1-like activity in cystic lesions of the jaw; Meghji S et al.; Odontogenic cyst capsules were cultured in vitro and the culture media analysed for bone-resorption and interleukin 1-like activity . Five cysts synthesised a non-dialysable bone resorbing factor with significant interleukin 1-like activity . One specimen thought to be a cyst with little interleukin 1 activity proved to be antral lining . The results indicate that interleukin 1 may play an important role in cyst expansion by its direct effects on fibroblast proliferation and bone resorption and by stimulating prostaglandin synthesis in stromal fibroblasts of the cyst capsule.

Eur J Immunol, 1989 Feb, 19(2), 245 - 51
T helper cells grown with hapten-modified cultured Langerhans' cells produce interleukin 4 and stimulate IgE production by B cells; Hauser C et al.; Hapten-specific CD4+ T helper (Th) lines generated by repeated stimulation with hapten-modified, cultured Langerhans' cells (cLC) release interleukin (IL 4) (B cell stimulatory factor 1) but not detectable IL 2 into the culture media . The growth of Th cells in response to hapten-modified cLC was inhibited by an anti-IL 4 monoclonal antibody (mAb) but not by mAb to either IL 2 or the p55 chain of the IL 2 receptor . Furthermore, these cells could be stimulated to proliferate by concanavalin A and IL 1 . These results indicate that IL 4 is the autocrine growth factor for these Th lines and that IL 1 plays a critical role in their growth . The Th cells exhibited 1,500-10,000 high-affinity IL 4 receptors cell . When cultured with syngeneic, hapten-modified, small resting B cells, Th cells caused specific IgE production of up to 20 ng/10(4) B cells . Thus, IL 4 producing Th lines appear to result from their selective stimulation by cLC, suggesting that T cell responses elicited in this way profoundly influenced the B cell isotype pattern.

Cell Immunol, 1989 Feb, 118(2), 413 - 24
Regulation of human T-cell production of interleukin 2 by Leu 11 (CD16) positive large granular lymphocytes; Toossi Z et al.; Peripheral blood large granular lymphocytes (LGL) expressing Leu 11 (CD16) antigen with potent natural killer cytotoxicity inhibited soluble antigen-induced T-cell production of interleukin 2 (IL-2) . Depletion of Leu 11-reactive cells from T-cells doubled IL-2 activity (P less than 0.05) . Leu 11-enriched cells did not express high affinity IL-2 receptors nor did they deplete IL-2 activity from culture media . Upon addition in low ratios to Leu 11-depleted cells, Leu 11-enriched fractions increased antigen-induced IL-2 production three-fold (P less than 0.05), whereas at higher ratios IL-2 production was suppressed P less than 0.01 . Additionally, adherent monocytes were increasingly accessory when added in graded numbers to Leu 11-depleted but not T-cell cultures . In the presence of small numbers (5%) of Leu 11-enriched cells, however, monocytes down-regulated IL-2 production of Leu 11-depleted cell cultures . Thus Leu 11-reactive lymphocytes have noncytotoxic functions and may play a major role in immunoregulation.

J Cell Biol, 1989 Feb, 108(2), 713 - 8
Transcriptional regulation of osteopontin production in rat osteoblast-like cells by parathyroid hormone; Noda M et al.; Osteopontin (OP) or bone sialoprotein is a recently characterized extracellular matrix protein which is abundant in bone and is produced by osteoblasts . Parathyroid hormone (PTH) is a potent calcitropic hormone which regulates osteoblastic function including the synthesis of extracellular matrix proteins . This study examines the effect of human PTH (hPTH-{1-34}) on the expression of this novel protein in rat osteoblast-like cells . hPTH(1-34) significantly decreased the amount of OP in culture media of the rat osteoblastic osteosarcoma cell line, ROS 17/2.8, detected by Western immunoblot analysis . hPTH(1-34) also suppressed the steady-state level of OP mRNA two- to threefold with an ED50 of approximately 3 X 10(-10) M . This inhibition was detectable at 24 h, reached its nadir at 48 h, and lasted at least up to 96 h . The hPTH(1-34) effects were mimicked by isobutylmethylxanthine, cholera toxin, 8-bromo-cAMP, forskolin, and isoproterenol . hPTH(1-34) suppressed by two- to threefold the rate of OP gene transcription, estimated by nuclear run-on assays . The suppression of OP mRNA levels by hPTH(1-34) was also seen when basal levels were increased by transforming growth factor type beta, or 1,25-dihydroxyvitamin D3, or were decreased by dexamethasone . A similar decrease in the steady-state level of OP mRNA by hPTH(1-34) was also observed in primary cultures of osteoblast-enriched cells from fetal rat calvaria . These findings indicate that hPTH(1-34) suppresses the production of the novel extracellular matrix protein, OP, in osteoblasts at least in part through transcriptional control.

J Biol Chem, 1989 Jan 25, 264(3), 1860 - 9
Independent regulation of collagenase, 72-kDa progelatinase, and metalloendoproteinase inhibitor expression in human fibroblasts by transforming growth factor-beta; Overall CM et al.; The effects of transforming growth factor-beta (TGF-beta) on fibroblast collagenolytic activity were investigated to determine if modulation of matrix metalloendoproteinase activity could augment the stimulation of connective tissue formation by TGF-beta . Quiescent human fibroblast cultures were incubated in the continuous presence of 1.0 ng/ml (40 pM) TGF-beta in culture medium supplemented with 0.2% (v/v) serum and containing {35S}methionine . Aliquots of conditioned cell culture media, harvested daily for 4 days, were processed individually to separate procollagenase and a 72-kDa progelatinase from metalloendoproteinase inhibitor (TIMP) and plasminogen activator inhibitor (PAI-1) using tandem minicolumns of heparin- and gelatin-Sepharose . The fractionated 54-kDa procollagenase was quantitated, after p-amino-phenylmercuric acetate activation, by functional assays using soluble {14C} glycine-labeled collagen as substrate . In cultures treated with TGF-beta, procollagenase expression was progressively decreased (approximately 50% on day 1, approximately 75% on day 2) to undetectable levels on days 3 and 4 . This decrease occurred despite a 1.6-fold increase in the synthesis of total secreted protein . Contrasting the effect on procollagenase, TGF-beta increased the synthesis of a 72-kDa progelatinase (characterized as a matrix neutral metalloproteinase and likely to be MMP-2) up to 1.8-fold, as determined by quantitation of affinity-purified radiolabeled protein and by enzymography . TIMP biosynthesis was analyzed by immunoprecipitation and quantitated by functional assays for biologically active TIMP following fractionation of the conditioned media . During the first 24 h TGF-beta had little apparent effect on TIMP activity in the medium although the TIMP mRNA transcript was induced 1.3-1.4-fold . Subsequently, TIMP levels were increased 1.7-fold relative to control cells on day 4 . This was accompanied by a 2.4-fold increase in TIMP mRNA, indicating that the regulation of TIMP mRNA and protein levels may be a secondary response to TGF-beta . In comparison, the synthesis of the Mr 48,000 PAI-1, analyzed by {35S} methionine labeling and immunoprecipitation, was elevated greater than 10-fold by TGF-beta at all time points with the highest levels occurring at day 2 . Thus, the effects of TGF-beta on procollagenase, 72-kDa progelatinase, TIMP, and PAI-1 were selective and showed temporal differences.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1989 Jan 24, 28(2), 438 - 42
Characterization of the bacterial cell associated calmodulin-sensitive adenylate cyclase from Bordetella pertussis; Masure HR et al.; Bordetella pertussis produces a calmodulin-sensitive adenylate cyclase that is associated with the whole bacteria and released into its culture media . Preparations of this enzyme invade animal cells, causing elevations in intracellular cAMP levels . Cell-associated adenylate cyclase accounted for 28% of the total adenylate cyclase activity while 72% was released into the culture supernatant . Over 90% of the cell-associated adenylate cyclase activity was sensitive to trypsin treatment of whole cells, indicating that the catalytic domain of the enzyme is localized on the outer surface of the bacterial cells . Enzyme activity was released from whole cells by treatment with SDS . This activity was resolved as a large form (Mr 215,000) by SDS-polyacrylamide gel electrophoresis . In contrast, the culture supernatant contained only the 45,000-dalton catalytic subunit . Enzyme activity released from spheroplasts by sonication was resolved into a large form (Mr 215,000) and a small form (Mr 45,000) . The appearance of the small form with spheroplast formation was probably the result of proteolytic degradation . Antibodies generated against the catalytic subunit purified from culture supernatants cross-reacted with and immunoprecipitated both the large and small forms of adenylate cyclase isolated from bacterial cells . Furthermore, incubation of the cell-associated enzyme with a crude bacterial extract resulted in a time-dependent disappearance of the 215,000-dalton form and a concomitant increase in the amount of the smaller 45,000-dalton form . There was also a parallel increase in the ability of the cell-associated preparation to elevate intracellular cAMP levels in N1E-115 mouse neuroblastoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunol Lett, 1989 Jan 15, 20(1), 41 - 6
A human embryo fibroblast-derived peptide suppresses the zymosan-induced biochemical activation in human polymorphonuclear leukocytes; Okai Y; Previously, a low-molecular-weight peptide was found in the serum-free culture media conditioned by a human embryo fibroblast cell strain (YH-1), which showed inhibitory effects on the proliferation and functions of B and T lymphocytes (Okai et al . (1987) Zool . Sci . 4, 99-105) . In this paper, the inhibitory effect of this peptide on the functional activation in human polymorphonuclear leukocytes (PMNs) is documented . This peptide suppressed the opsonized zymosan-induced generation of chemiluminescence and superoxide anion and concomitant RNA synthesis . However, this peptide did not show significant cytotoxic effects on human PMNs as judged by a cytoplasmic enzyme-releasing assay . These results indicate that a fibroblast-derived peptide reported previously has a suppressive activity on the functional activation of human PMNs . The immunological significance of this finding is discussed.

Science, 1989 Jan 13, 243(4888), 223 - 6
AIDS-Kaposi's sarcoma-derived cells express cytokines with autocrine and paracrine growth effects; Ensoli B et al.; When grown in vitro, cells from Kaposi's sarcoma lesions of AIDS patients (AIDS-KS cells) constitutively release several growth promoting activities . When inoculated into nude mice, the AIDS-KS cells induce a KS-like lesion of mouse origin . Here it is shown that the AIDS-KS cells express messenger RNA for a complex mixture of cytokines that correlate with several of the biological activities of these cells . Basic fibroblast growth factor, which is a potent angiogenic factor, and interleukin-1 messenger RNAs are expressed at very high levels and seem to account for a large proportion of the activities, since their corresponding proteins are released in biologically active form into the culture media where they induce autocrine and paracrine growth effects.

Reprod Nutr Dev, 1989, 29(1), 89 - 93
17 beta-estradiol secretion in normal and hypophysectomized chick embryos; Weniger JP et al.; Ovaries from decapitated, sham-operated and intact 18-day-old chick embryos were cultured in Medium 199 for 6 h, and the amount of 17 beta-estradiol released into the culture media was determined by radioimmunoassay . Ovaries from decapitated embryos secreted significantly lower amounts of 17 beta-estradiol than those from intact embryos, but there was no difference when they were compared to ovaries from sham-operated embryos in this respect . On an ovarian weight basis, 17 beta-oestradiol production was significantly different between the 3 groups of embryos, the ratio being highest in the decapitates . 17 beta-Estradiol concentration was the same in serum from both decapitated and intact female embryos . Considering these results, it is concluded that the hypophysis does not control 17 beta-estradiol secretion by the chick embryo ovary even near hatching time.

Nippon Sanka Fujinka Gakkai Zasshi, 1989 Jan, 41(1), 61 - 8
{Comparative studies on steroidogenesis and prostaglandins production by luteal cells in newly formed corpora lutea and early pregnancy}; Ichikawa F et al.; The present study was undertaken to assess the ability of cultured luteal cells from human corpora lutea (CL) in the mid luteal phase and the early pregnancy to secrete steroids and prostaglandins (PGs) . Luteal cells responded to hCG with a significant increase (2- to 4-fold) in progesterone (P) production . The addition of hCG to the culture media did not stimulate estradiol (E2) production . In contrast, both P and E2 secretion by luteal cells in early pregnancy were significantly lower than those found in the mid luteal phase . Exposure to hCG did not affect P production by luteal cells in early pregnancy . Arachidonic acid (AA) significantly stimulated PGE2 synthesis by luteal cells in the mid luteal phase in a dose-dependent manner . Both basal PGE2 production and the responsiveness to AA were maintained for the duration of the culture . However, hCG did not affect AA-stimulated PGE2 production . Both PGF2 alpha and 6-keto-PGF1 alpha production abruptly declined as the culture proceeded . PG synthesis by cultured luteal cells in early pregnancy was significantly lower than in the mid luteal phase . The ultrastructural characteristics of luteal cells in early pregnancy, which contained lipid droplets, granular and agranular endoplasmic reticulum and large spherical mitochondria, were maintained after 10 days in culture . The present results demonstrate that P and PGE2 production by cultured luteal cells predominate during the mid luteal phase . These data suggest that PGE2, but not PGF2 alpha, may be involved in the regulation of CL function in the menstrual cycle.

J Anim Sci, 1989 Jan, 67(1), 276 - 84
Uterine secretory alterations coincident with embryonic mortality in the gilt after exogenous estrogen administration; Gries LK et al.; Sixteen crossbred gilts were assigned randomly to receive either an i.m . injection of sesame oil (control) or estrogen (E), 5 mg of estradiol valerate, on d 9 and 10 of pregnancy . Gilts were unilaterally hysterectomized on either d 12 and 14 or 16 and 18 . Uterine horns were flushed with 20 ml of .9% sterile NaCl solution to recover conceptus tissue . Conceptuses and endometrial explants were cultured for 24 h with 100 microCi {3H} leucine in 15 ml of minimum essential media . After dialysis, culture media were submitted to 2D-polyacrylamide gel electrophoresis and incorporated proteins were analyzed by fluorography . Normal, intact conceptus tissue was recovered from control gilts . Estrogen-treated gilts flushed on d 12 and 14 contained intact conceptuses; however, uteri from two gilts on d 16 and three on d 18 contained degenerating conceptus tissue . Comparison of endometrial polypeptides synthesized in vitro indicated an alteration in E-treated gilts on d 12 through 18 . Although similar polypeptides were present, a band of polypeptides with a Mr of approximately 30,000 and pI from 7.9 to 8.9 and a larger, acidic polypeptide (Mr = 100,000, pI 3.5 to 5.0) were faint or absent in E-treated gilts . Conceptuses elongated normally in the altered uterine environment, but failed to survive past d 14 in E-treated gilts . Although loss of specific polypeptides in E-treated gilts coincides with conceptus death, their function in conceptus development or attachment is unknown.

J Recept Res, 1989, 9(1), 95 - 106
Potassium depletion and regulation of angiotensin II receptors in vascular smooth muscle cells; Paller MS; Mesenteric artery smooth muscle cells were grown in culture media containing high, normal, or low concentrations of potassium to study the effects on angiotensin II (Ang II) receptor regulation . Cell growth was similar among cells grown in the different culture media . Cells grown in high potassium media (K = 5.8 mEq/L) had an equilibrium dissociation constant, Kd, of 1.59 +/- 0.2 nM, whereas those grown in normal potassium media (K = 4.1 mEq/L) had a Kd of 1.79 +/- 0.2 nM and those grown in a low potassium media (K = 2.9 mEq/L) had a Kd of 1.19 +/- 0.12 nM (not significantly different, NS) . Binding capacity of smooth muscle cells grown in high potassium media was 81 +/- 16.7 fmol/mg prot, 95.1 +/- 12.4 fmol/mg prot in those grown in normal potassium media and those grown in low potassium media 86.4 +/- 24.1 fmol/mg prot (NS) . Binding of radiolabelled Ang II was reduced by approximately 70% in cells exposed to unlabelled Ang II for 30 or 60 minutes . However, this effect of exposure to Ang II to reduce subsequent binding of Ang II was identical in cells grown in high and low potassium medium . Therefore, we were unable to identify a direct effect of low potassium to induce changes in Ang II receptor binding affinity or binding capacity . Previously observed changes in these Ang II binding parameters in potassium-depleted rats was probably a consequence of other factors which were simultaneously altered by potassium deficiency.

J Toxicol Environ Health, 1989, 26(1), 83 - 99
Study of hepatotoxicity in isolated perfused liver versus cultures of rat hepatocytes; Fouad FM et al.; Isolated perfused liver and cultures of rat hepatocytes were assessed for the quantitative evaluation of hepatotoxicity . Release of de novo biosynthesized plasma proteins and acid hydrolases into perfusion or culture media was taken as an indication of the integrity of hepatocytes in both systems . The activities of six acid hydrolases, alpha-L-fucosidase, alpha-D-galactosidase, beta-D-galactosidase, beta-D-N-acetylgalactosaminidase, beta-D-N-acetylglucosaminidase, and cathepsin D, were assayed in collagenase-segregated hepatocytes and in monolayer cultures of rat liver cells obtained via collagenase perfusion of rat liver . In situ, liver perfusion with collagenase led to a loss of 45 +/- 5% of the total acid hydrolase activity in the mitochondrial-lysosomal pellet of the liver cells with concomitant increase of these enzymes in the cytosol . In monolayer cultures over a period of 30 h, increased activity of cathepsin D, beta-D-galactosidase, and beta-D-N-acetylglucosaminidase in the mitochondrial-lysosomal pellet and the cytosol fraction was evident with concurrent biosynthesis of plasma proteins . The use of radioactive tracing techniques with the isolated perfused liver revealed that the rate of catabolism of intracellular protein was approximately 5 times that of plasma protein synthesis . Both methods described here are suitable for the study of the effects of toxins on the function of hepatocytes.

J Cell Physiol, 1989 Jan, 138(1), 197 - 204
Butyric acid causes morphological changes in cultured chondrocytes through alterations in the extracellular matrix; Bretton RH et al.; Butyric acid induces characteristic changes in the morphology of chick embryo chondrocytes . Chick embryo chondrocytes when cultured in the absence of butyrate exhibit a spherical morphology and synthesize cartilage-specific chondroitin sulfate proteoglycan (CSPG) . When these cultures are initiated and maintained in the presence of butyric acid, chondrocytes exhibit a mesenchymal morphology, a 90% reduction in the synthesis of CSPG, and a 75% reduction in DNA synthesis . The reduced synthesis of CSPG and DNA was shown not to be dependent on the morphological change . Chondrocytes require CSPG in order to express a spherical morphology, since including chondroitinase ABC in the culture media caused the cells to spread . In addition, the treatment of chondrocytes with purified CSPG prior to culture in media containing butyric acid resulted in spherical cells . The butyrate-induced spreading was shown to require either serum or fibronectin and could be prevented with antiserum against chick cell-surface fibronectin (cFn) . Cell-surface fibronectin, which was present on both spherical and flattened chondrocytes, organized into fibrils beneath cells which spread . Increased fibronectin synthesis was not responsible for the butyrate-induced morphological change . From this evidence, it is concluded that the mechanism by which butyrate alters the morphology of these cells in culture involves inhibiting CSPG synthesis, thus preventing CSPG accumulation in the extracellular matrix (ECM) . The absence of CSPG in the ECM allows fibronectin to mediate spreading of chondrocytes in culture.

Reprod Fertil Dev, 1989, 1(3), 231 - 6
Alteration of extracellular cation concentrations and ratios in culture medium does not affect first cleavage division of hamster zygotes in vitro nor overcome the 'two-cell block'; Bavister BD et al.; In vivo fertilized hamster one-cell eggs (embryos) were cultured in a simple medium that was modified to provide a wide range of concentrations and ratios of the four major cation components (sodium, potassium, calcium and magnesium) while maintaining total osmotic pressure at 290 +/- 5 mosm . Embryos were cultured in these media to find the optimum cation concentrations for supporting the first cleavage division in vitro and to determine if physiologically abnormal cation concentrations and/or ratios in standard culture media could account for the 'two-cell block' to development in vitro in this species . Despite using a broad range of ratios for sodium:potassium (from 45:1 to 5:1) and for calcium:magnesium (from 17:1 to 1:1), there were no significant differences in the proportions of fertilized eggs that underwent the first cleavage division (approx . 60-80% across all treatments), and none of the two-cell embryos underwent further cleavage during extended culture . These data demonstrate that the first cleavage division of hamster embryos in vitro is insensitive to extracellular concentrations and ratios of the major cations, and that the non-physiological concentrations and/or ratios of these cations in the culture medium are not the primary reason for the failure of hamster zygotes to develop past the two-cell stage in vitro.

J Med, 1989, 20(3-4), 273 - 85
Functional characterization of mononuclear cells of normal and hypercholesterolemic rabbits; Brito B et al.; Cholesterol-fed rabbits are more susceptible to respiratory and skin infections, and show a higher index of mortality than rabbits given a normal diet . Several tests were employed to estimate the proportion of T and B lymphocytes (L-T and L-B) and aspects of their physiology in rabbits with hypercholesterolemia . The proportion of L-T and L-B in hypercholesterolemic rabbits (HChR) was found to be similar to that of normal rabbits (NR) . The proliferative response of mononuclear cells (MNC) from peripheral blood stimulated with mitogens in HChR decreased during the first ten days of the cholesterol rich-diet, being variable afterwards . The addition of HChR serum to the culture media decreased the proliferative response of MNC isolated from both HChR and NR and stimulated by Con A and PWM . A similar antibody response to sheep red blood cells (SRBC) and bovine serum albumin (BSA) antigens was found in HChR and NR.

Cancer Immunol Immunother, 1989, 29(4), 261 - 9
Serum levels of the low-affinity interleukin-2 receptor molecule (TAC) during IL-2 therapy reflect systemic lymphoid mass activation; Voss SD et al.; Expression of the low-affinity interleukin-2 (IL-2) receptor molecule (TAC) has been associated with lymphocyte activation, in vitro and in vivo {Greene WC (1987) Clin Res 35:439} . We have used an enzyme-linked immunosorbent assay (ELISA) to quantify the role of released and cell-bound IL-2 receptor following in vitro or in vivo activation of human lymphocytes with IL-2 . In vitro experiments, culturing fresh peripheral blood lymphocytes in 30 U/ml IL-2 (corresponding to the steady-state IL-2 concentration achieved in patients receiving IL-2 in our clinical trials), showed that the levels of IL-2 receptor released into the culture media exceeded the levels of cell-associated receptor, with both rising in parallel to the cytotoxic activity of the peripheral blood lymphocytes (PBL) against cultured tumor cells . In 12 patients receiving high-dose IL-2 for the treatment of various malignant neoplasms, the levels of IL-2 receptor released into the serum rose dramatically during the IL-2 infusion, and then fell following cessation of the IL-2 infusion . This heightened release of IL-2 receptor into the serum occurred during the episodes of profound lymphopenia that developed within hours after patients began an IL-2 infusion . Following each 4-day infusion of IL-2, a rebound lymphocytosis was observed, as has been previously reported . Serum IL-2 receptor levels do not rebound in parallel; rather, they reach a plateau near the end of the 4-day infusion and then decrease upon cessation of IL-2 . These changes in serum IL-2 receptor levels accompany changes in lytic activity of circulating PBL on Daudi target cells . These results suggest that lymphocyte populations exposed to IL-2 in vivo are activated to become cytotoxic, release TAC, and relocate in non-peripheral blood compartments . Following cessation of the IL-2 infusion these activated lymphocytes return to the peripheral circulation and do not secrete TAC as vigorously as while influenced directly by the IL-2 infusion.

Neirofiziologiia, 1989, 21(3), 416 - 20
{Effect of neurite-stimulating protein on the growth and proliferation of peripheral glia cells}; Chumasov EI et al.; Proliferative activity of peripheral glia was investigated in the organotypic culture of peripheral nerves of 9-10-day old chick embryos . The neurite-stimulating protein, a stimulator of the neurite growth in the organotypic culture of spinal ganglia, being added to the culture media sharply increased the mitotic activity of glia: on the 3d day its level was 3.5 times higher as compared with the control one.

Microbios, 1989, 57(230), 33 - 40
Riboflavin formation by mould fungi cultivated on hydrocarbon-containing media; Sabry SA et al.; The potentiality of some mould fungi, isolated from petroleum sludge to produce riboflavin when grown on hydrocarbon-containing media was tested . Aspergillus terreus was found to be distinguished by its capacity to produce riboflavin when cultivated on the different culture media tested . It was able to grow more luxuriantly and produce good riboflavin output on solar-containing medium . A solar concentration of 5% v/v favoured high riboflavin productivity . The maximal vitamin B2 yields were achieved after 20 days incubation at 30 degrees C . An initial pH value of 5.5-6.0 was found to be the optimum for growth of A . terreus and for riboflavin production.

Tumour Biol, 1989, 10(2), 95 - 102
Synthesis and release of glycoconjugates bearing N-linked oligosaccharides by ovarian carcinoma cells isolated from effusions; Allen HJ et al.; Ovarian carcinoma cell clusters were isolated from patient effusions . The cell isolates were incubated in vitro with radioactive glycoconjugate precursors . Radiolabelled glycoconjugates released to culture media were analyzed for molecular mass heterogeneity and lectin binding activity . From 10 to 50% of the released glycoconjugates were present as a heterogeneous array of glycoconjugates of molecular mass greater than 250 kilodaltons . The remaining glycoconjugates were dispersed in a molecular mass range extending down to approximately 15 kilodaltons . Concanavalin A-Sepharose affinity chromatography revealed the presence of N-linked oligosaccharides . Interaction of glycoconjugates with lentil and pea lectins indicated the presence of L-fucose residues linked to asparagine-bound N-acetylglucosamine . Precipitation of glycoconjugates with ricinus communis agglutinin I showed the presence of nonreducing terminal N-acetyllactosamine residues . Collectively, the data indicate that ovarian carcinoma cells release to culture medium fucosylated glycoconjugates bearing complex-type oligosaccharides . The synthesis and release of these glycoconjugates showed no significant differences among different histologic types of ovarian carcinoma; however, modulation as a function of tumor progression may occur.

J Steroid Biochem, 1989, 34(1-6), 279 - 84
Evidence for a direct effect of estrogen on bone cells in vitro; Ernst M et al.; Although the beneficial effects of estrogen in the treatment of postmenopausal osteoporosis are well documented, such effects were difficult to demonstrate in in vitro models . However, recent improvements in bone cell culture models (better defined osteoblastic cell populations, omission of Phenol Red from culture media) enabled several investigators to show albeit small, but reproducible, direct effects of estradiol in various osteoblastic cell types . Such findings were supported by the identification of low numbers of high-affinity estrogen receptors in bone cells derived from different mammalian species . The likely physiological relevance of the in vitro results is indicated by the specificity for 17 beta-estradiol, and the requirement for nanomolar concentrations of the hormone, consistent with a Kd of 0.6 nM for estradiol binding to its receptor {56} . In bone in vitro, estradiol may have anticatabolic effects by decreasing parathyroid hormone responsiveness, and anabolic effects by stimulating matrix synthesis and cell proliferation . Insulin-like growth factor-I is likely to be an autocrine/paracrine mediator for the anabolic effects and may, when associated with its binding proteins, effectively act in the bone compartment.

Tissue Cell, 1989, 21(5), 653 - 60
Culture media conditioned by wounded cells modify the fibronectin localization pattern of unwounded confluent PtK2 cells; Zhu QL et al.; A confluent PtK2 cell sheet was incised in a serum-free culture medium, at 15 min, 2 hr and 24 hr after wounding . The culture media were collected in the same way and used as conditioned media . Unwounded confluent cells were cultured in the conditioned medium for 24 hr . They showed a modification of fibronectin localization similar to that which we had previously observed in wounded confluent PtK2 cells: cells lost their normal fibronectin fibrils and were surrounded by fibronectin lace . This finding suggested that during wound healing, the cells released soluble chemical factors which could modify the fibronectin localization pattern of unwounded confluent cells . Subconfluent cells did not respond to conditioned media, showing that confluent cells and subconfluent cells had different susceptibilities.

Verh K Acad Geneeskd Belg, 1989, 51(3), 231 - 67
{I-cell disease: elucidation of the enzyme defect and its molecular biology significance}; Leroy JG; "I-Cell disease" (ICD) has received its name because of the innumerable intracytoplasmatic inclusions in connective tissue cells and in vitro fibroblasts derived from patients . It is a progressive disorder already recognizable in infancy . Severe disturbance of growth, coarsening facial features and moderate to severe psychomotor handicap are the most important clinical characteristics . ICD is fatal already in childhood . Prominent among many anatomo- and physiopathologic abnormalities is the paradox of a very reduced activity of many lysosomal hydrolases in connective tissue cells on the one hand and the strongly enhanced activity of these hydrolases in body fluids and culture media on the other hand . Similar, albeit less pronounced abnormalities are observed also in patients with pseudo-Hurler polydystrophy, (PHP) a related though milder disorder . In patients with either ICZ or PHP, phosphorylation of oligomannosyl type sidechains in acid hydrolases, known to be glycoproteins, does not occur . Because the mannose-6-phosphate (M-6-P) recognition marker necessary for normal transport of nascent hydrolases to the lysosomal compartment is not formed, these enzymes are not retained in the cells . The underlying cause is a functionally deficient N-acetylglucosaminephosphotransferase in ICD and PHP patients . The pathological consequences of this defect are more pronounced in fibroblasts than in parenchymatous cells, because the former lack alternative mechanisms by which hydrolases can reach lysosomes . The number of known secondary abnormalities in ICD remain useful for confirmation of the clinical diagnosis and for prenatal diagnosis . The elucidation of the metabolic defect in ICD, a rare monogenic disorder, has increased considerably knowledge on enzyme maturation and on the physiology of intracytoplasmic organelles.

J Med Assoc Thai, 1989 Jan, 72 Suppl 1, 174 - 6
Primary amoebic meningoencephalitis in Thailand: report of a case and review literatures; Sirinavin S et al.; Primary amoebic meningoencephalitis (PAM) which is caused by free-living amoeba, Naegleria fowleri, is a rare disease . We report the fifth case in Thailand in order to add more information . The patient was a previously healthy 4 1/2-year-old girl from Nakhon Pathom province . For several weeks before this illness she had swum in a water supply canal . She developed high fever with change in consciousness . Her cerebrospinal fluid contained numerous Naegleria fowleri which grew in culture media and mice inoculation . She did not respond to treatment with intravenous and intraventricular amphotericin B, and oral rifampicin . She died on the fifth day of illness . Water sample from the canal also grew N . fowleri . All five reported cases in Thailand were reviewed . It was found that none of them had been exposed to a common source . Four of the five cases were male, and four cases occurred during the summer months, March to May . These findings agree with worldwide information.

Life Sci, 1989, 44(6), 397 - 406
Estrogenic effects of phenol red on rat pituitary cell responsiveness to gonadotropin-releasing hormone; Dumesic DA et al.; This study investigated whether phenolsulfonphthalein (PR), a common pH indicator in tissue culture media, affects luteinizing hormone (LH) secretion from rat pituitary cells or 17 beta-estradiol (E2) augmentation of pituitary responsiveness to gonadotropin-releasing hormone (GnRH) . PR enhanced GnRH-stimulated LH secretion and shifted the GnRH dose-response curve leftward with a relative potency ratio of 0.24 +/- 0.09 (+/- SE; p less than 0.01) . The effect of E2 on LH release was significantly diminished by PR, which elevated GnRH-stimulated LH secretion in the absence of E2 . This phenomenon was elicited by PR from different sources and was inhibited by the antiestrogen Cl628 . Thus, PR exerted estrogen-like effects on rat pituitary cells and caused an underestimation of the degree to which E2 enhanced GnRH-stimulated LH secretion.

J Neurosci, 1989 Jan, 9(1), 183 - 94
Lipopolysaccharide-free conditions in primary astrocyte cultures allow growth and isolation of microglial cells; Gebicke-Haerter PJ et al.; Primary rat astrocyte cultures were used to isolate a macrophage population that does not adhere to the confluent glial cells . The cells multiplied vigorously in coculture with astrocytes during the 14 d culture period, provided that functionally active lipopolysaccharide (LPS) was either absent or present in very low concentrations . Based on morphological, immunocytochemical, and pharmacological data, it was concluded that the isolated cells were microglia, the resident macrophages of the brain . The findings characterized them as a distinct cell population that shares features both of peritoneal macrophages and of astroglial cells . Like peritoneal macrophages, the isolated cells were able to phagocytize as shown by their ingestion of latex beads and uptake of L-leucyl methylester . Furthermore, they were immunocytochemically stainable by a specific monoclonal antibody (ED 1) against a macrophage-specific antigen (Dijkstra et al., 1985) . They also synthesized prostaglandin E2 (PGE2) and secreted interleukin 1 (IL-1) upon stimulation with LPS . Upon stimulation with the ionophore A23187, PGD2, the predominant prostaglandin of the brain, was the major PG metabolite released by these cells . In contrast to peritoneal macrophages, microglial cells were able to multiply . Proliferation of microglial cells in coculture with astrocytes was suppressed when 2 ng LPS/ml or higher concentrations were added to astroglial culture media . These astrocyte cultures, which contained approximately 1% microglia, were used to investigate the influence of LPS on prostaglandin and IL-1 secretion in order to compare astroglial and microglial features . Increasing LPS concentrations induced increased PGE2 secretion, whereas PGD2 secretion was essentially unaffected by LPS . The critical influence of LPS contaminations in most of the commercially available animal sera used for astrocyte cultures on cellular composition in general and on metabolism of hormones and growth factors in particular is discussed.

Cytobios, 1989, 60(241), 115 - 26
Interaction between Leydig and Sertoli cells in vitro; Bilinska B; The interaction between Leydig and Sertoli cells grown in co-culture was studied . After 3 to 4 days in culture, Leydig and Sertoli cells formed monolayers . To distinguish functional Leydig cells from Sertoli cells, a histochemical test for delta 5,3 beta-HSD activity was performed, and cells which showed a positive reaction were defined as Leydig cells, in contrast to Sertoli cells which did not manifest enzyme activity . Testosterone and oestradiol levels in culture media were determined by radioimmunological assays . Sertoli cells in co-culture showed a tendency to organize themselves as in vivo, forming a kind of pseudo-wall of the tubule . This process becomes more evident with the time of culture . Co-cultures secreted more androgens than Leydig cells alone and more oestradiol than Sertoli cells alone . This influence was strengthened by the presence of follicle stimulating hormone (FSH) in the culture medium, which was not the case in cultures of Leydig and Sertoli cells cultured separately.

Connect Tissue Res, 1989, 23(1), 89 - 99
A histological and biochemical assessment of the cartilage matrix obtained from in vitro storage of osteochondral allografts; Amiel D et al.; Fresh osteochondral allografts were stored at 4 degrees C in tissue culture media at variable time periods (3, 7, 14 and 28 days) . Sterilely dissected tibial plateaus with a standardized 1/2 cm subchondral bone "shell" were obtained from canines 1-3 hrs post mortem . X-rays were taken to determine maturity of the animals . Only mature animals (closed epiphyses) were considered for the study . Histologically, safranin 0 (metachromatic stain for glycosaminoglycans) was observed in all experimental specimens . H&E stained sections showed at all time periods of 3, 7, 14 and 28 days that the cell morphology and arrangements were similar in the superficial and deep areas of the cartilage obtained from the stored osteochondral allograft when compared to the control articular cartilage . The cells were in lacunae and arranged in clusters . Biochemically, glycosaminoglycans and collagen content showed no difference at the 95% level of confidence during the duration of the study (28 days) when compared to the 0 day control cartilage . Collagen typing, based on the assessment by HPLC of the CNBr peptides showed the major presence of type II collagen (no evidence of dedifferentiation was observed) . No type I was found to be present . Some apparent variations in the proportions of minor collagen components were noted--e.g . at 14 days the cartilage appeared to contain increased amounts of type XI but little or no type IX collagen (HMW, LMW) when compared to the day 0 control . At 28 days a shift to a larger amount of type IX collagen occurs, especially in the LMW component, with a small amount of type XI collagen when compared to normal day 0 articular cartilage . Cell viability, i.e., the ability of the allograft tissue to incorporate 35SO4 in the synthesis of glycosaminoglycans, was intact up to 28 days of storage.

Acta Microbiol Bulg, 1989, 24, 76 - 81
{Citric acid biosynthesis by Aspergillus niger strain IM-13 on a nutrient medium with methanol}; Georgieva M et al.; The dynamics of citric acid biosynthesis and some morphological changes of Asp . niger IM-13 in culture media containing starch hydrolysate and 3% methanol were traced . The culture developed in filamentous form and little fine pellets . Deformed hyphae with characteristic extensions were observed . The changes in Asp . niger IM-13 metabolism were studied by comparing the biomass accumulation, the utilization of reducing substances, the citric acid biosynthesis, pH and the protein content of the control and the experimental variants . A suppression of growth in the period from the 48th hour of cultivation up to the end of the process, suppression of protein synthesis in the micelium and increased synthesis of citric acid were established.

Bull Cancer, 1989, 76(8), 805 - 11
{Hormonal regulation of GCDFP-15 secretion in breast tumors . Study of cultured cells}; Boutteville C et al.; We studied the hormonal regulation of the Gross Cystic Disease Fluid Protein 15 kd (GCDFP-15) which is secreted by mammary epithelial cells . Its precise role is still unknown . For some authors, it is associated with a risk of breast cancer, and for others with a functional differentiation of the cells . We measured the secretion of GCDFP-15 by an immunoenzymatic method in culture media and cytosols of mammary epithelial cells MCF-7, MDA-MB-231 and HBL-100 . We also measured the GCDFP-15 secreted by explants of human tumors in organ cultures . We compared the basal values of GCDFP-15 with those obtained after estradiol, dihydrotestosterone and tamoxifen stimulations . Our results show a decrease of GCDFP-15 production with estradiol and an increase with dihydrotestosterone and tamoxifen . These variations are not systematically correlated with variations of cell growth . This study highlights the interest of GCDFP-15 as a tumoral marker; the study of GCDFP-15 level variations could be useful in the monitoring of cancer response to hormonal and/or chemotherapeutic treatment.

Arch Exp Veterinarmed, 1989, 43(4), 623 - 35
{The detection of in vitro capacitation of bull sperm by heparin treatment}; Blottner S et al.; The capacitating effect of heparin upon spermatozoa from original and deep-frozen semen was characterised, using new methods for detection of inducible acrosomal reaction and heparin-mediated sperm aggregation, and was compared with frequently used capacitation by media of high ion strength . Heparin treatment was undertaken also by means of two culture media, "defined medium" (DM) and TCM 199, with 10% fetal calf serum . Higher motility was maintained by means of 10 I.U . of heparin/ml (= 77 micrograms/ml) which also proved helpful in achieving higher capability of inducible acrosomal reaction, as compared to pretreatment, using media of high ion strength . This applied to both fresh and deep-frozen sperm . The highest level of inducible acrosomal reaction was achieved after 2 hours of heparin action on fresh sperm and 30 minutes of action on deep-frozen sperm . That highest value was at its maximum, when TCM with 10% fetal calf serum had been used . This was the medium, after all, in which photometrically recorded aggregation of motile spermatozoa was at its fastest rate, reaching its maximum after about 60 minutes . The photometrically recorded activated motility of spermatozoa occurred more frequently in TCM, as compared to DM . Preparation of bull sperm in TCM 199 with fetal calf serum and heparin may be recommended as an effective and time-saving method for in vitro capacitation.

Neuroscience, 1989, 31(3), 649 - 61
Basic fibroblast growth factor promotes in vitro survival and cholinergic development of rat septal neurons: comparison with the effects of nerve growth factor; Grothe C et al.; The effects of basic fibroblast growth factor and nerve growth factor on survival and transmitter expression of cultured rat embryonic (E18) septal neurons were studied . Two different culture media were used: (i) a horse serum-containing Leibowitz L-15 medium and (ii) a serum-free N1-supplemented Dulbecco's modified Eagle's medium . Addition of basic fibroblast growth factor to either culture medium enhanced neuronal survival in low density cultures after 4 days . The effects of basic fibroblast growth factor were dose-dependent and blocked by anti-basic fibroblast growth factor antibodies . In serum-containing L-15 medium nerve growth factor also promoted neuronal survival . Basic fibroblast growth factor and nerve growth factor supported neurons comprised both cholinergic and GABAergic subpopulations . The effects of basic fibroblast growth factor and nerve growth factor were not additive . In high density cultures using serum-containing L-15 medium survival of septal neurons was four times higher than in low density cultures after 4 days . Addition of basic fibroblast growth factor or nerve growth factor did not further augment neuronal survival in high density cultures . Maintenance of septal neurons at high density was not affected by antibodies to basic fibroblast growth factor and/or nerve growth factor . Addition of basic fibroblast growth factor or nerve growth factor to serum-containing L-15 medium at high cell density significantly enhanced choline acetyltransferase activity 3- and 7.5-fold, respectively, without affecting cell survival . In conclusion, next to nerve growth factor, basic fibroblast growth factor, which has been located in the hippocampal target area of septal neurons, appears to be another potent trophic factor for septal neurons.

Tissue Cell, 1989, 21(4), 479 - 94
Microtubules, organelle transport, and steroidogenesis in cultured adrenocortical tumor cells . 1 . An ultrastructural analysis of cells in which basal and ACTH-induced steroidogenesis was inhibited by taxol; Benis R et al.; In adrenocortical cells, the first step in the enzymatic processing of cholesterol to steroid end products occurs in the mitochondria . ACTH increases mitochondrial cholesterol and steroidogenesis . In cultured mouse adrenocortical tumor cells, microtubule-based organelle motility may increase the proximity of mitochondria to the SER, lipid droplets and endoscome-derived lysosomes, thereby facilitating the transfer of cholesterol from these organelles to the mitochondrial outer membrane . ACTH may increase opportunities for the transfer by promoting organelle motility and by increasing the number of lysosomes . Taxol, a microtubule polymerizer, inhibits basal and ACTH-induced steroidogenesis in these cells, presumably at the step where mitochondria obtain cholesterol . We examined the ultrastructure of taxol-treated, unstimulated and ACTH-stimulated cells, seeking alterations which conceivably could interefer with the proposed organelle transport and encounters, and thus correlate with taxol's inhibition of steroidogenesis . Primary cultured cells were incubated in serum-containing medium for 4 hr with and without ACTH (10 mU/ml), with 10 micrograms/ml and 50 micrograms/ml of taxol, and with ACTH and taxol 10 or taxol 50 simultaneously . Culture media were analyzed for the presence of secreted steroids at the end of 1, 2, and 4 hr of incubation . At the end of the fourth hour, unstimulated cells and cells treated with ACTH, taxol 50, and both agents simultaneously, were fixed and processed for EM . Taxol inhibited basal and ACTH-induced steroidogenesis in a dose-dependent fashion . In both unstimulated and ACTH-stimulated cells, taxol 50 formed numerous microtubule bundles, but did not markedly change the distribution of mitochondria and lipid droplets . SER tubules, and clusters of Golgi fragments, endosomes, and lysosomes appeared to be translocated towards the cell periphery along some of the microtubules . Taxol permitted an ACTH-induced cell rounding and microfilament rearrangement considered to facilitate organelle motility . Our data indicate that taxol disrupts the formation of lysosomes by these adrenal cells, but it seemed unlikely that taxol's ultrastructural effects could prevent organelle transport proposed to cause meetings between mitochondria and the SER or lipid droplets, or prevent ACTH-caused increases in these encounters . Taxol may instead prevent the transfer of lipid droplet or SER-contained cholesterol to adjacent mitochondria, by a means not detectable in our electron micrographs.

Connect Tissue Res, 1989, 20(1-4), 289 - 94
Transforming growth factor-beta regulation of collagenase, 72 kDa-progelatinase, TIMP and PAI-1 expression in rat bone cell populations and human fibroblasts; Overall CM et al.; Quiescent cultures of normal fetal rat calvarial bone cell populations (RC I and RC IV) and human fibroblasts were incubated with 1.0 ng/ml TGF-beta and the conditioned culture media were processed individually to separate collagenase and 72 kDa-progelatinase from TIMP, the tissue inhibitor of matrix metalloendoproteinases, using mini-columns of heparin- and gelatin-Sepharose . Collagenase synthesis was decreased progressively by TGF-beta in fibroblasts despite a 1.6-fold increase in secreted protein levels and a approximately 1.8-fold increase in 72 kDa-progelatinase synthesis . The human fibroblasts and the osteoblast-enriched RC IV cells showed a greater TGF-beta-induced stimulation in 72 kDa-progelatinase levels over controls compared with the RC I cells . In contrast to RC IV cells, in which TIMP mRNA levels were increased 2.9-fold by TGF-beta, the constitutive level of TIMP transcripts in the RC I cells was greater than 20-fold over that of the RC IV cells, but was not elevated by TGF-beta . TGF-beta also increased TIMP expression in fibroblasts approximately 1.7-fold and PAI-1 levels approximately 5-fold in RC IV cells, and greater than 10-fold in fibroblasts.

Adv Exp Med Biol, 1989, 247B, 1 - 8
Human kallistatin, a new tissue kallikrein-binding protein: purification and characterization; Wang MY et al.; A new and specific tissue kallikrein-binding protein was identified in mammalian serum and in secreted transformed-cell culture media (Chao et al., Biochem . J . 239: 325-331, 1986) . We have designated this kallikrein-binding protein as "kallistatin" . Human kallistatin has been purified from serum, using chromatographic steps including DEAE-Sephadex, hydroxylapatite, Cibacron blue-Sepharose, Sephacryl S200, and preparative polyacrylamide gel electrophoresis . The purified kallistatin consists of a single polypeptide chain with an apparent molecular weight of approximately 54 kDa and isoelectric point of approximately 5.0 . Kallistatin was eluted as a single peak on reverse-phase HPLC . The purified kallistatin and 125I-labelled human tissue kallikrein form a approximately a 92 kDa SDS- and heat-stable complex . The complex formation is pH dependent and is inhibited by 0.1% (W/V) of deoxycholate or SDS but not by 0.5% (W/V) of Triton X-100, digitonin, Lubrol or CHAPS . A approximately 54 kDa protein was identified in partially purified kallistatin by polyclonal anti-kallistatin antibodies in Western blot analysis and by its binding to 125I-labelled-human tissue kallikrein in ligand blotting . The role of kallistatin in regulating tissue kallikrein activity and metabolism may now be evaluated.

Microbiol Immunol, 1989, 33(9), 733 - 45
Effects of infection by HIV-1, cytomegalovirus, and human measles virus on cultured human thymic epithelial cells; Numazaki K et al.; A tissue culture system for the growth of human fetal and infantile thymic epithelial (TE) cells has been established and characterized . We have investigated the effects of infection of these cells by human cytomegalovirus (CMV), measles virus, and human immunodeficiency virus type-1 (HIV-1) . In the case of CMV, morphological changes were apparent by 2-4 days after viral inoculation of infantile TE cells . CMV-related antigens were detected by immunofluorescence after 12 days, and progeny infectious CMV was recovered from culture media after 18 days . Following infection by measles virus, distinctive, multinucleated giant TE cells appeared in both cultures of fetal and infantile TE cells . Measles virus-inoculated TE cells displayed an altered phenotype, as revealed by reaction with monoclonal antibodies with specificity for a variety of TE markers . Finally, infection of TE cells by HIV-1 resulted in cellular disarrangement, increased numbers of Hassall's corpuscles, and multinucleated giant cells . An increase in the number of cells reactive with monoclonal antibodies, specific for Hassall's corpuscles, was observed in the case of cells infected by either measles virus or HIV-1 . These findings suggest that a variety of different viruses can successfully infect thymic epithelial tissue . Because of the important role of the thymus in development of the immune system, it is reasonable to conclude that viral infection of thymic tissue might play an important role in virus-mediated suppression of immune responsiveness.

Virologie, 1989 Jan-Mar, 40(1), 35 - 8
Epstein-Barr virus (EBV) . VII . Established lymphoid cell line (IVPat-88) obtained from synovial fluid of a patient with aseptic arthritis; Patrascu IV et al.; Attempts have been made to culture mononuclear cells from synovial fluid of 8 patients with arthropathy, and have led to the development of the lymphoid cell line IVPat-88 . Cell line has been propagated by serial passages for more than 14 weeks in continuous culture . The cells grew as single, free-floating individuals, or in dense clumps without adherence to glass or plastic surface . All these cells were identified as altered lymphoblasts because of their growth pattern and uniform morphology, and the presence of Epstein-Barr Viral Capsid Antigen (VCA) in 5 to 10% of the cells . The cell concentration varied during the period of culture from about 300,000 to 1,700,000 cells per ml, and mean doubling time during phases of active growth was 42 and 60 hours in MEM and RPMI 1640 tissue culture media, respectively . The methods used and the characteristics of the cell line are described.

J Inherit Metab Dis, 1989, 12(2), 139 - 51
Correction of sphingomyelinase deficiency in Niemann-Pick type C fibroblasts by removal of lipoprotein fraction from culture media; Thomas GH et al.; The average sphingomyelinase activity of fibroblasts obtained from 12 Niemann-Pick type C patients was 37.9% of that of normal fibroblasts (27.2 versus 72 nmol (mg protein)-1 h-1) when the cells were cultured in minimum essential medium containing 13% fetal bovine serum . Following replacement of the above medium with medium in which the lipoprotein fraction had been removed from the fetal bovine serum, the sphingomyelinase activity rose over a 7-day period from about 1/3 of normal to normal or above . Upon reintroduction of medium containing 10% fetal bovine serum which had not been extracted, the sphingomyelinase activity of the Niemann-Pick type C cells again fell within 48 h to 30% of the normal controls . In contrast, cell lines from patients with either Niemann-Pick Type A or B were not influenced by the presence or the absence of lipoprotein, i.e . lacked sphingomyelinase activity under all culture conditions examined . Histochemical staining with filipin showed an inverse relationship between the sphingomyelinase activity and intracellular, free, unesterified, cholesterol level . Moreover, immunochemical staining with an antibody against a lysosomal membrane protein provided direct evidence that the accumulation of unesterified cholesterol in cells cultured in regular (non-extracted) medium occurred within lysosomes and/or related organelles.

J Clin Invest, 1989 Jan, 83(1), 90 - 4
Differential regulation of protein kinase C and (Na,K)-adenosine triphosphatase activities by elevated glucose levels in retinal capillary endothelial cells; Lee TS et al.; Elevated cellular sorbitol levels resulting from conversion of increased glucose by aldose reductase might deplete cellular myoinositol content, which could then lower inositol phosphates (InsPs) and diacylglycerol levels, key regulators of protein kinase C (PKC) . Secondary to altered PKC activity, other cellular enzymes such as (Na,K)-ATPase could be affected . To test this hypothesis we examined the association between PKC activity, (Na,K)-ATPase activity, and sorbitol, myoinositol, and InsP levels in cultured bovine retinal capillary endothelial cells, a cell type prominently involved in diabetic retinopathy . Elevating glucose concentration in culture media from 100 to 400 mg/dl led to a 100% increase in sorbitol levels, which could be inhibited completely by sorbinil, an aldose reductase inhibitor . In contrast, no changes were observed in myoinositol or InsP levels . Subfractionated PKC activities showed a 100% increase in the membranous pool with a parallel decrease in the cytosolic fraction . Adding sorbinil did not affect PKC activity, whereas the PKC agonist, phorbol myristate acetate (PMA), stimulated translocation of PKC . Ouabain-inhibitable (Na,K)-ATPase activity was decreased 70% by elevated glucose levels . This decrease could be prevented by adding either PMA or sorbinil . Thus, in retinal capillary endothelial cells elevated glucose concentration can affect PKC and (Na,K)-ATPase activities, probably via different mechanisms.

Int J Biochem, 1989, 21(3), 313 - 6
Excretion of acetylated and free polyamines by polyamine depleted Chinese hamster ovary cells; Hyvonen T; 1 . Cultured Chinese hamster ovary cells (CHO) and their ornithine decarboxylase deficient mutant cells (C55.7) were found to excrete small amounts of N8-acetylspermidine and free polyamines, putrescine and spermidine into the culture medium . 2 . The concentration of N8-acetylspermidine in the control cells was 2-3% of that of spermidine . In the medium, however, the amount of N8-acetylspermidine was about 2-fold that of spermidine and 2- to 3-fold higher than the intracellular amount . N1-acetylspermidine or acetylated spermine were never detected in the cells or in the media . 3 . Confluent CHO cells treated with 2 mM difluoromethylornithine stopped the excretion when the intracellular spermidine concentration had decreased to 20% of control while there was no decrease in spermine concentration . At low cell density, neither polyamine depleted CHO cells nor the C55.7 cells excreted any polyamines into the culture media.

J Pharm Biomed Anal, 1989, 7(5), 593 - 600
A sensitive immunoradiometric assay for the quantification of murine monoclonal antibodies in human serum; Apathy JM et al.; The clinical investigation of murine monoclonal antibodies (MoAbs) as potential immunotherapeutic agents necessitates their quantification in human serum . The present paper reports the development of a sensitive, non-competitive "sandwich" immunoradiometric assay specifically optimized for measurement of murine IgG in human serum . Affinity-purified goat anti-mouse IgG antibody covalently bound to acrylic microspheres serves as the solid-phase antibody and 125I-labelled goat anti-mouse IgG antibody functions as tracer . All assay reagents were obtained from commercial sources . The assay is sensitive (capable of detecting 20 ng ml-1 of murine IgG), specific (less than 0.0001% cross-reactivity with human IgG), reproducible (intra-assay % RSD less than 6.3%), and rapid (a 100-tube assay can routinely be processed in 3 h) . The assay has a working concentration range of 20-2000 ng ml-1 and is suitable for measuring MoAbs of any antigenic specificity . The assay was validated for use with human serum and cell culture media by comparison studies with flow cytometric and immunonephelometric methods and by high-performance size-exclusion chromatographic studies . Application of this assay should facilitate further investigation of murine MoAbs as potential immunotherapeutic agents.

Reprod Fertil Dev, 1989, 1(2), 89 - 98
Effects of macromolecules recovered from uterine luminal fluid on the metabolism of {U-14C}glucose by mouse morulae and early blastocysts in vitro; Khurana NK et al.; Day-4 mouse embryos grew well in culture media supplemented with macromolecular components of uterine fluids recovered on day 3, 4 or 5 of pregnancy and pseudopregnancy . Addition of these components to media during a 2-h pulse culture had no significant effect on the incorporation of glucose carbon by morulae/early blastocysts . However, various fractions of uterine luminal macro-molecules significantly increased the turnover of glucose carbon incorporated into acid-soluble and acid-insoluble glycogen, into nucleic acids and into proteins during a 24-h chase culture . These effects were due mainly to components with a molecular weight between 1000 and 10,000 Da and the activity was most marked in fluids collected on day 5 of pregnancy or pseudopregnancy . Oxidation of glucose during a 4-h incubation was inhibited in the presence of certain uterine macromolecules but most consistently by the large molecular weight component (greater than 300,000 Da) . Some differences were noted in the inhibitory activity of macromolecules obtained from pregnant and pseudopregnant sources . There was little evidence of an effect of uterine-fluid components on lactate production from glucose.

J Physiol, 1989 Jan, 408, 199 - 222
The role of potassium channels in Schwann cell proliferation in Wallerian degeneration of explant rabbit sciatic nerves; Chiu SY et al.; 1 . Patch clamp studies of whole-cell ionic currents and biochemical studies of proliferation were carried out on Schwann cells of myelinated axons in explant segments of sciatic nerves of adult rabbit maintained in culture for 0-10 days . 2 . Schwann cell proliferation, as assayed by {3H}thymidine incorporation and by electron microscopic autoradiography, showed an increase following nerve explant . Proliferation proceeded in parallel with a gradual hyperpolarization of the resting potential and an increase in K+ currents in Schwann cells of myelinated axons . 3 . The relation between K+ channels and proliferation was studied by incubating explant nerves in the presence of various K+ channel blockers . Quinine, TEA and 4-aminopyridine (4-AP), which blocked K+ currents in Schwann cells, were found also to block Schwann cell proliferation in a dose-dependent fashion and over similar concentrations . Electron microscopy showed that TEA did not retard myelin and axonal break-down which is thought to be the source of mitogens for Schwann cell proliferation . 4 . The relation between resting potential and proliferation was studied by incubating explant nerves in depolarizing culture media . Depolarizing monovalent cations (K+ and Rb+) led to a marked inhibition of Schwann cell proliferation . However, Cs+ and NH4+, which did not depolarize Schwann cells in patch clamp studies, also inhibited proliferation . Gramicidin and veratridine also inhibited proliferation . 5 . The results suggest that the expression of K+ channels is functionally important for Schwann cell proliferation in Wallerian degeneration . A possible link between K+ channel and proliferation might be via a hyperpolarization of the resting membrane potential which occurs when Schwann cells proliferate.

J Orthop Res, 1989, 7(5), 637 - 44
Histological and biomechanical assessment of articular cartilage from stored osteochondral shell allografts; Kwan MK et al.; Normal and stored articular cartilage from the medial tibial plateaus of mature canine knee joints were evaluated histologically and biomechanically . The medial plateaus from the right knee (control) were assessed fresh, while the left (stored) were preserved in culture media at 4 degrees C for 3, 7, 14, or 28 days and then evaluated . Biomechanically, confined compression tests were performed on all specimens to determine the aggregate modulus and apparent permeability of the articular cartilage . Histologically, Safranin O- and hematoxylin and eosin (H&E)-stained sections were evaluated . All stored cartilage specimens had an aggregate modulus on average lower than normal, but the differences were not significant (p greater than 0.10) . The apparent permeability was on average higher than but also not significantly different from normal (p greater than 0.10) . Time in storage (up to 28 days) did not have a significant effect on the biomechanical properties of stored cartilage normalized by control values (p greater than 0.50) . Safranin O and H&E histological evaluation also showed no overall changes in cell appearance or staining of the stored cartilage when compared with control for the time periods studied.

Med Microbiol Immunol (Berl), 1989, 178(2), 89 - 98
Replication of cytomegalovirus in human thymic epithelial cells; Numazaki K et al.; Cytomegalovirus (CMV) has often been cited as a cause of immune suppression in children, yet little is known of the mechanisms through which this agent might affect immune function . We have succeeded in using CMV to productively infect cultured human fetal and infantile thymic epithelial (TE) cells . Morphological changes were apparent by 2-4 days after viral inoculation . CMV-related early antigen (EA) and late antigen (LA) were detected by immunofluorescence after 8 days, and progeny infectious CMV was recovered from culture media after 12-17 days . TE cells that reacted with monoclonal antibodies specific for keratin and for GQ ganglioside were predominant throughout the culture period . In contrast, infection by CMV resulted in a significant decrease in numbers of cells reactive with monoclonal antibodies specific for mesoderm-derived components . Inoculation of TE cells with CMV also caused a diminution in levels of detectable interleukin-1 (IL-1)-related antigen by 17 days after infection.

Tumori, 1988 Dec 31, 74(6), 669 - 74
Assay of GCDFP-15 by ELISA: an available method for in vitro studies of functional differentiation in human breast cancer; Revillion-Carette F et al.; An enzyme-linked immunosorbent assay (ELISA) was applied to a light protein, isolated from human breast cyst fluid (BCF) termed "gross cystic disease fluid protein - 15 Kda" (GCDFP-15), a potential differentiation marker in in vitro human breast cancer studies . The detection limits of this procedure, performed in microtiter plates, were 0.5 to 250 ng/well corresponding to 10 ng/ml to 5 micrograms/ml of sample or antigen solution . Possible cross-reaction with various antigens, especially those found in culture media, were investigated . The correlation coefficient between enzymoassay and radioimmunoassay was 0.978 . The results showed that quantification of GCDFP-15 by ELISA is a specific and highly sensitive method . This procedure may be of interest in in vitro studies on the functional differentiation of breast cancer cells.

J Biol Chem, 1988 Dec 5, 263(34), 18574 - 7
Cyclic AMP-mediated stabilization of osteocalcin mRNA in rat osteoblast-like cells treated with parathyroid hormone; Noda M et al.; Osteocalcin (bone gamma-carboxyglutamic acid-containing protein) is exclusively produced by osteoblasts, which are the major target cells of parathyroid hormone (PTH) in bone . This study examined the effect of human (h) PTH(1-34) on osteocalcin gene expression in the rat osteoblast-like osteosarcoma cells ROS17/2.8 . hPTH(1-34) increased in a dose-dependent manner the steady state levels of osteocalcin mRNA 2- to 3-fold with an ED50 of about 5 X 10(-10) M . This effect was detectable at 12 h, peaked at 24 h, and lasted at least up to 48 h . Forskolin, cyclic 8-bromo-AMP, isobutylmethylxanthine, cholera toxin, and (-)-isoproterenol similarly elevated osteocalcin mRNA . hPTH(1-34) did not alter the transcriptional rate of the osteocalcin gene, estimated by nuclear run-on assays, but increased the stability of osteocalcin mRNA . hPTH(1-34) also increased 2- to 3-fold the osteocalcin level in the culture media determined by radioimmunoassay . PTH, thus, promoted osteocalcin gene expression in these cells at least in part through mRNA stabilization via cyclic AMP mediation, a mechanism known only in few systems.

Biol Reprod, 1988 Dec, 39(5), 1183 - 92
Two-cell block to development of cultured hamster embryos is caused by phosphate and glucose; Schini SA et al.; The failure of hamster 2-cell embryos to develop in vitro (2-cell block) was examined with experiments in which concentrations of glucose and phosphate in the culture medium were varied . Embryos were cultured in a protein-free modified Tyrode's solution that normally contains 5.0 mM glucose and 0.35 mM sodium dihydrogen phosphate . In the presence of 0.35 mM phosphate but without glucose, 23% of 2-cell embryos reached the 4-cell stage or further after culture for 1 day and 27% after 2 days . Glucose inhibited embryo development even at 0.1 mM (4% development to greater than or equal to 4-cells after culture for 2 days); there was no dose-related inhibition above this glucose concentration . In a second experiment, phosphate levels were varied in the absence of glucose . Phosphate was highly inhibitory to development, with 97% of 2-cell embryos reaching the 4-cell stage or further after culture for 1 day in the absence of phosphate compared to 9-21% in the presence of 0.1-1.05 mM phosphate . After culture for 2 days, 26% of embryos reached the 8-cell stage or further when phosphate was absent compared to 0% development to 8-cells with 0.1 mM phosphate or higher . In a factorial experiment, phosphate blocked development when glucose was present or absent, whereas glucose did not block embryo development in the absence of phosphate . However, 2-deoxyglucose (a non-metabolizable analogue of glucose) inhibited embryo development in the absence of phosphate . These data show that the in vitro block to development of hamster 2-cell embryos is caused at least in part by glucose and/or phosphate . Deletion of these compounds from the culture medium eliminates the 2-cell block to development in virtually all embryos, and approximately 25-75% of embryos develop to the 8-cell or morula stages in vitro . The observations provide a possible explanation for the 2-cell and 4-cell blocks that occur in conventional culture media: stimulation of glycolysis by glucose and/or phosphate may result in inefficient adenosine triphosphate (ATP) production . The data indicate marked dissimilarities in the regulation of in vitro development of early cleavage stage hamster embryos compared with embryos of inbred mice, since the latter have an inactive glycolytic pathway prior to the 8-cell stage of development and will grow from 1-cell to blastocyst with both phosphate and glucose in the culture medium.

Neurosci Res, 1988 Dec, 6(2), 115 - 22
Myelin formation in rat dorsal root ganglion cultured in a serum-free medium: influence of various culture media on myelin formation; Takahashi K et al.; Survival and myelination in dorsal root ganglion culture were observed using 6 kinds of modified commercial serum-free media: MEM, neMEM, alpha-MEM, DMEM, F12 medium and DF medium (see Materials and Methods) . The results showed that the survival rates of dorsal root ganglia cultured in alpha-MEM and DF medium are the highest and that the rates of myelinated ganglia are the highest when alpha-MEM is used as culture medium, as compared with other serum-free media.

In Vitro Cell Dev Biol, 1988 Dec, 24(12), 1159 - 64
Culture conditions found to minimize false positive diagnosis of lysosomal storage disorders; Arnon J et al.; The effect of culture conditions on the ultrastructure and enzyme activities of cultured skin fibroblast cells relevant to the diagnosis of lysosomal storage disorders are reported . The parameters examined were: pH of the culture media, type of media, increasing cell passage, and day of harvest . Ultrastructural changes were defined in terms of the number of lysosome-like inclusion bodies per cell according to a method devised in our laboratory and proven reliable in the detection of affected individuals . Our biochemical results included determination of enzyme activities of beta-hexosaminidase, alpha-mannosidase, beta-glucuronidase-lysosomal enzymes, arylsulfatase C, a microsomal marker, and 5' nucleotidase, a plasma membrane marker . Our results indicate that the cellular ultrastructure is more sensitive than enzyme activity to changes in culture conditions . The resulting ultrastructural "artifacts" observed under certain conditions were severe enough to result in a mistaken diagnosis . Due to certain difficulties we had previously encountered in heterozygote cultures (for lysosomal storage disorders) of amniotic cells, we decided to examine heterozygote cultures of skin fibroblasts . From these (preliminary) studies it seems that an elevation in the pH over the physiologic levels in the culture media may help to define between normal individuals and affected heterozygotes . On the basis of our results, we recommend that to minimize false positive ultrastructural results for the diagnosis of lysosomal storage disorders, cultures be grown in minimal essential medium, the pH of the medium carefully monitored to remain below 7.4, examining the cultures not later than cell Passage 8 and no later than Day 10 after subculture.

Exp Mol Pathol, 1988 Dec, 49(3), 316 - 29
The fibroblast-like nature of myofibroblasts; Oda D et al.; The myofibroblast is found in normal tissue as well as in a wide variety of pathological processes . We have cultured myofibroblasts and dermal fibroblasts and have found that they secrete similar type-specific procollagens into the culture media . These were primarily type I and III procollagens with a predominance of type I procollagen . These patterns are distinctly different from those of smooth muscle cells, which synthesize predominantly type III procollagen . Cultured fixed cells were also examined by immunohistochemistry . Both myofibroblasts and fibroblasts stained positively with antibodies to type I and III procollagens . Reaction to type V procollagen antibodies was prominent only in the myofibroblast, as was immunostaining with anti-muscle actin antibodies . Immunostaining with desmin antibodies was negative in both cell types . By electron microscopy, the myofibroblast had well-developed dense microfilament fibers of 40-80 degrees that were prominent in the long axes of the cells near the cellular margins . Although the myofibroblast has properties of both smooth muscle cells and fibroblasts, it appears to be most likely a modified fibroblast that has undergone differentiation, probably in response to specific signals from the extracellular matrix.

Experientia, 1988 Dec 1, 44(11-12), 1007 - 10
Vitamins and other metabolites in various sera commonly used for cell culturing; Baker H et al.; Many cell culture media use different sera to enhance growth . We assayed vitamins and some related metabolites in different sera and identified the concentration of: thiamin, biotin, folates, riboflavin, pantothenates, nicotinates, vitamins B6, B12, A, E, C, and carotenes and some related metabolites: biopterins, free inositol, free and total choline, total carnitines in chicken, horse, rabbit, goat, pig, calf, newborn calf, fetal calf and human sera . Results indicate that vitamin and metabolite content of different sera vary . Such variations could produce fluctuant effects on cell culturings if the metabolite content of the serum is not documented.

Cancer Res, 1988 Dec 1, 48(23), 6855 - 62
Immunohistochemical and biochemical study of a cathepsin B-like proteinase in human colonic cancers; Keppler D et al.; An immunohistological study was carried out on 51 human colorectal adenocarcinomas and eight samples of histologically normal colonic mucosa removed far from tumors, using anti-rabbit cathepsin B and anti-human cathepsin B immunoglobulins . Positive reactions were obtained on tumor cells and macrophage-like cells . However, as these immunoglobulins could not discriminate between cathepsin B and cathepsin B-like proteinases, and as they cross-reacted with cathepsins H and L, a partial characterization of the proteinase activities was performed in order to identify the type of enzyme present in the positive cells . The levels of cathepsins H and L were very low in extracts of colorectal tumors and normal colonic mucosa . A peculiar cathepsin B-like proteinase activity with pH optimum at 6.8 was found in tumor extracts together with the lysosomal cathepsin B, whereas normal colonic mucosa showed only cathepsin B activity (pH optimum, 6.0) . These results indicate that lysosomal cathepsin B is responsible for staining of macrophage-like cells found in the lamina propria of colonic mucosa and in the peritumoral stroma . Immunohistochemical staining of colonic tumor cells observed in 29/51 cases seems on the other hand to be primarily due to a cathepsin B-like proteinase . Three colonic tumor cell lines, Colo-205, HT-29, and SW-1116, were also studied using the same methods . These cells produced a latent cathepsin B-like proteinase which, after activation, was similar to that found in tumor extracts . This latent proteinase was detected mainly in the culture media . The cultured colonic tumor cells, after staining by anti-cathepsin B antibodies, showed strongly positive granules . In conclusion, this work demonstrates that malignant colonic cells are the source of a cathepsin B-like proteinase, with optimal activity near neutrality . Its secretion into the extracellular space indicates furthermore, that it may be an important component of the "proteinase cascade" associated with tumor invasion and metastasis.

Am J Obstet Gynecol, 1988 Dec, 159(6), 1484 - 90
Fatty acid content of yolk sac and embryo in hyperglycemia-induced embryopathy and effect of arachidonic acid supplementation; Pinter E et al.; Using the postimplantation rat conceptus model, we analyzed with gas-liquid chromatography, the fatty acid composition in major lipid groups (phospholipids, triglycerides, nonesterified fatty acids, and cholesterol esters) of yolk sacs and embryos cultured for 48 hours under control, hyperglycemic, and arachidonic acid-supplemented hyperglycemic conditions . In all experimental conditions the yolk sacs had greater fatty acid content than the embryos in all lipid groups except in nonesterified fatty acids . The fatty acid level in embryonic nonesterified fatty acids was significantly higher (p less than 0.05) in hyperglycemia-exposed embryos than found with arachidonic acid supplementation . Total yolk sac triglycerides were greater with added glucose (p less than 0.05) than with the addition of arachidonic acid to the same medium . Oleic acid, a fatty acid associated with essential fatty acid deficiency, was increased in the embryonic phospholipids and nonesterified fatty acids of conceptuses exposed to excess glucose, as well as in the culture media of this group, compared with the control or arachidonic acid-supplemented, hyperglycemic group (p less than 0.05) . The results of this study demonstrate that diabetes-related embryopathy is associated with quantitative and qualitative abnormalities in major lipid groups . Furthermore, the elevation in embryonic oleic acid level suggests that the teratogenic mechanism could be related to a deficiency in essential fatty acids . The pattern of essential fatty acid deficiency and embryopathy was preventable with arachidonic acid supplementation in this experimental model.

Infect Immun, 1988 Dec, 56(12), 3015 - 20
Evidence for occurrence of passively adsorbed I antigen activity on a cultured strain of Mycoplasma pneumoniae; Uemura K et al.; The aim of this study was to investigate whether I antigen occurs in association with Mycoplasma pneumoniae in a form that may be immunogenic during natural infection or experimental immunization . I antigen activity was detected by radioimmunoassay in suspensions of M . pneumoniae MY11965 and in the soluble phase of mycoplasma lysates prepared with Triton X-100 . There was evidence for the occurrence of I antigen in at least two macromolecular forms . The first form partitioned in the lipid phase following chloroform-methanol extraction and chromatographed on thin-layer chromatograms as a ceramide decasaccharide . The second form was associated with the residue after lipid extraction and was solubilized by treatment with sodium dodecyl sulfate or pepsin; this component was tentatively designated a glycoprotein or polysaccharide and was not investigated further . In a lipid extract from mycoplasmas that had been surface labeled by the galactose oxidase-NaB3H4 method, two 3H-labeled glycolipids were detected as minor components which chromatographed on thin-layer chromatograms in the region of an authentic I-active ceramide decasaccharide . However, no significant radioactivity was incorporated into glycolipids after metabolic labeling with {3H}glucosamine . These observations suggested that the mycoplasmas contained surface-associated glycolipids with I antigen activity that were of exogenous origin . This was supported by the observations that horse, rabbit, and fetal calf sera contained I antigen activity and that the I antigen activity in M . pneumoniae cultures reflected the levels found in the sera included in the culture media . From rabbit serum, which expressed the highest antigen activity, an I-active glycolipid was isolated that chromatographed as a ceramide decasaccharide . I-active substances passively adsorbed onto M . pneumoniae are potentially immunogenic . However, we consider these unlikely to be the main stimulus for autoantibody production in natural infection, since the autoantibodies elicited are restricted to the I carbohydrate antigen and there is a lack of antibodies to other glycolipids that may be adsorbed from serous and cellular components of the host tissues . In our view, the more likely stimulus is the specific complex formed between the mycoplasma and the sialo-oligosaccharide receptors of the Ii antigen type, as suggested previously.

Acta Ophthalmol (Copenh), 1988 Dec, 66(6), 687 - 91
Long-term follow-up of cryopreserved corneal endothelium . A specular microscopic study; Ruusuvaara P et al.; This paper deals with the long-term survival of corneal transplant endothelial cells . Endothelia of 17 cryopreserved corneal transplants were photographed with a specular microscope, first 1.5 years (mean) following transplantation, and again 10 years later . All transplants were absolutely clear . The mean cell loss was 23% in 12 years . After 2 to 14 years post-operatively the cell loss was noticed to be only 13.6% (from 990 +/- 255 cells/mm2 to 855 +/- 213 cells/mm2) . It seems that endothelial cells of cryopreserved corneal grafts, although few in number, are still quite resistant and viable after a long follow-up time and survive like endothelial cells after preservation in tissue culture media.

J Trop Med Hyg, 1988 Dec, 91(6), 292 - 5
Evaluation of the laboratory diagnosis of vaginal trichomoniasis in Khartoum; Omer EF et al.; A total of 403 vaginal discharge specimens were investigated for Trichomonas vaginalis using fresh wet-mounts, culture and Papanicolaou staining . By fresh wet-mounts, 58 specimens (14.4%) were found harbouring the parasite . On culture 67 isolates (16.7%) were identified . Both wet-mount and cultural procedures detected 18.3% positive isolates . Papanicolaou staining detected 40 positive cases (9.9%) from the patients investigated . Two culture media were tried in this study to isolate T . vaginalis, namely Lumsden's medium and Diamond's medium . Their efficacy was found to be 16.9% and 14.8%, respectively.

J Chromatogr, 1988 Nov 18, 432, 223 - 31
Rapid method for the determination and quantification of bromosulphophthalein and metabolites in cultured hepatocytes, culture media and bile by high-performance liquid chromatography; Van 't Klooster GA et al.; An ion-pair high-performance liquid chromatographic method for the rapid, selective and sensitive analysis of samples containing bromosulphophthalein (BSP) and its conjugates is presented . The method is useful for analysis in bile, culture media and cultured hepatocytes . Two sample preparation methods are described . Even though BSP recovery from albumin binding is complete, only a small percentage of free BSP can be detected in cells, possibly owing to a conjugation-related pool of BSP in cells . As BSP-glutathione recovery is complete, the method offers a useful tool to investigate impairment of glutathione conjugation.

Biochem J, 1988 Nov 15, 256(1), 35 - 40
The small dermatan sulphate proteoglycans synthesized by fibroblasts derived from skin, synovium and gingiva show tissue-related heterogeneity; Larjava H et al.; Dermatan sulphate proteoglycans (DSPGs) synthesized in the presence of 35SO4 were characterized in culture media of fibroblast lines obtained from skin, synovium, and gingiva . The molecular mass of DSPG varied from 95-130 kDa as estimated by SDS/polyacrylamide-gel electrophoresis . Gingival fibroblasts constantly produced larger DSPGs than skin fibroblasts . This was due to the larger dermatan sulphate (DS) chains, which also showed tissue-related heterogeneity in the distribution of 4- and 6-sulphated disaccharide units . The N-glycosylated cores (44 and 47 kDa) obtained following chondroitinase ABC treatment were of identical size in all tissues . The cores from the different tissues were also of the same size (38 kDa) when addition of the N-linked oligosaccharides was inhibited by tunicamycin or when they were removed by N-glycanase treatment . No evidence for low-molecular-mass sulphated oligosaccharides was found . All tissues contained two mRNA species (1.6 and 1.9 kb) for the DSPG core protein . These data suggest that the pattern of transferase activities involved in the construction of DS chains differs from one tissue to another . This variation may modulate the functions of DSPG in the extracellular matrix.

Tsitologiia, 1988 Nov, 30(11), 1355 - 63
{Effect of cultivation conditions on the karyotype structure of a cell subline of the kangaroo rat kidney}; Polianskaia GG et al.; Variations in cultivation conditions were found to exert influence on the distribution of cells for chromosome number by changing the modal class . The change of the HMEM medium for the EMEM medium during 2-6 passages results in the appearance of a new modal class with 16 chromosomes . The change in the chromosome number is preferably due to the loss of one X chromosome within the main structural variant of the karyotype (MSVK) . On the other hand, the change of the HMEM medium for the F12 medium during 4-6 passages does not affect the cell distribution for the chromosome number . A comparative analysis of the total frequency of the MSVK cells and that of MSVK cells of the modal class showed that the karyotypic changes took place in all the variants, both in the modal class and beyond it due to other additive SVK . An exception is the variant NBLD (change of HMEM for the F12 during 6 passages) . In this case chromosome changes occur mostly in the modal class, primarily due to the redistribution of chromosomes in groups . In all the variants there is an insignificant frequency of chromosomes, morphologically different from the MSVK . This confirms the findings according to which chromosomal changeability in the NBLD may be associated mostly with the change in the number of homologous chromosomes rather than with chromosomal aberrations . The frequency of chromosomal aberrations is the same in all the variants examined . The dependence of karyotypic characteristics on culture media mentioned above indicate that care should be taken in choice of culture conditions for permanent cell lines.

Mol Cell Biochem, 1988 Nov, 84(1), 29 - 40
Cholesteryl ester handling by RAW264 macrophages: response to native and acetylated low density lipoprotein; Berg KA et al.; The effects of LDL and Ac-LDL on the growth properties, morphology, and cholesteryl ester (CE) metabolism of the RAW264 macrophage cell line have been characterized . Cells were grown in media supplemented by a defined media (DM) mixture or fetal bovine serum (FBS) . The addition of LDL or Ac-LDL to the culture media did not significantly alter cell growth properties . Cytoplasmic deposition of CE was observed by fluorescence microscopy in macrophages treated with LDL or Ac-LDL but not in untreated controls . Dose-response studies have shown that cholesteryl ester (CE) can accumulate in RAW264 treated with LDL . Cellular cholesterol content saturated at 4 hours with 50 micrograms/ml LDL; this effect may be associated with receptor saturation . Dose-response studies conducted with Ac-LDL in DM have shown dramatic increases in total cell cholesterol content . However, deposition of CE was not observed below Ac-LDL concentrations of 100 micrograms/ml . This indicates that a critical concentration of Ac-LDL must be reached to trigger deposition in DM . In contrast, no critical concentration of Ac-LDL was observed in macrophages grown in medium supplemented with 10% FBS . Cholesterol esterification in response to LDL and Ac-LDL was examined by 14C-oleic acid incorporation into CE . These results confirmed the mass cellular cholesterol and CE measurements . Kinetic studies conducted with RAW264 cells treated with 50 or 100 micrograms/ml Ac-LDL resulted in a cholesterol efflux from the cells at 6-12 hours of incubation . Therefore, these studies show that (1) the nature of CE deposition is highly dependent upon the incubation media and (2) CE deposition is very sensitive to Ac-LDL concentration under certain conditions.

J Infect, 1988 Nov, 17(3), 215 - 20
Effect of blood culture media on the in vitro recovery of Mycoplasma hominis; Davies S et al.; Despite the prevalence of Mycoplasma hominis few cases of septicaemia due to this organism have been reported . The ability of various blood culture media to sustain the growth of an inoculum of M . hominis was therefore studied . Of the media tested, an 'in-house' Hartley's digest broth with 0.1% glucose was the most efficient . The investigation also demonstrated that growth of M . hominis is adversely affected by the concentration of liquoid (sodium polyanetholsulphonate) present in blood culture media.

Biol Reprod, 1988 Nov, 39(4), 779 - 86
Noninvasive measurement of glucose uptake by two populations of murine embryos; Butler JE et al.; Glucose uptake was measured by a noninvasive fluorescence technique on a total of 165 morula- and blastocyst-stage murine embryos in two different culture media . Eighty-four embryos were tested in M2 medium, and the remaining 81 embryos were tested in M16 . Embryos assayed in M2 took up significantly less glucose over the 4-h assay period than did embryos assayed in M16 . The lower uptake of glucose by embryos in M2 corresponded with a decrease in the quality of embryos cultured overnight in M2 as judged by morphological criteria . Embryos that were judged to be degenerate or had gross abnormalities took up significantly less glucose than did normal embryos . Glucose uptake in both populations of embryos covered a wide range of values and was normally distributed . A significant effect between mothers was noted in glucose uptake for embryos assayed in both M16 and M2 media . The possible uses of noninvasive measures of glucose uptake as a test of embryo viability or for optimizing culture conditions are discussed.

J Pharmacol Exp Ther, 1988 Nov, 247(2), 685 - 9
Inhibition of taurocholate transport by cyclosporin A in cultured rat hepatocytes; Kukongviriyapan V et al.; Cyclosporin A, a powerful immunosuppressant, has been shown to interfere with the transport of bile salts and other substrates in isolated rat liver cells and to suppress bile flow and bile salt secretion in situ . Cyclosporin A was added to primary hepatocyte cultures just before taurocholate to study the immediate effects on transport . In prolonged exposure experiments cyclosporin A was dissolved in culture media . Efflux was studied by preloading cultured cells with {14C}taurocholate and then changing to sodium-free buffer containing no taurocholate . Cyclosporin A inhibited the uptake and efflux of taurocholate in a competitive manner when added just before determination of transport . Hepatocyte cultures which were pre-exposed to cyclosporin A (1-25 microM) for 18 hr and then washed, also showed a significantly lower taurocholate uptake . The longer the pre-exposure time the greater was the suppression . However, the inhibition could be reversed gradually by incubation of cultured cells in fresh media, complete reversal being attained within 3 hr . These results indicate that cyclosporin A competitively inhibits the taurocholate transport system . The interaction between cyclosporin A and transport components is rapid and long lasting but reversible.

Xenobiotica, 1988 Nov, 18(11), 1255 - 70
Metabolism of 7,12-dimethylbenz(a)anthracene by different types of cells in the human ovary; Bengtsson M et al.; 1 . The metabolism of 7,12-dimethylbenz(a)anthracene (DMBA) by primary cultures of human ovarian cells has been studied to identify the cell type(s) responsible for biotransformation of this carcinogen . The rate of DMBA metabolism was maximal in granulosa cells prestimulated in vivo with antiestrogen, hMG (human menopausal gonadotropin) and hCG (human chorionic gonadotropin), i.e., treatments required for maximal oocyte maturation and, thus, granulosa cell proliferation . In cells from unstimulated ovaries, the metabolism was maximal in granulosa-lutein cells isolated from corpus luteum . 2 . Steroid (progesterone and estradiol) levels were determined in the spent culture media or in media in parallel with DMBA metabolism to find out whether elevated steroid levels in vivo are required for the rapid metabolism of DMBA . In granulosa cell cultures from stimulated cycles, the concentrations of both progesterone and estradiol were at least 2 or 3 times higher, respectively, than in any of the other cell types tested . In cell cultures derived from unstimulated ovaries, the progesterone and estradiol concentrations were highest in granulosa-lutein cell cultures . 3 . Incubations of granulosa cells with DMBA in the absence or presence of gonadotropins, testosterone or anti--hCG were performed to investigate possible hormonal requirements for the cytochrome P-450 system(s) which metabolize DMBA . No change in the rate of metabolism was obtained with follicle stimulating hormone (FSH), luteinizing hormone (LH), hCG or testosterone . However, anti-hCG increased this activity about 70%, indicating a negative modulatory role of hCG on DMBA mono-oxygenase activity in human granulosa cells . 4 . DMBA mono-oxygenase activity in cell cultures was inhibited about 95% by alpha-naphthoflavone (ANF), an inhibitor of certain cytochrome P-450-catalysed activities . Benzo(a)pyrene (BP) was metabolized at the same rate as DMBA in granulosa cell cultures.

Invest Ophthalmol Vis Sci, 1988 Nov, 29(11), 1708 - 12
Arachidonic acid metabolism in human trabecular meshwork cells; Weinreb RN et al.; Prostaglandins and other eicosanoids in the trabecular meshwork may play important physiological and pharmacological roles in the aqueous outflow pathway . In the present studies, we employed {14C}-arachidonic acid to explore potentially important pathways for the production of eicosanoids in cultured human trabecular meshwork cells (HTM) . In these cells, we demonstrated that prostaglandin E2 (PGE2) and PGF2 alpha are major cyclooxygenase products, with some 6-keto-PGF1 alpha also detected . The amount of radiolabelled PGE2 formed was substantially higher than the PGF2 alpha formed in the early time periods . The amount of PGF2 alpha in the culture media increased at a time when the amount of PGE2 was declining, suggesting a possible metabolic conversion between the prostaglandins . HTM produced a range of products of the lipoxygenase pathway . Products co-eluting with 5, 12, and 15-hydroxyeicosatetraenoic acids (HETEs) were detected, with 12 and 15-HETEs predominating . A large amount of radiolabelled product was detected also in peaks co-eluting with leukotriene B4 (LTB4) and an LTB4 degradation product . Biosynthesis of lipoxygenase products was markedly inhibited by BW 755c and partially inhibited by dexamethasone . These data emphasize that HTM cells are capable of converting arachidonic acid into a wider variety of biologically active products than previously recognized.

Med Toxicol Adverse Drug Exp, 1988 Nov-Dec, 3(6), 449 - 62
Drug-induced agranulocytosis; Heimpel H; Agranulocytosis is a rare but potentially serious adverse side effect of many drugs . Although it was recognised as an idiosyncratic type of drug reaction more than 50 years ago, its pathogenesis is still not fully understood . Drug-related antibodies are responsible for the neutropenia in the so-called 'immune' or 'aminopyrine' type of agranulocytosis . In contrast to former assumptions, the disappearance of leucocytes is not only due to rapid destruction of circulation cells, but it can result also from failure of the production of granulopoetic cells . In some other groups of drugs there is no evidence of immune-mediated disease, but direct toxicity to bone marrow cells has been observed using biochemical methods or inhibition of the growth of granulopoetic colonies in semisolid culture media . Until now it has not been possible to define the enzymatic abnormality which could explain this metabolic type of idiosyncrasy . The quantification of the incidence of potentially drug-induced agranulocytosis in general, and in particular its association with single drugs, requires studies on large populations and the use of strict epidemiological methodology to prevent reporting of grossly biased results . Data from recent case control studies show definitely lower risks for some relevant groups of drugs than formerly appreciated . As expected, agranulocytosis has been observed in association with some recently introduced drugs . This underlines the necessity for continued postmarketing monitoring of potential haematological side effects and for further case control studies to furnish data to aid prescribing physicians and health authorities in decision-making.

J Protozool, 1988 Nov, 35(4), 470 - 5
Maintenance of integrity, viability, and adhesion of Entamoeba histolytica trophozoites in different incubation media; Cano-Mancera R et al.; We have determined the integrity, viability and adhesion of Entamoeba histolytica HK9 and HM1 trophozoites during their incubation in two basal culture media (TP and TYI) and three saline media ("maintenance medium" MM-1 and two others buffered with HEPES) . In basal culture media, more than 70% of the trophozoites maintained their integrity and adhesion to human red blood cells (RBC) for up to 4 h, and the proportion of those excluding Trypan blue decreased slowly after 2 h . In saline media, the number of ameba-RBC complexes reached a maximum after 20-30 min and then decreased rapidly (and fastest in MM-1), less than 10% of the amebae were intact after 3-4 h, and dye exclusion fell abruptly from the start of incubation . The number of ameba-RBC complexes formed and the rate of adhesion were highest in basal TP medium . Normal nonvacuolated refringent (NVR) trophozoites deteriorated progressively in all media--although much faster in the saline ones--to vacuolated refringent (VR), nonrefringent, and disrupted . Trypan blue was excluded by all NVR and a fraction of the VR trophozoites . Horse serum helped to maintain ameba integrity and viability, but inhibited adhesion in a concentration-dependent manner . We conclude that E . histolytica trophozoite integrity and adhesion are adequately preserved and should be characterized only in basal culture media, that refringence without vacuolization is a more stringent characteristic of ameba quality than Trypan blue exclusion, and that some serum component inhibits ameba adhesion.

Acta Endocrinol (Copenh), 1988 Nov, 119(3), 420 - 6
Monoclonal antibodies against rat adrenocortical cell antigens; Laird SM et al.; Cell lines secreting antibodies directed against rat adrenocortical cell antigens have been produced using hybridoma techniques . As tested by immunofluorescent staining, eleven of these antibodies were specific for rat adrenocortical cells in that they did not interact with rat adrenal medulla, liver, muscle, ovary or kidney cells . Two of these antibodies were studied in greater detail . One interacted with an antigen present only in adrenal fasciculata and reticularis cells (inner zone antibody, IZA) . The expression of the antigen with which this antibody interacted was increased by in vivo treatment with ACTH . The other antibody interacted with an antigen present in all three adrenocortical cell types (adrenocortical antibody ACA) . Expression of this antigen was unaffected by ACTH treatment . The effect of both antibodies on steroid biosynthesis by mitochondrial and microsomal preparations of zona glomerulosa and inner zone tissue was tested . For these experiments, the antibodies were purified from culture media by Protein-A-Sepharose affinity chromatography . ACA had no effect on steroidogenesis by any preparation . IZA specifically decreased the production of 18-hydroxydeoxycorticosterone from deoxycorticosterone by the inner zone microsomal and mitochondrial fraction . The results suggest that the IZA antigen is found predominantly in the rat adrenal inner zone and that the antibody may be useful in monitoring adrenocortical zonation . In addition, the antigen would appear to have a functional role in steroid metabolism.

J Neurosci, 1988 Nov, 8(11), 4307 - 18
Antibody to galactocerebroside alters organization of oligodendroglial membrane sheets in culture; Dyer CA et al.; Antibodies to galactocerebroside (GalC) cause major changes in the organization of the membrane sheets elaborated by murine oligodendroglia in culture . Exposure of oligodendroglia to rabbit anti-GalC IgG for 15 min followed by fluoresceinated second antibodies results in patches of surface GalC staining; when second antibodies are applied after 2 hr of anti-GalC, the pattern of staining on membrane sheets is solid and wrinkled . Anti-GalC exposure for 24 hr results in contracted membrane sheets . No membrane contraction is detected in cultures treated with anti-sulfatide IgM or anti-proteolipid protein IgG . In cultures exposed to anti-GalC continuously for 4-7 d, there is a marked decrease in numbers of extended membrane sheets with an accompanying increase in contracted sheets . This effect is reversible upon removal of anti-GalC from the culture media . By scanning electron microscopy, normally flat membrane sheets appear ruffled after 2 hr of anti-GalC treatment; by 24 hr, contracted membrane sheets consist entirely of bulbous protrusions . Oligodendrocyte membranes exposed to anti-sulfatide for 24 hr are not contracted but are covered with bulbous protrusions . The organization of underlying membrane structures was examined in relation to membrane patching and sheet contraction . In membranes with patching induced by exposure to anti-GalC for 15 min, the anti-GalC: GalC complexes are localized over cytoplasmic MBP domains, with the unstained areas located above cytoplasmic microtubular structures . Membrane sheets that are contracted in response to anti-GalC exposure for 6-24 hr show intense GalC staining over microtubular structures . Anti-GalC exposure does not change metabolism of GalC; in cultures incubated with 3H-galactose and anti-GalC for 24 hr, there are no alterations in GalC labeling compared with control cultures . In summary, these results provide direct evidence that interaction between surface glycolipids and external antibodies can initiate a sequence of events leading to dramatic changes within the oligodendrocyte.

J Biol Chem, 1988 Oct 25, 263(30), 15568 - 77
Expression of human preproapo AI and pre(delta pro)apoAI in a murine pituitary cell line (AtT-20) . A comparison of their intracellular compartmentalization and lipid affiliation; Fennewald SM et al.; The role of the NH2-terminal propeptide of human apolipoprotein (apo) AI in intracellular transport and lipid-protein interactions was examined by transfecting a murine anterior pituitary cell line (AtT-20) with the human preproapo AI gene and a mutant gene lacking the 18-base pair segment of exon III encoding its hexapeptide prosegment . ProapoAI was not processed to the mature apolipoprotein either prior to or after export from these cells making this an attractive model system for directly assessing structure/activity relationships of its propeptide . ApoAI was sorted into a regulated pathway for protein export . The signal responsible for this trafficking pattern was not contained in the prosegment since both pro- and mature ApoAI exhibited a similar rate of secretion, a similar magnitude of stimulation of export by the secretagogue 8-bromo-cyclic AMP, and similar targeting to dense core granules . This sorting behavior, exhibited by a protein which is not normally targeted to dense core secretory granules in its cells of origin, raises questions about the domains and mechanisms involved in protein sorting into the regulated and constitutive pathways of AtT-20 cells . Density gradient ultracentrifugation, immunoaffinity chromatographic and electron microscopic analysis indicated that approximately 10% of proapoAI and approximately 10% apoAI appeared in AtT-20 culture media in the form of two discrete classes of nascent lipoproteins: a small 6-8 nm spherical particle and a larger discoidal particle . These particles had morphologies identical with those secreted by Hep G2 cells which normally synthesize apolipoproteins . Although these results do not resolve the issue of whether or not a fraction of apoAI can act as an acceptor of cellular lipid during its transport through the secretory pathway, the data do show that this functional capability for lipoprotein assembly is not obviously regulated by its prosegment.

J Biol Chem, 1988 Oct 15, 263(29), 15159 - 65
Phosphatidylinositol linkage of a truncated form of the platelet-derived growth factor receptor; Orchansky PL et al.; The platelet-derived growth factor (PDGF) receptor is usually anchored to the plasma membrane through a membrane-spanning hydrophobic amino acid sequence that splits the molecule into two approximately equal pieces, an amino-terminal external domain that contains the binding site for PDGF and a carboxyl-terminal cytoplasmic domain that includes the tyrosine kinase coding sequences . Here we report the expression of a truncated PDGF receptor that consists of the extracellular domain without the transmembrane and cytoplasmic domains . Unexpectedly, this form of the receptor that lacks a hydrophobic membrane-anchoring sequence was bound to the membrane and was not secreted into the culture media . Conventional methods to dissociate noncovalent protein-protein interactions failed to release the protein from the membrane . When the transmembrane and cytoplasmic sequences were artificially deleted from the PDGF receptor, the truncated extracellular domain was anchored to the membrane through phospholipids and could be released by phospholipase C treatment . This truncated form of the receptor bound PDGF with an affinity 5-20-fold lower than the full-length receptor.

J Chromatogr, 1988 Oct 14, 431(2), 327 - 42
Purification of monoclonal antibodies against the low-density lipoprotein receptor by preparative isotachophoresis; Schmitz G et al.; A preparative free-flow isotachophoretic method for the purification of monoclonal antibodies from mouse ascites fluid and tissue culture media is described . This high-resolution method allows the direct separation of monoclonal antibodies from antibody-containing tissue culture media or ascites fluid and gives a better separation from the major contaminant protein fractions and a higher recovery of the monoclonal antibodies than anion-exchange chromatography . The purification can run continuously and without any time-consuming regeneration procedures; the monoclonal antibody is obtained under mild conditions in a small electrolyte volume.

J Immunol Methods, 1988 Oct 4, 113(1), 75 - 81
Production of streptavidin in a synthetic medium; Cazin J Jr et al.; A simple, inexpensive procedure for producing streptavidin has been described . The biotin-binding protein was produced by growing Streptomyces avidinii in a synthetic liquid culture medium containing L-asparagine as the sole nitrogen source . With this procedure, extraneous proteinaceous substances inherently present in culture media prepared with yeast extract or with peptones were not present to interfere with isolation and purification of streptavidin . When harvested after 7-8 days of incubation, the culture fluid was relatively free of contaminating cell breakdown products . Maximal production of streptavidin (100-120 mg/l) was obtained in 8-10 day cultures . For some applications, the culture fluid can be used directly as a source of streptavidin . Under the same conditions used to grow S . avidinii, 11 other actinomycete strains and 134 eumycetes were found to lack the capacity to produce detectable amounts of an extracellular biotin-binding protein.

J Endocrinol, 1988 Oct, 119(1), 133 - 9
Comparison of some biological effects of epidermal growth factor and commercial serum albumin on the induction of alpha-lactalbumin in rat and rabbit mammary explants; Nicholas KR et al.; Commercial preparations of serum albumin from six species can markedly enhance the prolactin-independent induction of alpha-lactalbumin in mammary explants from pregnant rats, and evoke such induction in the tissue from virgin rats . These effects are similar to those of epidermal growth factor (EGF) reported previously . The stimulatory activity of bovine serum albumin (BSA) resides in a putative impurity in the albumin . Charcoal extraction and gel filtration of the BSA results in complete loss of activity . Of the five milk protein mRNAs studied, only alpha-lactalbumin mRNA is induced by insulin, glucocorticoid and serum albumin in the absence of prolactin . Despite these similarities, the biological effects of serum albumin and EGF on mammary tissue diverge in some respects . They appear to operate by different mechanisms since their effects on the rat system are additive . Furthermore, while both inhibit prolactin-mediated induction of alpha-lactalbumin in rabbit mammary explants, cortisol converts EGF into a stimulatory agent, but merely blocks the inhibitory effect of serum albumin . The results emphasize that commercial serum albumin is not to be regarded simply as an inert protein additive to culture media.

J Lipid Res, 1988 Oct, 29(10), 1359 - 66
Alpha-tocopherol is secreted from rat liver in very low density lipoproteins; Cohn W et al.; Three separate studies were carried out to test the hypothesis that rat liver secretes vitamin E (alpha-tocopherol) within very low density lipoproteins (VLDL) . i) When the clearance of plasma chylomicrons (CM) and VLDL was blocked by the administration of Triton WR-1339, alpha-tocopherol concentrations increased linearly with time in both classes of triacylglycerol-rich lipoproteins, although accumulation rates within VLDL exceeded those within CM . For fasted rats, appearance of alpha-tocopherol in VLDL persisted at slightly reduced rates . alpha-Tocopherol and triglycerides in the VLDL fraction responded to Triton WR-1339 administration by coordinate increases . In contrast to the situation in serum, alpha-tocopherol concentrations decreased in the liver following injection of Triton . ii) In order to inhibit the secretion of hepatic lipoproteins containing apolipoprotein B (apoB), rats were fed a diet containing orotic acid . This resulted in a reduction of apoB and alpha-tocopherol concentrations in serum and VLDL, whereas the vitamin E content of liver was increased . iii) In primary cultures of hepatocytes, alpha-tocopherol was secreted into the culture media predominantly within VLDL . We, therefore, conclude that the liver secretes alpha-tocopherol within VLDL and in this way contributes to the maintenance of serum vitamin E concentrations.

Curr Eye Res, 1988 Oct, 7(10), 961 - 7
Superoxide dismutase activity and growth of retinal pigment epithelial cells are suppressed by 20% oxygen in vitro; Akeo K et al.; Despite knowledge of the toxicity of oxygen to the retina, its effects on the retinal pigment epithelium have not been considered . We examined the effect of 20%, 10% and 5% oxygen on growth and superoxide dismutase (SOD) activity of porcine retinal pigment epithelial cells (RPE) . Growth of RPE cells was very significantly lower in 20% oxygen than in either 10% or 5%; optimal growth occurred at 10% oxygen, the concentration most like their environment in vivo . Inclusion of SOD and catalase in the media very significantly stimulated growth in 20% oxygen . The SOD activity of RPE cells was significantly related to ambient oxygen . In first passage (P1) cells, SOD activity was 44% lower on day 7 than on day 1 of culture in 20% oxygen (p less than or equal to 0.05) . Transfer of cells growing in 20% oxygen to 5% oxygen arrested the decrease in SOD and resulted in significantly higher SOD levels . In fourth passage (P4) cells grown in 20% oxygen, SOD was 25% and 44% lower than cells in 10% and 5% oxygen, respectively . After one week, SOD levels in the P4 cells were significantly higher than in P1 . A statistical model of SOD activity in RPE cells indicated significant negative correlations with both oxygen concentration and the cell number . Growth of RPE cells was significantly influenced by oxygen level, days of culture and passage number, but not SOD activity . We conclude that traditional culture conditions support generation of free radicals in tissue culture media that suppress both growth and superoxide dismutase activity.

J Reprod Immunol, 1988 Oct, 14(1), 9 - 25
Assessment of immunoregulation by cultured, pre-attachment bovine embryos; Croy BA et al.; The possibility that the viability of bovine embryos might be predicted by measuring their release of immunoregulatory substances during culture has been investigated . Bovine embryos between days 2 and 19 of gestation were cultured for 24-48 h, the embryo-conditioned medium was harvested and studied for suppression of PHA-stimulated bovine leukocyte cultures . Medium incubated in the absence of any conditioning tissues served as control . Artefactual immunosuppression was detected in incubated control material that could be attributed, in part, to the mixing of different tissue culture media, the type of plastic-ware employed for incubation and supplementation of media with additional L-glutamine . It was observed that day-2 to day-9 bovine embryos, cultured in medium able to support the lymphocyte proliferation assay, did not release immunosuppressive substances . Medium conditioned by day-10 to day-12 embryos produced variable immunosuppression while that conditioned by trophoblastic vesicles derived from day-14 to day-19 embryos was consistently highly suppressive . Since bovine embryo transfer is normally conducted at 6-8 days of gestation, it is unlikely that measuring the immunosuppressive products released from bovine embryos will be of value for predicting their viability.

In Vitro Cell Dev Biol, 1988 Oct, 24(10), 1053 - 6
Production of IGF-II-related peptide by an anaplastic cell line (AT-3) established from the Dunning prostatic carcinoma of rats; Matuo Y et al.; AT-3 cells, one of anaplastic cell lines established from the Dunning prostatic carcinoma of rats, were able to grow under serum-free conditions in a state of suspension detached from a substratum . Radioimmunoassay using monoclonal antibody against rat insulin-like growth factor II (IGF-II) revealed the presence of IGF-II-related peptide in acid-ethanol extracts of lyophilized serum-free media conditioned by AT-3 cells . The peptide contents in the culture media increased with increase in cell number; 71 ng at 3.0 X 10(6) cells and 449 ng at 4.6 X 10(7) cells . IGF-II-related peptide was hardly detectable in acid-ethanol extracts of AT-3 cells harvested after 13-days culture . These results indicate that AT-3 cells produce IGF-II-related peptide and may release it into the culture media.

J Med Chem, 1988 Oct, 31(10), 1978 - 83
Bis(4-hydroxyphenyl){2-(phenoxysulfonyl)phenyl}methane: isolation and structure elucidation of a novel estrogen from commercial preparations of phenol red (phenolsulfonphthalein)
Bindal RD, Katzenellenbogen JA.
Commercial preparations of phenolsulfonphthalein (Phenol Red), a pH indicator dye widely added to cell culture media, have weak estrogenic activity that can be accounted for by a minor lipophilic impurity (ca . 0.002%) . We have isolated this impurity, determined its structure to be bis(4-hydroxyphenyl){2-(phenoxysulfonyl)phenyl}methane, and synthesized it from phenolsulfonphthalein . This compound binds to the estrogen receptor with an affinity 50% that of estradiol; it stimulates the proliferation and increases the progesterone receptor content of estrogen-responsive breast cancer cells in vitro, and it stimulates uterine weight gain in rats in vivo, but shows a potency in these assays only 0.1-0.2% that of estradiol . We suggest how this novel estrogen may be generated during the preparation of phenolsulfonphthalein.

J Virol Methods, 1988 Oct, 22(1), 1 - 11
Suppression of viral replication by guanidine: a comparison of human adenoviruses and enteroviruses; Hurst CJ et al.; A comparison was made between the relative sensitivities of laboratory strain human adenoviruses and enteroviruses, and recently isolated human enteroviruses, to the presence of guanidine hydrochloride in cell culture media . The concentration of guanidine hydrochloride used was 100 micrograms per ml . Representatives of all six human Adenovirus subgenera were unaffected in their replication at this concentration of guanidine . The different human Enterovirus types examined varied in their sensitivity, with suppression ranging from less than 1 to 3 log10 units for laboratory strains, and from 2 to 7 log10 units for recently isolated viruses . The findings suggest a novel role for antiviral drugs; serving as an adjunct in facilitating selective isolation of specific virus groups which may be present as part of mixed viral populations.

Arch Pathol Lab Med, 1988 Oct, 112(10), 987 - 96
Morphology and increased growth rate of atherosclerotic intimal smooth-muscle cells; Yoshida Y et al.; Atherosclerotic intimal smooth-muscle cells (SMCs) in vitro showed higher growth activity than did medial SMCs obtained from either atherosclerotic or normal aortas . Using an electron microscope, it was proved in primary cultures by an explant method that intimal SMCs had rich organelles and fewer filaments in their cytoplasms . They were regarded as synthetic phenotype . In contrast, most medial SMCs had rich filaments and fewer organelles . They were regarded as contractile phenotype . When atherosclerotic intimal and normal medial SMCs were plated on type I collagen gel, cytoplasmic cyclic adenosine monophosphate concentration increased and DNA synthesis was suppressed . Intimal SMCs cultured on the gel showed contractile phenotype . Dibutyryl cyclic adenosine monophosphate added to culture media decreased DNA synthesis and altered cellular phenotype to a contractile state . Intimal SMCs were more resistant to injury by hyperlipidemic low-density liproprotein and homocysteine . Lysosomal enzyme activity was enhanced in intimal SMCs.

Genetica, 1988 Sep 30, 77(2), 123 - 31
Stable polymorphism for mutant eye colour genes in populations of Drosophila melanogaster in two different media; Najera C et al.; In previous work analyzing variability of eye colour alleles existing in natural populations of D . melanogaster, it was observed that the number of females heterozygous for some eye colour alleles was greater in a wine cellar population than in populations outside this cellar . In order to determine which mechanisms caused these eye colour alleles to be favored in the heterozygotes, the changes in the frequency of four eye colour alleles frequently seen in the cellar population (se77o, sf77m, cd77o and multichromosomal 77o) was studied in artificial populations . Two different culture media, one supplemented with 10% ethanol and the other without ethanol were used . It was found that each of the four mutants reached similar equilibrium frequencies in both media, though the safranin allele (sf77m) equilibrium frequency was significantly higher in the alcohol medium . A significant excess of heterozygotes were also observed in these populations.

Clin Chim Acta, 1988 Sep 30, 177(1), 11 - 20
Effect of colchicine on collagen synthesis by liver fibroblasts in murine schistosomiasis; Mansour MM et al.; Colchicine, an antimicrotubular agent, was shown to block the transcellular movement of certain structural macromolecules such as collagen . In the present study, the effect of colchicine on collagen synthesis and secretion by monolayer cultures of fibroblasts from livers of mice infected with Schistosoma mansoni was investigated . The effect of colchicine on proliferation of these fibroblasts was studied as well . Collagen and non-collagen protein synthesis was measured by incubating cultures with {14C}proline and measuring the incorporation of radioactivity into these protein fractions in both culture media and cell layers . Proliferation was measured by {3H}thymidine uptake . The isolated fibroblasts actively formed collagen and secreted most of it into the culture medium; 10-20% of the collagenase-sensitive radioactive protein remained in the cell layer . The addition of colchicine to culture medium led to selective inhibition of collagen formation with negligible effects on non-collagen protein synthesis . Fibroblast proliferation was also reduced by colchicine treatment . Both inhibition of collagen synthesis and inhibition of fibroblast proliferation were dose-dependent . Comparison of medium and cell layer collagen radioactivity confirmed inhibition of synthesis rather than only inhibition of secretion . These data suggest that colchicine has a specific effect on synthesis of collagen and proliferative activity by fibroblasts from S . mansoni-infected liver and may, therefore, be useful in modulating schistosomal hepatic fibrosis.

J Biol Chem, 1988 Sep 25, 263(27), 13916 - 21
Transcriptional regulation of osteopontin production in rat osteosarcoma cells by type beta transforming growth factor; Noda M et al.; Type beta transforming growth factor (TGF beta) was shown to regulate the production of several extracellular matrix proteins . Osteopontin (OP) is a recently discovered bone matrix protein which was shown to promote the attachment of osteoblastic rat osteosarcoma ROS 17/2.8 cells to their substrate . We examined the effects of TGF beta on OP production and OP mRNA in ROS 17/2.8 cells . Four-day treatment with 4 ng/ml TGF beta 1 increased substantially the level of osteopontin in the cell culture media, as estimated by immunoblotting . Metabolic labeling showed that this effect was associated with a 3-4-fold increase in OP biosynthesis . TGF beta 1 also increased, in a dose-dependent manner starting at 0.4 ng/ml, the steady-state level of OP mRNA . The increase in OP mRNA was first detected 48 h after the addition of TGF beta 1 and lasted at least until 120 h . The half-life of OP mRNA, estimated in the presence of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, was about 10 h and was not altered by TGF beta 1 . On the other hand, the increase in OP mRNA was blocked by actinomycin D . Nuclear run-on assays indicated that TGF beta 1 increased the rate of transcription of the OP gene . Examination of hormonal interactions showed that TGF beta 1 opposed or compensated for the reduction in OP mRNA produced by dexamethasone and that TGF beta 1 did not further augment OP mRNA levels which had been increased by 1,25-dihydroxyvitamin D3 treatment . TGF beta 2 had similar effects on OP gene expression as TGF beta 1 . In conclusion, TGF beta promotes the production of osteopontin in the osteoblastic osteosarcoma cells through a pathway which is at least in part mediated by transcriptional events.

Biochem Biophys Res Commun, 1988 Sep 15, 155(2), 786 - 93
Effect of cytokines and growth factors on the expression of elastase activity by human synoviocytes, dermal fibroblasts and rabbit articular chondrocytes; Redini F et al.; Human synoviocytes, rabbit articular chondrocytes and human skin fibroblasts in culture were examined for their ability to express elastase activity . Latent enzyme activity degrading insoluble elastin was detected in the culture media of the three cell types and was completely abolished by metal chelating agents . Triton X-100 cell extracts were found to degrade a synthetic elastase substrate, N Succinyl-(Ala)3p-nitroanilide (SANA) . The SANA-degrading activity of cell extracts could be attributed to a metalloprotease for fibroblasts and synoviocytes (100%) and to a metalloprotease associated with a cysteine protease for chondrocytes (70 and 30% respectively) . This SANA-degrading activity was partly due to the combined action of an endo and an exopeptidase . Tumor Necrosis Factor-alpha (TNF-alpha) and Interferon-gamma (IFN-gamma) significantly enhanced the elastin degrading activity present in the culture media of both synoviocytes and chondrocytes . Interleukin-1 beta significantly increased the secretion of elastase by chondrocytes . By contrast, Transforming Growth Factor-beta (TGF-beta) reduced by 80 per cent the secretion of elastinolytic activity by chondrocytes but had not effect on other cell types.

Cancer, 1988 Sep 15, 62(6), 1171 - 8
Effect of differentiation-inducing agents on oncogene expression in a chronic myelogenous leukemia cell line; Eisbruch A et al.; K562 is a Philadelphia (Ph) chromosome-positive chronic myelogenous leukemia (CML) blast crisis cell line representing a pluripotent precursor cell . At the molecular level, K562 cells express high levels of the aberrant bcr-abl product, p210bcr-abl, believed to be critical to the pathogenesis of CML . The authors demonstrate that exposure of K562 cells to hemin causes a state of partial, reversible erythroid maturation, accompanied by a marked decrease in p210bcr-abl . The change in bcr-abl expression may be mediated at the translational level since steady state amounts and enzymatic activity of the bcr-abl protein are reduced whereas bcr-abl mRNA levels are unaltered . The decrease in p210bcr-abl phosphokinase enzymatic activity can be detected within 2 hours after addition of hemin to the culture media, indicating that changes in expression of this oncogene probably occur before or concurrent with differentiation . No change in bcr-abl protein occurred in a CML cell line (KBM-5) which did not undergo differentiation after exposure to hemin, consistent with a direct relationship between altered p210bcr-abl expression and hemin-induced erythroid differentiation . Importantly, the marked diminution in bcr-abl protein was not associated with a disruption in K562 growth rates, indicating that the proliferative capacity of these cells may be independent of the bcr-abl product . In contrast to hemin, cytosine arabinoside (Ara-C) caused terminal erythroid differentiation of K562 cells, characterized by irreversible hemoglobin accumulation and cytostasis; and no change in bcr-abl protein expression was observed . The distinct effects of Ara-C and hemin could reflect the existence of pleiotropic differentiation pathways . Both Ara-C and hemin-exposed cells showed a decrease in c-myc and c-myb transcripts, suggesting that altered levels of these proto-oncogenes may be associated with erythroid maturation, regardless of the rate of cell division . K562 cells provide a useful model for analyzing the interaction between oncogene expression and CML cell growth and differentiation.

Cancer Res, 1988 Sep 15, 48(18), 5193 - 202
Biosynthesis and secretion of laminin and laminin-associated glycoproteins by nonmalignant and malignant human keratinocytes: comparison of cell lines from primary and secondary tumors in the same patient; Frenette GP et al.; Laminin biosynthesis was compared in four pairs of human squamous cell carcinoma cultures derived from primary and recurrent or metastatic tumors in four patients with cancer of the larynx and hypopharynx to determine if changes in laminin production accompany tumor progression . Laminin profiles of the malignant cells were compared with laminin biosynthesized by nonmalignant human keratinocytes . Pulse-chase biosynthetic labeling of the cultures with {35S}methionine established that all of the squamous carcinoma cell lines synthesize immunoreactive A (Mr 400,000), B1 (Mr 205,000), and B2 (Mr 200,000) laminin subunits; assemble them to form the intact laminin molecule (Mr 950,000); and secrete a portion of the laminin they produce into the culture media . One aspect of laminin expression unique to keratinocytes, both malignant and nonmalignant, was the occurrence of three additional glycoprotein forms (Mr 195,000, 170,000, and 160,000) in the laminin immunoprecipitates . In contrast to the laminin subunits, these glycoproteins were not immunoreactive with the anti-laminin antiserum on Western blots . Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis without and with reduction of disulfide bonds revealed that the laminin immunoprecipitates contained a family of oligomeric molecules . These ranged in apparent molecular weight from 370,000 to 950,000 and were composed of laminin subunits and the glycoprotein forms linked by interchain disulfide bonds . The malignant keratinocyte cell lines from different patients were distinguishable in terms of the array of laminin and glycoprotein forms displayed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the rate of {35S}methionine incorporation into laminin during the pulse-labeling, the fraction of {35S}methionine-labeled laminin secreted into the medium during the chase incubation, and the absolute amount of laminin secreted into the culture medium as determined by enzyme-linked immunosorbent assay . However, cell lines established from primary and metastatic or recurrent cancer in the same patient were indistinguishable in their profile of laminin biosynthesis and secretion . In comparison to primary cultures of nonmalignant foreskin basal keratinocytes, the malignant cells secreted into the culture medium a larger fraction of the laminin that they produce . This is an indication that the malignant keratinocytes in culture deposited a less stable basal lamina-like extracellular matrix than their malignant counterparts . The diminished integrity of the basal lamina matrix may be an important factor in the invasive growth of human epithelial cancer.

Biochemistry, 1988 Sep 6, 27(18), 6751 - 8
Monoclonal antibodies to human fibroblast procollagenase . Inhibition of enzymatic activity, affinity purification of the enzyme, and evidence for clustering of epitopes in the NH2-terminal end of the activated enzyme; Birkedal-Hansen B et al.; This study describes 11 monoclonal antibodies (Mabs) against human fibroblast collagenase that (i) inhibit the specific catalytic activity of the enzyme and/or (ii) react with one or more forms of the enzyme on Western blots . Each of the Mabs specifically immunoprecipitated the Mr 57,000/52,000 procollagenase from {35S}methionine-labeled culture medium . Five Mabs, designated VI-3, VI-4, 2C5, 4A2, and 7C2, inhibited the activity of fibroblast-type collagenase against soluble monomeric collagen and against reconstituted collagen fibrils but did not inhibit the genetically distinct human PMN leukocyte collagenase . The interstitial collagenase produced by human mucosal keratinocytes (SCC-25) was also inhibited, whereas the corresponding enzyme from rat was not . Assignment of epitopes to structural domains within the molecule based on immunoperoxidase staining of Western blots of collagenase and its autocatalytic fragments revealed that 9 of 11 epitopes, including those recognized by 4 inhibitory Mabs, were clustered in a 169-residue domain, which constitutes the NH2-terminal part of the Mr 46,000/42,000 active enzyme . One Mab (X-2a) specifically recognized the Mr 57,000/52,000 zymogen species and failed to react with the active Mr 46,000/42,000 form . The inhibitory Mab VI-3 was used for immunoaffinity purification of procollagenase from culture media with a recovery better than 80% and a yield of approximately 1.4 mg of enzyme/L of medium.

Am J Obstet Gynecol, 1988 Sep, 159(3), 629 - 35
Lipid and glucose metabolism in human placental culture; Ogburn PL Jr et al.; Placental culture models have been used to increase the understanding of endocrinology and pathophysiology in pregnancy . This article describes glucose and lipid metabolism in several of these models . Of special interest is the availability of arachidonic acid for the production of prostanoids . Ten placentas were collected at the time of cesarean section in term pregnancies without labor . Minced villous tissue was incubated for 48 hours in media with a glucose concentration of 100, 200, or 500 mg/dl . Tissue was dispersed in the media or was left as a single clump during the incubation . Glucose levels in the culture media were measured at 8, 20, 32, and 48 hours . Tissue lipid levels were measured before and after incubation in seven placentas . At 8 hours, glucose utilization ranged from 2.38 +/- 0.40 to 9.44 +/- 1.22 mumol/gm tissue/hr (mean +/- SEM) . By 48 hours the cumulative glucose utilization ranged from 1.56 +/- 0.09 to 6.87 +/- 0.38 mumol/gm tissue/hr . Tissue lipid analysis showed most of the fatty acids to be in the phospholipids initially (4477 +/- 179 micrograms/gm tissue) . Subsequent to incubation for 48 hours, phospholipid levels fell to a range of 2686 +/- 90 to 3466 +/- 157 micrograms/gm tissue in various culture conditions (p less than 0.005 compared with initial values) . Whereas phospholipid levels decreased during incubation, levels of triglycerides and nonesterified fatty acids increased significantly in placental tissue . Arachidonic acid, the precursor of prostaglandins, thromboxane, and prostacyclin, makes up about one quarter of the fatty acid in the initial placental phospholipid . Arachidonic acid follows the pattern of total fatty acids during incubation; it is released from phospholipid and is converted to nonesterified fatty acid and triglyceride . We may conclude from this study that each placenta has a unique glucose utilization rate and a unique capacity to produce triglyceride . In tissue culture, arachidonic acid is released to its nonesterified state much more quickly than it can be converted to prostanoids by cyclooxygenase . The choice of initial glucose concentration, tissue preparation (dispersed in media or left as single clump), and time of incubation all may determine the rate of glucose metabolism, the rate of phospholipid breakdown, the rate of triglyceride production, and the quantity of nonesterified arachidonic acid in placental tissue culture.

J Steroid Biochem, 1988 Sep, 31(3), 287 - 93
Lipophilic impurities, not phenolsulfonphthalein, account for the estrogenic activity in commercial preparations of phenol red; Bindal RD et al.; Previously, we found that Phenol Red, a pH indicator dye commonly used in tissue culture media, had weak estrogenic activity, demonstrable by competitive binding to the estrogen receptor, stimulation of the growth rate of human breast cancer (MCF-7) cells, and elevation of progesterone receptor levels in these cells . We have now examined in more detail the source of this estrogenic activity, present in commercially available preparations of Phenol Red . By high performance liquid chromatography and solvent partitioning, we find that the receptor binding and growth promoting activity does not correspond to the indicator dye itself (phenolsulfonphthalein), but rather to more lipophilic impurities present in these preparations . There are numerous such impurities, many of which show some competitive binding activity, but the major receptor binding activity is accounted for by a single impurity component . Commercial preparations of Phenol Red can be purified by ether extraction of the sodium salt, whereby 95-99% of the lipophilic estrogenic impurities are removed, and the growth stimulating activity towards MCF-7 cells is reduced.

J Mol Cell Cardiol, 1988 Sep, 20(9), 825 - 35
Determinants of cardiomyocyte development in long-term primary culture; Piper HM et al.; The influence of cell attachment to substrates and of medium composition on development of cardiomyocytes from adult rats in cultures up to 9 days old was investigated . Cardiomyocytes prevented from attaching to a culture substratum deteriorated within 3 days in medium 199 (M199) with or without fetal calf serum (FCS) . Rapid attachment during the first 4 h after plating could be attained equally well on FCS or laminin coated surfaces . In M199 without FCS, attached cardiomyocytes on FCS coated dishes were able to retain their overall elongated morphology, but the number of cells remaining attached constantly decreased during the first 9 days in serum free culture . Attached on laminin the rate of loss from serum free cultures was lower . In the presence of 20% FCS, attached cardiomyocytes spread extensively after day 3, both on FCS and on laminin coated dishes . In serum containing media many cells pass through a spherical intermediate state before spreading extensively . Almost all cardiomyocytes cultured with 20% FCS on untreated tissue culture plastic gradually become spherical before attaching . With 20% FCS in culture media, the number of cells remaining in culture after 9 days was similar whether cells were rapidly attached to FCS treated or laminin coated substrata, or were plated on culture plastic, i.e., 52, 63, and 45% of the maximal number attached on day 1 . By day 9 in all three culture types cells were spread and were beating spontaneously . These results indicate that adult cardiomyocytes do not establish in a stable morphological state in long-term cultures, in other than a surface attached spread cell form . For this stability the presence of yet unidentified components of fetal calf serum is required.

Eur J Cancer Clin Oncol, 1988 Sep, 24(9), 1445 - 55
Two new human tumor cell lines derived from squamous cell carcinomas of the tongue: establishment, characterization and response to cytotoxic treatment; Gioanni J et al.; Two new permanent cell lines derived from squamous cell carcinomas of the tongue, CAL 27 and CAL 33, have been established in culture . Both cell lines were isolated in standard culture media without epidermal growth factor or fibroblast feeder layer to avoid obtaining clones of more differentiated cells . Analysis of the morphology, ultrastructure, karyotype and immunohistochemical properties of these two cell lines demonstrated that they are both well characterized, uncontaminated by HeLa cells, and do in fact correspond to transformed epithelial cells that have conserved certain characteristics of the original Malpighian epithelium . CAL 27 and CAL 33 have relatively long doubling times (35 and 43 h respectively) . Their response to 14 drugs used for cancer chemotherapy was evaluated by a short term assay based on tritiated thymidine incorporation after exposure to the drugs . CAL 27 was more resistant than CAL 33 in all cases but one . Although cytogenetic examination revealed both lines to be malignant, neither CAL 27 nor CAL 33 produced colonies in soft agar; both lines were tumorigenic after inoculation into nude mice . This study clearly demonstrates the diversity of cancers of a given histologic form, in agreement with the diversity noted previously in vivo . Isolated without the use of any selection criteria, these cell lines constitute appropriate models for the study of human tumors.

In Vitro Cell Dev Biol, 1988 Sep, 24(9), 927 - 30
Cytokinetics and SCE baselines in rat and human lymphocytes during successive divisions in the presence of different culture media; Sinha AK et al.; Peripheral blood cultures were set up from male rats and humans in TC199, RPMI 1640, and minimal essential medium in the presence of 5-bromo,2-deoxyuridine and harvested at 48, 72, and 96 h . Mitotic indices were compared in the different media at all three harvest times, but cytokinetic patterns and baseline sister chromatid exchange (SCE) frequencies were evaluated exclusively in the 72- and 96-h cultures . In general, lymphocyte division kinetics, as determined by average lymphocyte division (ALD) numbers, were comparable between rat and human lymphocytes cultured in any of the three media and harvested at either of the culture times . The numbers of SCEs were distributed between and within the two systems independent of ALD numbers or the harvest time . Overall, no influence of media was detected on the distribution of SCEs . Despite a number of similarities in growth characteristics between rat and human lymphocytes, the rat lymphocyte test system has distinct advantages over that of the human because of the easy access to rat blood samples and the absence of the many restrictions applicable to humans.

Am J Pathol, 1988 Sep, 132(3), 474 - 8
Perturbation of cultured human endothelial cells by atherogenic levels of low density lipoprotein; Holland JA et al.; Cultured human umbilical vein endothelial cells (EC) exposed to atherogenic levels of low density lipoprotein (LDL) for protracted periods demonstrated no measurable evidence of overt cytotoxicity, but were perturbed as indicated by an increase in prostacyclin (PGI2) production . Confluent EC were incubated with high LDL concentrations (240 or 330 mg/dl cholesterol) for 1 to 12 days . LDL was added to culture media containing 25% human lipoprotein-deficient serum to determine the effects of LDL independent of other lipoproteins . LDL did not injure EC as assessed by cell count, vital dye exclusion, 51chromium release, and lactate dehydrogenase release . Although high concentrations of LDL did not cause EC cytotoxicity, such LDL concentrations did results in increased PGI2 generation . PGI2 accumulation in postincubation media was increased two-to-fivefold in otherwise unstimulated cells as measured by radioimmunoassay of the stable PGI2 breakdown product, 6-keto-PGF1-alpha . This elevation persisted for the entire 12-day exposure to high LDL concentrations . These results indicate that prolonged exposure to atherogenic concentrations of LDL does not effect EC viability, but does cause an endothelial perturbation as demonstrated by an increased PGI2 production.

Brain Res Bull, 1988 Sep, 21(3), 353 - 61
Measurement of intracellular Ca2+ in cerebellar Purkinje neurons in culture: resting distribution and response to glutamate; Connor JA et al.; Ca ion levels in cerebellar Purkinje neurons, in culture, have been measured using the fluorescent indicator, fura-2, and digital imaging . Cells were loaded with the indicator both by injecting the free acid form and by allowing the membrane permeant form (/AM) become deesterified and trapped . The two methods gave significantly different results in that the /AM loaded cells showed localized regions of high Ca2+ in the soma whereas the injected cells did not . Resting levels in the remainder of the cytoplasm were similar however, as were the excursions in Ca2+ induced by electrical or chemical stimulation . Comparison of the data from the two methods suggests that qualitative measures of Ca in intracellular stores can be derived from the /AM loading method . Injected cells showed high Ca2+ levels in the soma that persisted for 3-8 minutes following removal of the injection electrode . The dendrites of these cells however maintained low Ca2+ levels and differences of several hundred nM in Ca2+ were maintained between the soma and initial dendrite segment, demonstrating directly the large Ca pumping capacity of the dendrites . Localized regions of high Ca2+ in dendrites could be generated by applying glutamate from a microelectrode in TTX-Krebs saline . When studied in culture media with 4.7 mM K, the Purkinje neurons showed a biomodal distribution of Ca2+ with 35 to 40% showing stable Ca2+ levels between 250 and 350 nM, and the remainder 80 to 130 nM Ca2+ . Granule neurons on the same coverslips had Ca2+ level in the lower range in greater than 95% of the examples observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuroendocrinology, 1988 Sep, 48(3), 229 - 34
Stimulatory effect of isoproterenol but not of dibutyryl cyclic AMP on N-acetyltransferase activity and melatonin content of Syrian hamster pineal gland in organ culture; Santana C et al.; The purpose of this study was to compare the response of Syrian hamster pineal glands in organ culture either to isoproterenol, a beta-adrenergic agonist, or to dibutyryl cyclic AMP . When pineal glands were collected at night, hamsters were exposed to light for 30 min to depress pineal N-acetyltransferase (NAT) activity and melatonin values to low levels . Pineal glands were removed and placed in organ culture containing either isoproterenol or dibutyryl cyclic AMP and subsequent changes in NAT activity and melatonin levels were measured . At night, isoproterenol (10(-7) or 10(-6) M) induced an increase in the NAT activity and melatonin levels in both pineals and culture media . However, dibutyryl cyclic AMP was either ineffective or minimally effective in stimulating these parameters at either different incubation times (2, 4, and 6 h) or drug concentrations (0.1, 0.5, and 1.0 mM) . Conversely, when rat pineal glands were incubated with either isoproterenol (10(-7)) or dibutyryl cyclic AMP (0.5 mM) dramatic rises in NAT activity and melatonin levels were observed . In another experiment, hamster pineal glands were collected from animals killed either late in the light period (19.00 h) or in the latter half of the dark period . Isoproterenol promoted NAT activity and melatonin production only in glands collected in the latter half of the dark phase.

Blood, 1988 Sep, 72(3), 978 - 82
Establishment and characterization of an amylase-producing human myeloma cell line; Matsuzaki H et al.; Two stable lines of IgA lambda-producing plasma cells (KHM-1A and KHM-1B) that were free of the Epstein-Barr virus were established from a patient with multiple myeloma complicated by hyperamylasemia . Surface marker studies of the two cell lines showed that the cells had no surface immunoglobulins but were positive for cytoplasmic immunoglobulins (IgA lambda) and for HLA-DR and PCA-1 . Secretion of IgA monoclonal immunoglobulin by the two lines was detected by a plaque-forming cell assay and by an enzyme-linked immunosorbent assay of culture media . KHM-1B cells also secreted alpha-amylase, but no such activity was detected in the culture-conditioned supernatant fluid of KHM-1A.

Diabetes, 1988 Sep, 37(9), 1279 - 86
Structure, function, and immunogenicity of human insulinoma cells; Thivolet CH et al.; Dissociated human insulinoma cells were plated onto plastic multiwell dishes . Cells were maintained for 1 mo on plastic with three passages . Cultures consisted of small colonies with some areas of stratification and few intercellular spaces . Ultrastructural studies indicated that cultured cells had epithelial features with desmosomes at cell-to-cell contacts and intermediate filaments in addition to secretory granules in the cytoplasm . Insulin and C-peptide were released in equimolar amounts in culture media . When challenged for 30 min with 16.7 mM glucose, 1 mM 3-isobutyl-1-methylxanthine, 4 mM tolbutamide, or 10(-6) M glucagon, insulinoma cells responded by a 1.5-, 1.5-, 2-, or 3-fold increase, respectively, in insulin release above baseline levels . A 15-min challenge with 10(-5) M isoproterenol increased insulin secretion by 1.85-fold . By indirect immunofluorescence, an anti-insulin antibody reacted positively with cell cytoplasm, whereas anti-somatostatin and anti-glucagon antibodies did not . Insulinoma cell surface expressed class I MHC molecules but not class II molecules . Immediately after isolation, crude insulinoma cells were contaminated by 2% of DR+ cells from nonislet components that disappeared after several weeks in culture . The ability of insulinoma cells to stimulate allogenic T-lymphocyte proliferation was assessed by {3H}thymidine incorporation in mixed culture combinations . Crude insulinoma cells elicited a strong lymphoproliferative response with a stimulation index ranging between 3.5 and 7, whereas no stimulation was found after 1 mo in culture . It is postulated that absence of class II-positive cells in the stimulatory cell preparation conditioned this immune tolerance across the major histocompatibility barrier.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Biophys Res Commun, 1988 Aug 30, 155(1), 398 - 404
Rat clusterin isolated from primary Sertoli cell-enriched culture medium is sulfated glycoprotein-2 (SGP-2); Cheng CY et al.; Clusterin, a glycoprotein originally isolated from ram rete testis fluid, is a dimer composed of monomers with non-identical NH2-terminal amino acid sequences . In view of its possible role in cell-cell interactions in the seminiferous epithelium, we sought to identify such a protein in the rat . Using the bioassay developed for the ovine protein, rat clusterin was purified to apparent homogeneity by HPLC from primary Sertoli cell-enriched culture media . This protein is also a heterodimer consisting of monomers of Mr 43,000 (alpha) and Mr 35,000 (beta) . NH2-Terminal amino acid sequence analysis indicated that the alpha subunit has a sequence of NH2-SLMPLSHYGPLSFHNMFQPFFDMIHQAQQA and the beta subunit, NH2-EQEFSDNELQELSTQGSRYVNKEIQNAVQG . These two subunits show marked similarity with the corresponding subunits of ram clusterin isolated from rete testis fluid . Using an antibody against the alpha subunit of rat clusterin, a cDNA clone was isolated from a rat testicular lambda gt11 cDNA library . Analyses of the amino acid sequence derived from the isolated rat clusterin cDNA and of the NH2-terminal amino acid sequences indicate that rat clusterin is identical to a Sertoli cell glycoprotein previously designated sulfated glycoprotein-2.

Brain Res, 1988 Aug 16, 458(1), 115 - 22
Facilitation of noradrenergic character of sympathetic neurons by co-culturing with heart cells; Wakade AR et al.; Noradrenergic properties of peripheral sympathetic neurons obtained from 10-12-day-old chick embryos were examined under various culture conditions . Sympathetic neurons supported by nerve growth factor in serum-free or serum-containing medium took up significant and almost equivalent amounts of {3H}norepinephrine . The uptake was markedly enhanced when neurons were co-cultured with heart cells, either in the absence or presence of nerve growth factor, for 3 days . The facilitatory effect of heart cells on the uptake was persistent only if the nerve growth factor was present . In its absence there was a gradual decrease in the uptake . Endogenous norepinephrine content was increased by several fold when sympathetic neurons were grown with either heart cells or in a medium conditioned by the heart cells . Sympathetic neurons initially selected in culture by nerve growth factor in regular medium and then exposed to a conditioned medium for 3 days exhibited a marked facilitation of {3H}norepinephrine uptake . The number of surviving neurons was almost constant when culture media were changed . Choline acetyltransferase activity of neurons grown in heart-conditioned medium plus nerve growth factor was not significantly higher than that of neurons grown in regular medium plus nerve growth factor . The overall conclusion of the study is that the noradrenergic character of sympathetic neurons can be further enhanced by heart cells or a medium conditioned by these cells.

J Biol Chem, 1988 Aug 15, 263(23), 11227 - 36
Inter- and intraclonal variability of polypeptides synthesized in a rat hepatoma cell line . Quantitative two-dimensional gel analysis; Miller MJ et al.; To examine the degree of clonal heterogeneity in the synthesis of polypeptides in neoplastic cells, single-cell subclones from the rat hepatoma cell line H4-II-E were isolated . Polypeptides from the clones were resolved on high resolution two-dimensional polyacrylamide gels (PAGE), and quantitatively analyzed with a computerized two-dimensional PAGE analysis system developed in this laboratory . Only four qualitatively different spots were found which were synthesized in one of the subclones in four out of five experiments . In contrast, 5-20% of the spots showed statistically significant quantitative differences when any one subclone was compared to any other . These differences were generally quite small, averaging about 1.5-fold in intensity, although variations of fourfold or more were observed . Different cultures of the same subclone showed quantitative differences of the same order as seen in different subclones, indicating that this variability was primarily intraclonal in nature, i.e . associated with the cultures rather than the subclones . The distribution of quantitatively variable spots indicates that 50% or more of the polypeptides in these cells may display intraclonal variability . Similar results were obtained with a second set of subclones derived from these primary ones . Time course studies were conducted where cells were maintained continuously for 12 weeks, with samples taken for two-dimensional PAGE analysis once a week . The fraction of polypeptides that vary significantly generally increased with time between sampling points . Experiments with independent cultures grown in parallel indicate that about 4% of this variability can be correlated to the age of the culture media, although the majority appears due to uncontrolled and/or random differences that arise between cultures . These results indicate that independent cultures quickly develop detectable quantitative differences in the expression of a large fraction of their polypeptides . These differences cannot, at present, be associated with the observable biology of the cells and probably reflect time-associated variations in the balance of cellular macromolecular synthesis which arise in tissue culture cells.

FEBS Lett, 1988 Aug 15, 236(1), 47 - 52
Tumor necrosis factor inhibits collagen and fibronectin synthesis in human dermal fibroblasts; Mauviel A et al.; Tumor necrosis factor (TNF) caused inhibition of collagen production by confluent cultures of human dermal fibroblasts in a dose-dependent manner . Concomitant increase of prostaglandin E2 release was observed as a result of TNF-induced cell activation . However, a blockade of the cyclooxygenase pathway of arachidonate metabolism by indomethacin did not abrogate the inhibitory effect of TNF on collagen synthesis, suggesting that this effect could be independent of prostaglandin metabolism . Gel electrophoresis of the newly synthesized macromolecules from the culture media showed that both type I and type III collagens as well as fibronectin were affected by the inhibition . Electrophoresis of cell layer-associated proteins demonstrated that the reduction in amounts of collagen and fibronectin in the medium did not result from an intracellular accumulation of these macromolecules . Production of procollagens was reduced in parallel to that of collagens, suggesting that the effect of TNF is exerted before the processing steps of procollagens . These results clearly show that TNF could play a role in modulation of matrix deposition by fibroblasts during inflammatory processes.

Biochem J, 1988 Aug 15, 254(1), 51 - 7
Immunochemical characterization and biosynthesis of pI-6.4 esterase, a carboxylesterase of rat liver microsomal extracts; Robbi M et al.; Rat liver pI-6.4 esterase was purified from microsomes (microsomal extracts) and used to generate antibodies in the rabbit . Two active enzyme forms, similarly sensitive to endo-H (endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96), but differing slightly in polypeptide chain length, were present in the preparation . In microsomes, immunoblots revealed a single form, with Mr congruent to 62,000, identical with the large component of the purified enzyme, indicating that the second component is an artefact . Rabbit reticulocyte lysates and wheat germ extracts programmed with RNA extracted from total or bound polysomes synthesized a single immunoreactive 61 kDa polypeptide, which was not formed with RNA extracted from free polysomes . The immunoreactive product synthesized in the presence of dog pancreas microsomes was slightly larger (62 kDa); like the authentic enzyme, it bound to concanavalin A and was decreased in molecular size to 60 kDa by the action of endo-H . Thus the enzyme is synthesized with a short cleavable sequence and bears at least one high-mannose oligosaccharide chain . Metabolic labelling in hepatocytes cultured with {35S}methionine also generated a single immunoreactive polypeptide of 62 kDa, which was decreased to 60 kDa in size by treatment with endo-H or addition of tunicamycin to the culture medium . This confirms the molecular homogeneity and the glycosylation of the enzyme in the intact cell . Culture media contained no pI-6.4-esterase-related protein, whether tunicamycin was present or not . The processing steps in the synthesis of pI-6.4 esterase are thus, as for other esterases of the endoplasmic reticulum {Robbi & Beaufay (1986) Eur . J . Biochem . 158, 187-194; (1987) Biochem . J . 248, 545-550} indistinguishable from those occurring early in the synthesis of secretory proteins . Glycosylation is apparently not the sorting signal responsible for their retention in the endoplasmic reticulum.

Eur J Pharmacol, 1988 Aug 2, 152(3), 341 - 6
Quisqualate, high calcium concentration and zero-chloride prevent kainate-induced toxicity of cerebellar granule cells; McCaslin PP et al.; Kainic acid (KA; 100 microM), results in the death of all cultured rat cerebellar granule cells (18-22 days in vitro) within 30 min . Changes in the cells are evident within 2 min of applying the excitatory amino acid (EAA) and include an apparent cellular granulation with a loss of cell body birefringence at 10 X magnification . Quisqualic acid (QA; 25 microM) completely prevents this KA-induced neurotoxicity . In addition, cells are protected from toxicity by increasing calcium concentrations to 10 mM . Moreover, following a 30 min exposure and after washing the cells free of these compounds, cells placed in culture media remain alive 24 h later . Interestingly, neurons die when placed in a balanced salt solution which lacks calcium even when no KA is present . This death is also dependent on the presence of chloride and is prevented with the non-selective EAA antagonist, kynurenic acid, but is not prevented by QA . Collectively, these data suggest that the activation of the EAA receptor by KA in cerebellar granule cells is at least partially regulated by calcium and chloride and is suppressed by QA . Furthermore, placing granule cells in zero-calcium results in neuronal death which appears to be mediated by EAA mechanisms.

Am J Hematol, 1988 Aug, 28(4), 227 - 31
Serum-free culture of human hemopoietic progenitors in attenuated culture media; Sonoda Y et al.; To elucidate the precise mechanisms of molecular and cellular regulation of hemopoiesis, it is necessary to develop a chemically defined culture assay for purified hemopoietic progenitors . To approach this long-term goal, we attempted to develop a serum-free culture system for enriched human progenitors that permits expression of all hemopoietic lineages and stages of development . Preliminary studies indicated that alpha-medium was superior to Iscove's modified Dulbecco's medium (IMDM) and that culture under low (5%) oxygen condition was better than an ambient level of oxygen . We developed an attenuated (modified quarter-strength) alpha-medium and compared the colony-supporting ability of the three media by plating 1,000 bone marrow null cells per dish in the presence of a combination of recombinant human colony-stimulating factors (CSFs) . The numbers of colonies supported in alpha-medium and attenuated alpha-medium were approximately 70% of those in serum-containing cultures . IMDM failed to support colony formation . While, in general, the colony sizes were smaller in the serum-free cultures than in the serum-containing cultures, a variety of types of single lineage and multilineage colonies were seen in serum-free culture . A linear relationship between cell number and colony formation was seen in 100-2,000 cells per dish . Serum-free cultures of enriched human progenitors should be an important tool for analysis of the mechanisms of recombinant CSFs.

Endocrinology, 1988 Aug, 123(2), 913 - 21
Development of a primary culture system for rainbow trout corpuscles of stannius and characterization of secreted teleocalcin; Gellersen B et al.; A primary culture system has been established for rainbow trout corpuscles of Stannius (CS) . A RIA has also been developed to monitor and characterize the teleocalcin secreted by these cultures . The primary cultured CS cells actively synthesized and secreted teleocalcin for up to 39 days when maintained in amphibian and mammalian culture media . Numerous teleocalcin cells were also evident after immunocytochemical staining of the CS cultures . When the cultures were labeled with L-{35S}methionine, de novo synthesized and secreted teleocalcin was judged to be a glycosylated protein on the basis of Concanavalin-A-Sepharose binding and was similar in size to the intracellular form of the hormone . This culture system may prove to be ideal for identifying those factors that regulate teleocalcin secretion.

J Biochem Biophys Methods, 1988 Aug, 16(4), 311 - 8
alpha-Oligodeoxynucleotide stability in serum, subcellular extracts and culture media; Bacon TA et al.; Degradation of a synthetic alpha-oligodeoxynucleotide was studied in order to compare its survival with naturally occurring beta-oligodeoxynucleotides in five systems used for antisense hybridization arrest experiments . In contrast to beta-oligodeoxynucleotides, alpha-oligodeoxynucleotides were not detectably degraded over 24 h at 37 degrees C in HeLa cell postmitochondrial cytoplasmic extract or RPMI 1640 with 10% fetal bovine serum, and showed significant survival after 24 h at 37 degrees C in rabbit reticulocyte lysate, fetal bovine serum and human serum.

J Reprod Immunol, 1988 Aug, 13(3), 221 - 34
Immunosuppressive effects of rabbit blastocoelic fluid and embryo culture medium; Pandian AM et al.; In order to understand the mechanism of the feto-maternal immune relationship, we assayed the immunosuppression activities of fresh blastocoelic fluid and decomplemented peripheral serum collected from day-9 pregnant white New Zealand rabbits and of rabbit embryo culture medium (ECM) . Because the viability of the human lymphocytes was not affected by either of these biological fluids and since they were easy to obtain in sufficient quantities, they were used uniformly in all the experiments . Immunosuppressive effect was calculated by the relative inhibition of proliferation of Con A-stimulated lymphocytes . The immunosuppressive effect of blastocoelic fluid of the 9-day pregnant rabbits was significantly higher than that of autologous decomplemented serum (P less than 0.001) . The inhibition by the serum was non-specific because sera from non-pregnant animals as well as sera from different stages of pregnancy and pseudo-pregnancy showed the same level of inhibition . The ECM of 6.5-7-day-old embryo showed a pronounced immunosuppressive effect . When embryos of 1,3 and 5 days were cultured and their culture media were assayed only with 5-day-old embryo the effect had begun to appear, but it was far less than that of 7-day-old embryo (P less than 0.02) . The suppressive activity of both the blastocoelic fluid and ECM was not due to cytotoxic effect, since this fluid supported the in vitro growth of single-cell rabbit embryos up to the stage of blastocyst . These results suggest that the immunologic tolerance of the embryo might be due to the immunosuppressors secreted by the embryo and that there might be a localized effect at the implantation site rather than a maternal systemic immunosuppressive effect.

J Neurosci, 1988 Aug, 8(8), 2844 - 58
Heparan sulfate proteoglycan and laminin immunoreactivity on cultured astrocytes: relationship to differentiation and neurite growth; Ard MD et al.; Extracellular matrix (ECM) produced by Schwann cells is known to promote growth of several types of neurites (Ard et al., 1987) . Whether a similar material produced by astrocytes may be available to promote neurite growth during CNS development is now open to question . The present study was undertaken to define conditions under which cultured astrocytes deposit the ECM components laminin and heparan sulfate proteoglycan (HSPG), and to relate this deposition to the ability of astrocytes to support neurite growth . The use of 2 different culture media permitted the growth of astrocytes either with or without these ECM components . Neonatal rat cortical astrocytes were cultured by the method of McCarthy and de Vellis (1980) and studied by immunocytochemistry and electron microscopy . Astrocytes grown in serum-containing medium for 5 or 9 d after subculturing were shown to have fibrillar patches of ECM containing both HSPG and laminin immunoreactivity . Immunoreactivity for the 2 molecules was usually colocalized . In contrast, astrocytes subcultured for 5 d in defined medium showed no immunocytochemical staining for either laminin or HSPG and had no ECM visible in EM . Formation of stellate processes was increased when cells were grown in defined medium compared with that seen in serum-containing medium, and growth of the population was slower . In 3 other conditions, attainment of stellate morphological differentiation by the astrocytes was correlated with diminution in immunostaining for ECM components . (1) In older cultures (30-42 d after subculturing), stellate, mitotically quiescent cells showed relatively little HSPG or laminin immunoreactivity . (2) Cultures initially maintained in serum-containing medium and then converted to defined medium lost much of their immunoreactivity for ECM components and developed longer processes . (3) When neurites from fetal rat dorsal root ganglion explants grew across monolayers of astrocytes in serum-containing medium, diminution of ECM immunostaining and development of stellate processes were seen in areas directly contacted by the neurites . ECM-containing laminin and HSPG did not appear to be necessary for neurites to interact with astrocytes . In defined medium, in which no ECM was detected, dorsal root ganglion neurites were found in contact with astrocyte surfaces rather than on the rat tail collagen substratum on the culture dish.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuroendocrinology, 1988 Aug, 48(2), 160 - 6
Human recombinant interleukin-1 beta and -alpha, but not recombinant tumor necrosis factor alpha stimulate ACTH release from rat anterior pituitary cells in vitro in a prostaglandin E2 and cAMP independent manner; Kehrer P et al.; The pituitary-adrenal axis is known to be stimulated during the acute-phase response . As cytokines play a central role in mediating the constellation of host response occurring during the acute-phase response it was of interest to assess the ability of cytokines to stimulate ACTH secretion from normal pituitary cells in culture . We used human recombinant interleukin-1 beta and -alpha (hrIL1 beta, hrIL1 alpha) and human recombinant tumor necrosis factor alpha (hrTNF alpha) to analyze the ability of these cytokines to induce ACTH secretion from normal rat anterior pituitary cells in culture . We also investigated the possible roles of prostaglandin E2 (PGE2) and cAMP in the cellular transduction mechanism . After 3 days of incubation primary cultures of rat anterior pituitary cells were stimulated for 24 h with either hrIL1 beta, hrIL1 alpha or hrTNF alpha alone or with the addition of dexamethasone or indomethacin . The culture media were analyzed for ACTH, PGE2 and cAMP content . At doses ranging from 0.03 to 30 nM, hrIL1 beta stimulated the release of ACTH and PGE2 in a dose-dependent manner . In contrast, at doses ranging from 3 to 60 nM, hrTNF alpha was unable to stimulate ACTH secretion although it stimulated PGE2 synthesis . Time-course experiments demonstrated that hrIL1 beta (3 nM) stimulates ACTH production over a period of 8, 16 and 24 h, but not after a period of 4 h . In these experiments, hrIL1 beta failed to cause any change in the secretions of growth hormone and luteinizing hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Lab Invest, 1988 Aug, 59(2), 173 - 80
Morphologic, immunohistochemical, and ultrastructural studies of the production of hepatitis B virus in vitro; Gerber MA et al.; Well differentiated human hepatoblastoma Hep G2 cells after transfection with cloned hepatitis B virus (HBV) genomes produce replicative HBV DNA intermediates, high levels of HBsAg, HBeAg and HBcAg as well as mature Dane particles . To analyze the replication cycle of HBV, we studied the expression of HBV antigens with monoclonal antibodies by immunomorphologic methods in the transfected cells at various time intervals after plating . HBcAg and HBeAg were detected in the cytoplasm and less frequently in the nuclei of transfected cells . The percentage of positive cells increased with time after plating and reached a plateau of about 50% positive cells at 10 days . HBsAg and the large and middle HBsAg polypeptides were observed in the cytoplasm of transfected cells and a maximum of 20 to 30% positive cells was reached during the 3rd week after plating . Examination of viable cells in suspension revealed HBcAg/HBeAg and HBsAg expression on the cell surface . Electron microscopy demonstrated characteristic core particles in the nuclei and cytoplasm and Dane particles in cytoplasmic vesicles and culture media of transfected cells . The HBV producing cells did not show any evidence of a cytopathic effect . These observations demonstrate significant similarities between the HBV DNA transfected cells and infected human hepatocytes which support active HBV replication in vivo . Taken together, the results suggest that the cultured cells may serve as a model to elucidate a number of unsolved problems of the molecular and cellular pathobiology of hepatitis B.

Hypertension, 1988 Aug, 12(2), 117 - 21
A monoclonal antibody to alpha-human atrial natriuretic polypeptide; Mukoyama M et al.; A monoclonal antibody to alpha-human atrial natriuretic polypeptide (alpha-hANP), KY-ANP-I, has been produced by fusion of a nonproducing mouse myeloma cell line, X63-Ag8.653, with spleen cells from BALB/c mice immunized with synthetic alpha-hANP conjugated to bovine thyroglobulin using the carbodiimide coupling procedure . Hybridomas were screened for antibody production by radioimmunoassay using culture media and 125I-alpha-hANP . They were cloned by the limiting dilution technique, expanded in culture, and injected intraperitoneally into BALB/c mice . The obtained antibody belonged to the immunoglobulin G1 subclass . Analysis by a Scatchard plot revealed a high affinity for alpha-hANP, with an association constant of 3.1 x 10(10) M-1 . With this monoclonal antibody, a specific radioimmunoassay for alpha-hANP has been established . The antibody in mouse ascites was available for radioimmunoassay at a final dilution of 1:10(6) . Values of IC10 and IC50 in this radioimmunoassay were 3 and 30 fmol/tube, respectively . The radioimmunoassay showed a cross-reactivity of 0.9% with alpha-rat ANP . alpha-hANP-(8-22) and alpha-ANP-(1-6) exhibited less cross-reactivity than alpha-rat ANP on a molar basis . There was no cross-reaction with alpha-ANP-(17-28) . Thus, the recognized epitope must be located in the N-terminal half of the ring structure of alpha-hANP including Met12 residue . This radioimmunoassay could detect gamma-hANP and beta-hANP as well as alpha-hANP . The monoclonal antibody was also useful for immunohistochemical studies . ANP-positive cells were finely stained in the human atrium using the avidin-biotin-peroxidase complex technique.(ABSTRACT TRUNCATED AT 250 WORDS)

Anticancer Drug Des, 1988 Aug, 3(2), 117 - 27
Evaluation of N-ras oncogene anti-sense, sense and nonsense sequence methylphosphonate oligonucleotide analogues; Tidd DM et al.; We have investigated the potential for using anti-sense non-ionic methylphosphonate oligonucleotide analogues to study the relationship between oncogene expression and maintenance of the transformed phenotype in malignant cells . Our results confirmed that the methylphosphonates are resistant to biochemical degradation and are devoid of non-specific toxicity towards cultured human HT29 cells . At low temperature (less than 5 degrees C) both N-ras anti-sense and nonsense analogue 9-mers formed 1:1 complexes in solution with an N-ras sense phosphodiester oligodeoxynucleotide 20-mer, but these were largely dissociated at 25 degrees C . Only a fraction (10-20%) of the anti-sense molecules formed stable sequence specific hybrids (Tm 34 degrees C) with the 20-mer . The biological activity of the oligonucleotide analogues was tested in cell culture at 37 degrees C using T15 cells, a line of NIH 3T3 cells transfected with multiple copies of the human N-ras oncogene under control of the glucocorticoid inducible MMTV promoter . On balance the N-ras anti-sense methylphosphonate 9-mer (20-80 microM) had no effect on these cells . In only one of five experiments was an apparent reduction in dexamethasone-induced p21N-ras protein accumulation observed in the presence of the oligonucleotide analogue . Also without effect was an anti-sense 20-mer consisting of a phosphodiester sequence bounded by two methylphosphonate linkages at each end (25-50 microM in culture media; 4.8 microM by microinjection) . We conclude from these experiments that, in order to achieve pronounced effects on oncogene expression, it may be necessary to use longer anti-sense methylphosphonate chains, affinity purified for their ability to hybridize with the target sequences.

Cancer Res, 1988 Aug 1, 48(15), 4294 - 8
Preclinical pharmacology of arabinosyl-5-azacytidine in nonhuman primates; Heideman RL et al.; The plasma and cerebrospinal fluid (CSF) pharmacokinetics of arabinosyl-5-azacytidine (AAC) were studied in rhesus monkeys following a 15-min, 1-h, or 12-h i.v . infusion of 200 mg/kg . No clinically significant toxicity was observed with these schedules . The plasma elimination of AAC is rapid and characterized by a triphasic decay with t1/2 alpha = 3.6-5.4 min, t1/2 beta = 18-24 min, and t1/2 gamma = 94-144 min for the above infusion schedules . The CSF penetration of AAC as measured by the CSF:plasma Css ratio for the 12-h infusion was 0.15 . The stability of AAC in pooled plasma, phosphate buffered saline, and RPMI 1640 culture media at 37 degrees C was compared with the terminal half-life of AAC observed in vivo . The shorter in vitro AAC half-life in plasma with or without tetrahydrouridine versus that in phosphate buffered saline suggests that the terminal half-life of AAC in vivo is most likely a result of enhanced nucleophilic attack and hydrolytic degradation of the unstable triazine ring in plasma . A triexponential equation modeling the disappearance of AAC was constructed from the in vivo experimental data . Use of this equation in computer-aided simulations of current Phase I doses and schedules of AAC correctly predicts the human plasma concentrations which have been observed . The preclinical pharmacokinetic data provided here may be useful in helping to develop rational human studies with specific concentration x time goals.

Gene, 1988 Jul 30, 67(2), 279 - 86
Secretion of Escherichia coli chloramphenicol acetyltransferase by mammalian cells; Bunker CA et al.; We show here that expression of the Escherichia coli cat gene in mammalian cells results in accumulation of enzymatically active CAT in the culture media as well as in the cytoplasm . We call the extracellular product secreted CAT (sCAT) . Three to four days after introduction of cat-expressing plasmids into mouse L cells by transient transfection, total extracellular sCAT activity exceeds total cytoplasmic CAT activity . As sCAT levels increase, substantially more CAT is found outside the cells than inside at later times . Comparison of different populations of cat-expressing cells shows that, at any given time, the level of sCAT is proportional to the level of intracellular CAT . Thus, assay of sCAT provides a convenient, non-invasive alternative to assay of intracellular CAT . The molecular sizes of sCAT and intracellular CAT are indistinguishable, suggesting that the protein is not cleaved or glycosylated during secretion . Several observations, including a lack of sensitivity to drugs which inhibit Golgi activity, suggest that CAT may be secreted via an unusual pathway.

J Immunol, 1988 Jul 15, 141(2), 690 - 8
Effects of transforming growth factor-beta on human lymphokine-activated killer cell precursors . Autocrine inhibition of cellular proliferation and differentiation to immune killer cells; Kasid A et al.; With subpopulations of human lymphoid cells that were enriched for lymphokine-activated killer (LAK) cell precursors, studies were performed to examine the effects of transforming growth factor-beta (TGF-beta) on their IL-2-dependent growth and differentiation to killer cells . The majority of the LAK precursor cells appeared to reside in nonadherent, non-T, and non-B lymphocyte populations that expressed CD11 and CD16 Ag . These cells were induced to proliferate and become LAK cells by high concentrations of rIL-2 alone in the apparent absence of any prior activation with mitogen or Ag . The partially purified lymphocyte subpopulations generated varying but several-fold greater levels of LAK killing on a per cell basis than did unfractionated lymphocytes . The exogenous addition of TGF-beta to the LAK precursor cultures, markedly inhibited IL-2-stimulated growth as well as the development of LAK activity in a dose-dependent manner . The antimitotic effect of TGF-beta was reversible; inhibition of proliferation could be largely restored by increasing the concentration of IL-2 in culture . In contrast, TGF-beta inhibition of cytotoxicity was relatively independent of the concentration of IL-2 . Further, LAK precursors constitutively expressed TGF-beta mRNA and high affinity receptor for TGF-beta . Activation of LAK precursors with IL-2 alone, resulted in a three- to fivefold up-regulation of intracellular TGF-beta mRNA and TGF-beta biologic activity secreted in the culture media . Furthermore, Northern blotting revealed that the resting LAK precursors did not express the Tac-mRNA . Receptor binding studies with 125I-IL-2 suggested the presence of a single class of IL-2R with an apparent Kd of intermediate range (beta-chain of IL-2R) on the unstimulated cells . Stimulation with high concentrations of Il-2 induced Tac-mRNA (both the 3.5- and 1.5-kb transcripts) and resulted in the expression of high affinity IL-2R (Kd approximately 10(-11) M) on these cells . Suppression of IL-2-dependent responses by TGF-beta was accompanied by a selective down-regulation of the 1.5-kb Tac-mRNA as well as by reduction in high affinity IL-2R . The results suggest a negative autocrine control of TGF-beta on IL-2-dependent growth and differentiation of human LAK cells, possibly related to regulate the killer activation function.

J Cell Physiol, 1988 Jul, 136(1), 182 - 7
Bone marrow stromal proteoglycan heterogeneity: phenotypic variability between cell lines and the effects of glucocorticoid; Bentley SA et al.; Hematopoiesis in vivo is dependent upon the interaction of hematopoietic stem cells with a complex microenvironment, of which stromal proteoglycans are an important functional component . Certain bone marrow stromal cell lines provide a microenvironment that supports hematopoiesis in vitro, a function that is dependent upon glucocorticoid supplementation . Proteoglycan synthesis in the hematopoietic-supportive D2XRII, Bl6 and 14F1 bone marrow stromal cell lines was studied by 35S-sulfate precursor labelling and ion-exchange separation, followed by isopyknic CsCl density centrifugation and gel filtration HPLC . The effects of glucocorticoid were also investigated . A similar pattern of proteoglycan heterogeneity was observed in all three cell lines, although there was considerable quantitative variation . All cultures synthesized three species of chondroitin/dermatan sulfate (CS/DS) proteoglycans: DS1, excluded from a Bio-Sil TSK-400 HPLC column, and DS2, eluting at Kd = 0.31, were present mainly in the culture media . The smallest (DS3) eluted at Kd = 0.63 and was present mainly in the cell layers . CS/DS species were the major proteoglycans in all cultures . Hydrocortisone-free cultures also synthesized heparan sulfate (HS) proteoglycans, including a cell-associated form (HS1), partially excluded from the TSK-400 column, and a secretory form (HS2), eluting at Kd = 0.15 . D2XRII cells also secreted an apparently-unique, high-density proteoglycan, Kd = 0.65, into the culture medium . Hydrocortisone at 10(-6) M virtually abolished HS proteoglycan synthesis in all three cell lines, and altered the pattern of CS/DS proteoglycans in the culture media, increasing the quantity of DS1 and DS3, and reducing the quantity of DS2.

Carcinogenesis, 1988 Jul, 9(7), 1121 - 7
In vivo and in vitro characteristics of early carcinogen-induced premalignant phenotypes in cultured rat tracheal epithelial cells; Steele VE et al.; The initial stages of neoplastic transformation in respiratory tract epithelial cells were defined and studied by characterizing a series of morphologically transformed cell colonies from carcinogen-exposed rat tracheal epithelial (RTE) cell cultures both in vivo and in vitro . RTE cells were isolated from Fischer 344 rats, plated on collagen-coated dishes, and exposed to 7,12-dimethylbenz{a}anthracene on day 1 for 24 h . Between days 26 and 30, single colonies of morphologically altered cells were isolated and classified into three major classes based on cell density . Following replating, the cells were tested for their ability to grow on various substrates and in various culture media . Generally, Class II and III cells exhibited a higher colony forming efficiency when replated on various substrates . Class III cells appeared to grow better than Class I or II cells in complete medium, while Class I cells grew better in medium without 3T3 conditioning factors . At early passage levels, the population doubling times were longer for Class I cells than for Class II cells . Class III cells had the shortest population doubling times . The various cell lines were also placed into denuded tracheal grafts . Untreated cells produced a normal mucociliary epithelium, while Class I cells produced a simple cuboidal epithelium . Class II and III cells formed a highly atypical and usually malignant epithelia . Inoculation of the three classes of cells into nude mice provided confirming evidence of the benign nature of Class I cell lines and the malignant nature of some Class II cell lines and all of the Class III cell lines.

Mol Cell Endocrinol, 1988 Jul, 58(1), 65 - 72
Paracrine control of immature Sertoli cells by adult germ cells, in the rat (an in vitro study) . Cell-cell interactions within the testis; Le Magueresse B et al.; Enriched populations of germ cells prepared from adult rats were found to influence 20-day-old rat Sertoli cell secretory activity by stimulating androgen-binding protein (ABP) and inhibiting oestradiol-17 beta production in the presence of follicle-stimulating hormone (FSH) as well as of dibutyryl cyclic AMP (dbcAMP) . Among the different populations tested in coculture, pachytene spermatocytes were the most effective at stimulating ABP and inhibiting oestradiol production, whereas early spermatids had relatively less effects . Cytoplasts from elongated spermatids only slightly stimulated ABP secretion . The influence of germ cells upon Sertoli cells may be mediated via paracrine component(s) detected in nonconcentrated conditioned culture media . The stimulatory (ABP) and inhibitory (oestradiol) effects of pachytene spermatocyte and early spermatid-spent media were reversible (change of media), dose related, specific (no effect of cytoplast, peritubular cell, rat liver epithelial cell or 3T3 cell-conditioned media) and strictly proportional to the cell viability estimated at the end of the incubation periods . Furthermore, the nature of the germ cell factor(s) influencing Sertoli cell secretory function is likely to be proteinaceous since both germ cell-spent media effects were trypsin and heat (100 degrees C; 3 min) sensitive and retained by molecular weight (MW) greater than 10,000 cut-off dialysis membranes . It is hypothesized that germ cells, in particular pachytene spermatocytes and early spermatids, may influence Sertoli cell function during sexual development in the rat.

J Virol, 1988 Jul, 62(7), 2258 - 64
Purification and characterization of the external envelope glycoprotein from two human immunodeficiency virus type 1 variants, HTLV-IIIB and HTLV-IIIRF; Pyle SW et al.; External envelope glycoprotein from cell membranes and culture media of H9 cells infected with human immunodeficiency virus type 1 (HIV-1) isolate HTLV-IIIRF was isolated by immunoaffinity chromatography and compared with similar materials isolated from another variant, HTLV-IIIB . Envelope glycoprotein from IIIB and IIIRF appears to be identical, whether isolated from infected cell membranes or culture media . The molecular size of the IIIRF external envelope glycoprotein was 110 kilodaltons, whereas the relative size of IIIB gp120 was 123 kilodaltons . Amino-terminal sequence analysis of purified external envelope glycoprotein isolated from infected cell membranes or culture fluids revealed identical single sequences for the first 20 amino acids for each variant . The sequences obtained for IIIB gp120 were identical to those reported for the BH10 clone of the IIIB isolate, and the sequences determined for IIIRF gp110 matched the amino acid sequence predicted for the HAT3 clone of the Haitian HIV isolate . The amino-terminal sequences of external envelope glycoproteins isolated from either HIV-1 variant corresponded to the sequence starting at the proposed proteolytic cleavage site for the processing of the signal peptide of gp160 . Immunization with external envelope glycoprotein isolated from either of the two HIV-1 variants yielded goat antibodies that primarily precipitated the homologous antigen . Sequential immunization of a single goat with gp120 and then gp110 resulted in the generation of antibodies that precipitated external envelope glycoprotein from both variants.

Jpn J Cancer Res, 1988 Jul, 79(7), 836 - 42
Isolation of virus-producing transformants from human gastric cancer cell line, HGC-27, infected with human T-cell leukemia virus type I; Akagi T et al.; A human anaplastic gastric cancer cell line, HGC-27, showed marked degeneration with formation of multinucleated syncytia and cell detachment of nearly all cells which began 24 hr after and reached a maximum 2 to 3 days after co-cultivation with X-irradiated MT-2 cells, HTLV-I producing human cord leukocytes . Less severe degeneration without formation of syncytia was also observed in the cultures inoculated with cell-free MT-2 culture media . Morphologically altered cells began to proliferate and formed piled up colonies in some of the cultures co-cultivated with X-irradiated MT-2 cells after a long culture period . The two clones designated HGC/MT2 (Cl-1) and HGC/MT2 (Cl-2) were separated by cell cloning . HGC/MT2 (Cl-1) and HGC/MT2 (Cl-2) cells were positive for HTLV-I gag proteins (p19 and p24) and pX gene products, p40x, as demonstrated by immunohistochemistry and immunoblotting analysis, contained HTLV-I provirus DNA, and consistently produced type C virus particles.

Br J Cancer, 1988 Jul, 58(1), 17 - 21
Macromolecular osteolytic factor synthesised by squamous carcinoma cell lines from the head and neck in vitro is interleukin 1; Meghji S et al.; Three human cell lines derived from oro-pharyngeal squamous cell carcinomas of the head were investigated for bone-resorbing activity in vitro . Culture media from all three spontaneously produced a non-dialysable osteolytic factor with activity in three in vitro assays for interleukin 1 (IL1), viz . the lymphocyte activating factor (LAF) assay, stimulation of collagenase synthesis by articular chondrocytes, and stimulation of prostaglandin E2 synthesis by fibroblasts . Addition of anti-human IL1 antibody to the culture media abolished all the bone-resorbing activity . Fractionation of the cell culture media by high performance liquid chromatography (HPLC) showed a single peak of activity in the chondrocyte assay with an apparent mol.wt of 15-17,000 . This co-eluted with activity in a preparation of IL1 from rat peritoneal macrophage cultures . These results indicate that IL1 is responsible for the prostaglandin-independent bone resorbing activity synthesised by these cells in vitro, and may contribute to the bone destruction associated with the tumour.

APMIS, 1988 Jul, 96(7), 589 - 95
An experimental model system for leishmaniasis . An ultrastructural study on cultured macrophages exposed to Leishmania parasites and sodium stibogluconate; Abok K et al.; To facilitate studies on the effect of chemotherapeutic agents on the host-parasite interaction in leishmaniasis, we have developed an experimental model for infecting mouse peritoneal macrophages in culture with recently-isolated Leishmania donovani promastigotes . As the drug action is often dependent on concentration, the distribution of sodium stibogluconate, which is the commonly used drug for treatment of leishmaniasis, was studied in various parts of the macrophages by energy dispersive X-ray microanalysis . The drug was found to accumulate in secondary lysosomes . The ultrastructural examination, using TEM and SEM, of macrophages, whose secondary lysosomes had been preloaded with gold particles, showed that leishmania parasites are phagocytosed and finally located in secondary lysosomes . Using flameless atomic absorption spectrophotometry, the concentration of Mn, Fe and Cu in promastigotes of Leishmania donovani, Leishmania aethiopica, Leishmania crithidia, Leishmania major and their culture media was estimated . Of the three transition metals, the parasites accumulated only Mn from the medium, which they may use in a primitive defense mechanism against reactive oxygen metabolites produced by macrophages during the respiratory burst associated with phagocytosis.

Arch Biochem Biophys, 1988 Jul, 264(1), 351 - 4
Induction of collagenase mRNA in lapine articular chondrocytes by synovial factors and interleukin-1; Lin CW et al.; The cDNA probe H-9, originally constructed to recognize a portion of the mRNA for lapine synovial collagenase, also hybridized with a RNA of the same size (approximately 2.0 kb) isolated from activated lapine articular chondrocytes . Primary, monolayer cultures of lapine articular chondrocytes did not contain detectable amounts of this RNA, nor did they secrete measurable amounts of collagenase into their culture media . Following exposure to synovial factors, the chondrocytes contained high levels of collagenase mRNA, while their conditioned media had considerable collagenolytic activity . Collagenase mRNA started to appear in chondrocytes 3-5 h after treatment with the synovial material . Maximum levels occurred after 12-24 h . Recombinant human interleukin-1 also induced the appearance of this mRNA . We conclude that chondrocyte collagenase is likely to be the same gene product as synovial collagenase, and that its regulation by lapine articular chondrocytes probably occurs at a pretranslational level.

Blood, 1988 Jul, 72(1), 273 - 81
An analysis of the multilineage production of human hematopoietic progenitors in long-term bone marrow culture: evidence that reactive oxygen intermediates derived from mature phagocytic cells have a role in limiting progenitor cell self-renewal; Meagher RC et al.; To better understand the limited hematopoietic life span of human marrow "Dexter" cultures, we developed a miniaturized, two-stage culture system with which in vitro production of hematopoietic progenitors could be reproducibly detected and quantified . Light-density, gradient-separated human marrow cells were inoculated into Leighton slide tubes, and adherent ("stromal") cell layers were allowed to develop on the removable coverslips within these tubes during an initial 4 weeks of culture . Once stromal cell layers were established, cultures were irradiated (800 cGy) to eliminate all residual hematopoietic progenitors . The cultures were then recharged with autologous, cryopreserved marrow cells (enriched for BFU-E and CFU-GM) to reconstitute stem cell populations and to initiate in vitro hematopoiesis . Most progenitor cells added to irradiated cultures were no longer detectable by clonal assays within one to four days after recharge . Nonetheless, stable populations of adherent BFU-E and CFU-GM became established in these cultures within 24 to 48 hours, and when the total numbers of progenitors (adherent and nonadherent) were measured at weekly intervals thereafter, it was evident that both BFU-E and CFU-GM were generated in vitro . However, progenitor cell production declined as neutrophils and macrophages accumulated in the cultures . Moreover, with this accumulation of mature myeloid cells, increasing levels of O2- and H2O2 could be detected in the cultures, and it was found that the addition of oxidant scavengers (catalase and mannitol) to culture media enhanced the weekly expansions of progenitor cell numbers that could be measured . These findings support the conclusion that reactive O2 intermediates generated by mature myeloid cells have a role in limiting the duration and extent of hematopoietic progenitor cell self-renewal in long-term "Dexter" cultures of human marrow.

Metabolism, 1988 Jul, 37(7), 664 - 8
Inhibition of thyroxine 5'-deiodination type II in cultured human placental cells by cortisol, insulin, 3', 5'-cyclic adenosine monophosphate, and butyrate; Hidal JT et al.; The regulation of conversion of thyroxine (T4) to 3,5,3'-triiodothyronine (T3) by the type II iodothyronine deiodinating pathway was studied in normal human placental cells cultured from the chorionic membrane . T4 5'-deiodination was measured in cell sonicates after intact cells were incubated with test agents for 24 to 48 hours . Stimulation of T4 5'-deiodination occurred to a similar degree after depriving cells of thyroid hormone in serum-free medium and in medium containing 10% calf serum . Cortisol at 10 to 100 nmol/L in serum-free medium inhibited T4 5'-deiodination up to 36%, and 1 to 100 nmol/L of insulin inhibited deiodination up to 50% . Dibutyryl-cyclic AMP (dbcAMP) inhibited deiodination, but this appeared to result from the inhibitory effects of butyrate . Addition to the culture media of 8-bromo-cAMP, cholera toxin, and theophylline each caused partial inhibition of T4 5'-deiodination, strongly suggesting an inhibitory effect of raised intracellular cAMP . Neither alpha- nor beta-adrenergic agonists had any effect when added to the culture medium, nor did glucagon or cysteamine . These results demonstrate a complex, multihormonal control of human placental type II iodothyronine deiodination, and suggest that changes in the activity of this pathway may result in altered intracellular, and conceivably circulating, T3 concentrations in states of cortisol excess and marked hyperinsulinism . The factor that regulates type II deiodination via cAMP remains to be identified.

Cancer, 1988 Jul 1, 62(1), 92 - 7
Demonstration of Chromogranin A in human neuroendocrine cell lines by immunohistology and immunoassay; Deftos LJ et al.; We have used immunohistology and radioimmunoassay procedures to study Chromogranin A (CgA) in human neuroendocrine tumor cell lines, especially small cell lung cancers (SCLC) . By immunohistology, CgA could be detected in 11 of 18 classical SCLC cell lines, in a medullary thyroid carcinoma (MTC) cell line, and in only one of 13 variant- or non-SCLC cell lines . By radioimmunoassay, CgA could be detected in the cells and culture media of all of the classical SCLC cell lines tested . Many of the classical SCLC cell lines also produced calcitonin (CT) . These studies demonstrate that CgA production is a common feature of SCLC cell lines, especially those with neuroendocrine characteristics.

J Immunol Methods, 1988 Jun 28, 111(1), 125 - 9
Mitogen-induced lymphocyte transformation in four different serum-free media; Blaehr H et al.; The proliferative responses of lymphocytes induced by the mitogens phytohemagglutinin (PHA), pokeweed mitogen (PWM) and concanavalin A (ConA) were tested in four different serum-free lymphocyte culture media in which serum was replaced by commercially produced serum substitutes . Two of the serum-free lymphocytes cultures (SF-X and FEB-100) achieved proliferative responses to PHA and PWM comparable with those obtained in medium containing fetal calf serum, but only when the cell number was increased, whereas none of the lymphocyte cultures in serum-free medium showed adequate proliferative responses to ConA stimulation . The essential factors in serum-free media for optimal growth and mitogen stimulation of lymphocytes include insulin, transferrin, electrolytes, glutamine and a high cell concentration.

J Immunol, 1988 Jun 15, 140(12), 4253 - 5
H-2-dependent binding of xenogeneic beta 2-microglobulin from culture media; Kievits F et al.; Sera of C57BL/6 mice contained lymphocytotoxic antibodies after injections with syngeneic lymphoblasts . The antibodies were directed against bovine beta 2-microglobulin, a component of the culture medium, and mimicked H-2-specific antibodies by a preferential recognition of target cells expressing certain H-2 Ag . This "polymorphic" reactivity pattern was due to a variable capacity of H-2 molecules associated with bovine beta 2-microglobulin.

J Biol Chem, 1988 Jun 15, 263(17), 8473 - 9
The kinetics of interleukin 1 secretion from activated monocytes . Differences between interleukin 1 alpha and interleukin 1 beta; Hazuda DJ et al.; We have performed pulse-chase experiments to investigate the secretion and processing of interleukin 1 (IL-1) by human peripheral blood monocytes . Polyclonal antisera generated against either recombinant IL-1 alpha (p15) or IL-1 beta (p17) could distinguish the two isoelectric forms in lysates and supernatants of lipopolysaccharide-activated monocytes . In agreement with previous results, no processed IL-1 (alpha or beta) is detected in cell lysates . Both the 31-kDa precursor and 17-kDa mature forms of IL-1 were present, however, in the culture media indicating that processing is not required for secretion . The relative amounts of the secreted 31- and 17-kDa forms of IL-1 remain constant with time throughout each experiment; in addition, 31-kDa IL-1 added to monocyte cultures is not processed to the mature 17-kDa form . Precursor IL-1 beta is however, processed to 17 kDa by monocyte extracts . Therefore, the maturation and secretion of IL-1 are intimately coordinated processes . The kinetics of IL-1 secretion are unique in comparison with other secreted proteins; release of both IL-1 alpha and IL-1 beta is delayed following synthesis, and large pools of precursor IL-1 accumulate intracellularly . The intracellular half-lives of IL-1 alpha and IL-1 beta are 15 and 2.5 h, respectively . This discrepancy in half-lives is a reflection of the different kinetics with which IL-1 alpha and IL-1 beta are secreted . IL-1 beta is released continuously beginning 2 h after synthesis, whereas the secretion of IL-1 alpha is delayed for an additional 10 h . The distinct kinetics of secretion demonstrated for IL-1 alpha and IL-1 beta suggest that the release of each pI species of IL-1 is controlled by a selective mechanism(s).

Biochem J, 1988 Jun 15, 252(3), 701 - 7
Polyamines and insulin production in isolated mouse pancreatic islets; Welsh N et al.; The aim of the present study was to evaluate the role of polyamines in the metabolism and insulin production of pancreatic-islet cells . For this purpose islets were prepared from adult mice and used either immediately or after tissue culture . There was a significant decrease in the islet content of spermidine during culture, although the effect was less pronounced in a high glucose concentration . Furthermore, a stimulatory effect of a high glucose concentration, as compared with low guclose, on the content of spermine was observed . To elucidate further the role of polyamaines in beta-cell physiology, the ornithine decarboxylase inhibitors difuoromethylornithine (DFMO) and methylacetylenic putrescine (MAP) and the S-adenosylmethionine decarboxylase inhibitor ethylglyoxal bis(guanylhydrazone) (EGBG) were added to the culture media . Addition of DFMO together with MAP decreased the cellular contents of putrescine and spermidine, whereas the content of sperimine was unaffected . When EGBG was added in combination with DFMO and MAP, there was a decrease in the content of spermine also . Cell viability in the islets depleted of their polyamine contents was not impaired, as assessed by determinations of oxygen-uptake rates and ATP contents . Depletion of putescine plus spermidine by addition of DFMO+MAP was associated with decreased biosynthesis of (pro)insulin and total protein . When the content of spermine was decreased also by the further addition of EGBG, the decrease in (pro) insulin biosynthesis was more pronounced and was paralleled by a decrease in the insulin-mRNA content . Under these conditions, the glucose-stimulated insulin release, the insulin content and the rates of islet DNA synthesis were also decreased . It is concluded that putrescine and spermidine are necessary for the maintenance of normal insulin and protein biosynthesis, whereas spermine may exert a role in some other cellular processes, such as DNA replication, RNA transcription and glucose-stimulated insulin release.

Am J Physiol, 1988 Jun, 254(6 Pt 2), F912 - 7
Atrial natriuretic peptide regulates release of Na+-K+-ATPase inhibitor from rat brain; Crabos M et al.; Tissue culture media from incubations of fragments of rat brain were collected and partially purified . These supernatants were effective in inhibiting the Na+-K+ pump as indicated by a 77% reduction of ouabain-sensitive 86Rb+ uptake into human erythrocytes . Release of the Na+-K+-ATPase inhibitor depended on the amount of tissue, the temperature, and the length of incubation . Atrial natriuretic peptide (ANP) injected intravenously, or included (10(-8) M) in the in vitro incubation of brain tissue, decreased the release of the Na+-K+-ATPase inhibitor by 74 and 42%, respectively . Control experiments using the neuropeptide arginine vasopressin showed no effect on release of the inhibitor . These studies indicate that ANP is capable of regulating the release from brain of a Na+-K+-ATPase inhibitor with similar chromatographic characteristics to the one previously obtained from extraction of bovine hypothalamus and raise the possibility that the two factors are interrelated in the regulation of fluid and electrolyte balance.

J Biol Chem, 1988 Jun 5, 263(16), 7567 - 73
Proliferative effects of insulin and epidermal growth factor on mouse mammary epithelial cells in primary culture . Enhancement by hydroxyeicosatetraenoic acids and synergism with prostaglandin E2; Bandyopadhyay GK et al.; Linoleate metabolism via the cyclooxygenase pathway enhances the proliferation of mammary epithelial cells in serum-free culture in the presence of epidermal growth factor and insulin (Bandyopadhyay, G.K., Imagawa, W., Wallace, D., and Nandi, S . (1987) J . Biol . Chem . 262, 2750-2756) . Prostaglandin E2 (PGE2) can fully substitute for linoleic acid provided endogenous hydroxyeicosatetraenoic acids (HETEs, lipoxygenase metabolites) are available . The PGE2 effect is partial if lipoxygenase activity is inhibited by nordihydroguaiaretic acid . Any combination of two HETEs out of three tested (5-, 12-, and 15-HETEs) stimulates growth synergistically with PGE2; and together (i.e . PGE2 + HETEs), they completely substitute for linoleate . In the absence of PGE2, maximal stimulation cannot be attained with HETEs . Exogenous 5-HETE, compared with 12- or 15-HETE, is preferentially incorporated by the mammary epithelial cells, and about 25-30% of it is retained esterified in phospholipids . The cellular level of nonesterified, free HETE is low . Radioimmunoassay revealed that the concentrations of 12- and 15-HETEs in the culture media (with or without added linoleate) were always higher than that of 5-HETE . Both intra- and extracellular free HETEs are rapidly metabolized by the cells . Since these cells are capable of producing eicosanoids from linoleate, periodic supplementation of the cultures with linoleate allows maintenance of higher HETE and PGE2 levels . Thus, it appears that not only are HETEs short-lived in the cell cultures, but cells handle 5-HETE differently than 12- and 15-HETEs . Whatever may be the pathways of interaction, synergism between HETEs and PGE2 seems to explain how linoleate stimulates the growth of mammary epithelial cells in the presence of epidermal growth factor and insulin.

Mol Cell Endocrinol, 1988 Jun, 57(3), 169 - 78
Estrogenic activity of phenol red; Welshons WV et al.; It has recently been reported that phenol red, a pH indicator present in most tissue culture media, is a weak estrogen that can stimulate some estrogen-sensitive cells . However, the relative impact of phenol red on various cell lines is controversial . We examined the effect of phenol red on several estrogen-responsive cell systems that we use to study estrogen action . These included estrogenic stimulation of progesterone receptor and growth in human breast cancer-derived MCF-7 cells, stimulation of growth in human breast cancer-derived T47D cells, stimulation of prolactin synthesis in primary cultures of immature rat pituitary cells, and stimulation of progesterone receptor in primary cultures of immature rat uterine cells . Estrogenic responses in MCF-7 cells were the most sensitive to the presence of phenol red, while the other three cell cultures showed lesser effects of the indicator . In addition to intrinsic differences in cell responses, there were several other factors involved . These included differences in the estrogenic activity of phenol red-containing media and phenol red itself from different commercial suppliers, and differences in the concentration of free phenol red in final media due to binding of the indicator by serum . Higher concentrations of serum reduced the impact of phenol red on estrogenic responses in primary pituitary cells . Phenol red added to rat uterine cytosol competed with estradiol for binding to the estrogen receptor (relative binding affinity (RBA) approx . 0.001), and the acidic and basic forms of the indicator showed similar activity . Some commercial phenol red samples inhibited cell growth at levels of 100 mg/l; these effects were toxic rather than antiestrogenic, because growth inhibition could not be competitively reversed by an excess of estradiol . The amount of the indicator bound to serum in the final media, the source of the phenol red and the sensitivity of different cell types to the indicator ultimately determine its influence to the response of cells in tissue culture.

J Steroid Biochem, 1988 Jun, 29(6), 641 - 8
Gerbil adrenal 11 beta- and 19-hydroxylating activities respond similarly to inhibitory or stimulatory agents: two activities of a single enzyme; Drummond TD et al.; A high level of steroid 19-hydroxylation is exhibited by adrenal mitochondria of the gerbil, Meriones, unguiculatus, that accounts for the ability of that species to produce nearly equal amounts of corticosterone and 19-hydroxycorticosterone (Proc . Soc . exp . Biol . Med . 165 (1980) 69-74) . Inhibitors of steroidogenesis and a polyclonal antibody against bovine cytochrome P-450(11 beta) were used to determine if the agents would effect differential or parallel suppression of 19- vs 11 beta-hydroxylation by gerbil adrenal mitochondria in vitro . The inhibitors (0.1-60 microM) tested (listed in order of decreasing effectiveness) were imazalil, metyrapone, miconazole and 4-hydroxyandrostenedione . With each inhibitor the degree of suppression of 11 beta-hydroxylation was accompanied by a parallel decline in 19-hydroxylation . The addition of the polyclonal antibody preparation also produced equivalent declines in the rates of the two hydroxylation reactions . The addition of ACTH 1 microM to primary cultures of gerbil adrenal cells brought about nearly equal increases in the secretion of 11 beta- and 19-hydroxylated steroids into the culture media . These results support the hypothesis that the 11 beta-hydroxylase of gerbil adrenal mitochondria has the capacity to carry out 11 beta- and 19-hydroxylations with nearly equal facility.

J Steroid Biochem, 1988 Jun, 29(6), 629 - 34
Contrasting effects of estradiol-17 beta and 17 alpha-ethinyl estradiol-17 beta on cultured whole embryos; Beyer BK et al.; Estradiol-17 beta (E2) and 17 alpha-ethinyl estradiol-17 beta (EE) were compared in terms of their relative capacities to alter growth and developmental patterns of cultured whole embryos during the early stages of organogenesis . Embryos exhibited a notable differential susceptibility to the embryotoxic effects of parents E2 vs EE when these estrogens were added directly to the media at the onset of the culture period . At initial concentrations of 0.1 mM, E2 failed to produce statistically significant effects whereas EE elicited marked embryotoxicity . Inclusion of a P-450-dependent biotransformation system in the culture media resulted in a significant attenuation of the embryotoxic effects of parent E2 vs EE when these estrogens were added directly to the media at the onset of the culture period . At initial concentrations of 0.1 mM, E2 failed to produce statistically embryotoxicity by hepatic S9 . The divergent results produced by the two steroids could not be attributed to differences in rates of catecholestrogen generation in the culture medium or by the conceptuses . The results demonstrate definitive dissimilarities between the effects of two steroidal estrogens on developmental parameters and document marked differences in the effects of biotransformation on their embryotoxic potential . The data strongly suggest that the embryotoxicity of these steroids is not mediated via interactions with estrogen receptors . Additionally, the data show that the differential capacity of these two steroids to produce embryotoxic effects is diametrically opposite to earlier reported patterns of their carcinogenic potential in the Syrian hamster kidney.

J Clin Invest, 1988 Jun, 81(6), 1746 - 51
Isolation and partial characterization of melanoma-associated antigens identified by autologous antibody; Vlock DR et al.; The study of the autologous immune response to cancer avoids the difficulties encountered in the use of xenoantisera and may identify antigens of physiological relevance . However, the low titer and incidence of autologous antibody to melanoma have hampered further evaluation . By utilizing acid dissociation and ultrafiltration of serum, we have been able to augment the detectable autologous immune response to melanoma in the majority of patients studied . In autologous system Y-Mel 84:420, serum S150 demonstrated a rise in titer from 1:32 in native sera to 1:262,044 after dissociation . The antigen detected by S150 was found to be broadly represented on melanoma, glioma, renal cell carcinoma, neuroblastoma, and head and neck carcinoma cell lines . It did not react with bladder or colon carcinoma, fetal fibroblasts, pooled platelets, lymphocytes and red blood cells, or autologous cultured lymphocytes . Using polyacrylamide gel electrophoresis, S150 detects a 66,000-mol wt antigen in spent tissue culture media and serum ultrafiltrate . In cell lysate two bands between 20,000 and 30,000 mol wt are detected by S150 . The 66,000-mol wt antigen is sensitive to trypsin digestion and but is resistant to pepsin and heat inactivation . Exposure of spent media to trypsin results in the development of a 24,000-mol wt band that appears to correspond to the antigen detected in the cell lysate . The difference between the antigens detected in the cell lysate as compared with spent media and serum ultrafiltrate may be due to degradation during cell lysis . We conclude that melanoma-associated antigens are present in the serum of patients with melanoma and are shed or secreted by their tumor cells.

Am J Kidney Dis, 1988 Jun, 11(6), 512 - 4
Coccidioidal peritonitis associated with continuous ambulatory peritoneal dialysis; Ampel NM et al.; We report the first three cases of peritonitis due to the fungus Coccidioides immitis occurring during continuous ambulatory peritoneal dialysis (CAPD) . At the time of diagnosis, none of the patients had evidence of active infection outside of the peritoneal cavity . Clues suggesting the diagnosis including a previous history of pulmonary coccidioidomycosis, an excess number of eosinophils in the peritoneal fluid, and failure to respond to therapy directed against bacteria . C immitis in peritoneal fluid was more readily isolated on specific fungal culture media than on routine bacterial culture media . In no instances did potassium hydroxide (KOH) preparations of the fluid reveal fungi . Coccidioidal peritonitis during CAPD appears to be a localized form of extrapulmonary coccidioidomycosis that has a relatively benign course once the peritoneal catheter is removed.

Endocrinology, 1988 Jun, 122(6), 2379 - 86
Estrogen receptor distribution in enucleated breast cancer cell lines; Welshons WV et al.; The intracellular location of estrogen receptors in hormone-responsive cells has been studied with a number of techniques which indicate that the unoccupied receptors are nuclear and not cytoplasmic proteins . We used cell enucleation of two human breast cancer-derived cell lines, MCF-7 and T47D, to determine whether the unoccupied receptors were also nuclear in these cells and to determine whether the weak estrogen phenol red, present in nearly all tissue culture media, affected the distribution of the receptors seen with this technique . Nucleoplasts prepared from the breast cancer cells contained most of the estrogen receptors that were present in whole cells . The cytoplast fraction, which contained some contaminating whole cells, also contained some receptors . However, incubating cells with estradiol before enucleation did not translocate any receptors out of the cytoplast fraction (to the nucleoplasts) . The unoccupied receptors appeared to be almost exclusively nuclear in these cells . The same results were obtained with either radioligand binding or enzyme-linked immunoassay used to measure estrogen receptor, and the distribution of receptors was unaffected by the presence of the pH indicator phenol red . In addition, we observed changes in the estrogen receptor content of incubated cytoplasts that were consistent with receptor synthesis, and this may prove to be a useful model system to characterize receptor synthesis and degradation.

Immunology, 1988 Jun, 64(2), 227 - 31
Malarial parasites induce TNF production by macrophages; Bate CA et al.; Mouse peritoneal macrophages incubated with erythrocytes infected with non-lethal or lethal variants of Plasmodium yoelii or with P . berghei, in the presence of polymyxin B to exclude the effects of any contaminating endotoxin, secreted a cytotoxic factor into the supernatant that was shown to be tumour necrosis factor (TNF) . No differences were observed in the ability of the three types of parasite to induce TNF production, which was maximal in the range of 0.2-5 infected erythrocytes per macrophages . TNF production was equivalent to that induced by lipopolysaccharide (LPS) and was enhanced by pretreatment of the macrophages with interferon-gamma (IFN-gamma) or with indomethacin . Culture media containing parasite products also induced macrophages to secrete TNF . The activity withstood boiling and was inhibited by malaria-specific antisera . Since heat-stable antigens are present in the circulation of patients with malaria, they may induced the secretion of TNF, a mediator of endotoxic shock, which could contribute to the pathology of the disease.

J In Vitro Fert Embryo Transf, 1988 Jun, 5(3), 149 - 52
The temporal effects of changes in in vitro fertilization culture media on the one-cell mouse embryo system; Davidson A et al.; The one-cell mouse embryo system has previously been shown to be more sensitive than the two-cell system to mild changes in in vitro fertilization (IVF) culture media . To determine whether this greater sensitivity is related to the developmental stage or to the length of exposure, one-cell embryos were collected and cultured in control media (Ham's F-10, 282 mOsm/liter), in media of altered osmolality (260, 300, and 316 mOsm/liter), or in media containing Cidex diluted 1:100,000 . The one-cell embryos were exposed to control or altered media in four patterns: control group--control medium for 96 hr; Group A--altered medium for the first 24 hr followed by control medium for 72 hr; Group B--control medium for the first 24 hr followed by altered medium for 72 hr; and Group C--control medium for the first 24 hr, altered medium for the next 24 hr, and control medium again for 48 hr . The percentage of embryos developing to blastocysts in Group A (exposed to adverse conditions only for the first 24 hr of culture) was significantly lower than in the control group under all conditions studied . In contrast, the percentage of blastocysts developing in Group B was significantly lower than in the control group only in medium of 315 mOsm/liter and was not different from that in controls under the other conditions studied . There was no difference between Group C and the control group . We conclude that the higher sensitivity of the one-cell system is an inherent property of the one-cell stage, as exposure of the embryo during this critical first 24-hr period proved to have the most profound consequences.

J Cell Biol, 1988 Jun, 106(6), 2127 - 37
Dexamethasone-dependent inhibition of differentiation of C2 myoblasts bearing steroid-inducible N-ras oncogenes; Gossett LA et al.; ras proteins are localized to the plasma membrane where they are postulated to interact with growth factor receptors and other proximal elements in intracellular cascades triggered by growth factors . The molecular events associated with terminal differentiation of certain skeletal myoblasts are inhibited by specific polypeptide growth factors and by constitutive expression of transforming ras oncogenes . To determine whether the inhibitory effects of ras on myogenic differentiation were reversible and to investigate whether muscle-specific genes remained susceptible to ras-dependent repression in terminally differentiated myotubes, the murine myoblast cell line, C2, was transfected with a plasmid containing a mutationally activated human N-ras oncogene under transcriptional control of the steroid-sensitive promoter of the mouse mammary tumor virus long terminal repeat . Addition of dexamethasone to myoblasts bearing steroid-inducible ras oncogenes prevented myotube formation and induction of muscle creatine kinase and acetylcholine receptors . Inhibition of differentiation by dexamethasone occurred in a dose-dependent manner and was a titratable function of ras expression . In the presence of dexamethasone, myoblasts bearing steroid-inducible ras genes retained their dependence on exogenous growth factors to divide and exhibited contact inhibition of growth at confluent densities, indicating that the inhibitory effects of ras on differentiation were independent of cell proliferation . Removal of dexamethasone from N-ras-transfected myoblasts led to fusion and induction of muscle-specific gene products in a manner indistinguishable from control C2 cells . Examination of the effects of culture media conditioned by ras-transfected myoblasts on differentiation of normal C2 cells yielded no evidence for inhibition of differentiation via an autocrine mechanism . In contrast to the ability of N-ras to prevent up-regulation of muscle-specific gene products in myoblasts, induction of N-ras in terminally differentiated myotubes failed to extinguish muscle-specific gene expression . Together, these results suggest that oncogenic ras proteins reversibly activate an intracellular cascade that prevents establishment of the differentiated phenotype . The inability of ras to extinguish muscle-specific gene expression in terminally differentiated myotubes also suggests that ras may interfere with an early step in the pathway of myoblasts toward the differentiated state.

J Biol Buccale, 1988 Jun, 16(2), 51 - 7
Fibronectin-degrading activity in human crevicular fluid, gingival explants culture medium and bacterial plaques; Pellat B et al.; 125I-fibronectin was incubated with extracts having presumably a proteolytic activity . Plaque from children without gingivitis, plaque from adults with chronic periodontitis, human gingival fluid and pooled culture media of human gingival explants were studied . Proteolysis was usually faster with plaque from patients with adult periodontitis than with plaque from children without gingivitis and the inhibition tests showed that several enzymes were implicated in the process . For the culture medium of gingival explants, the electrophoretic profile of the digestion products of fibronectin was different and showed a decreased activity . Nevertheless the gingival fluid gave a very similar degradation to bacterial plaque . The sulcular content was able to assume enzymatic activity capable of destroying fibronectin . These sulcular activities could be important for bacterial colonisation of sulcular surfaces and perhaps also for fibronectin destruction of periodontal tissues.

Hum Cell, 1988 Jun, 1(2), 207 - 17
Growth and hormonal responsiveness of human endometrial stromal cells in culture; Holinka CF; The present review describes and discusses published results on growth and hormonal responsiveness of human endometrial stromal cells in culture . The proliferative potential of serially subcultured cells, that is, the number of cell doublings before cells enter mitotic senescence and cease to divide, was unusually high in stromal cells from several endometrial specimens, a property that may reflect the unique proliferative capacity of human endometrium when compared to other adult tissues . Fluorescent visualization of microfilaments revealed distinct age-related changes in the distribution of cytoskeletal fibers . Addition of ovarian steroids to the culture medium of stromal cells resulted in significant morphologic changes . From comparative studies using different culture media it became evident that medium components remarkably influenced cell morphology during early culture periods in an irreversible manner . Cultured stromal cells yielded interesting results in experiments designed to define the role of polyamines in growth regulation . Proliferation was greatly inhibited when polyamine levels were reduced by specific inhibition of ornithine decarboxylase, the first and rate limiting enzyme in polyamine synthesis which produces putrescine by catalytic conversion from ornithine . The antiproliferative effects were reversed by addition of putrescine to the culture medium . These results clearly establish a causal link between polyamine depletion and growth deficiencies and reveal an essential function of polyamines in stromal cell proliferation . Hormonally regulated parameters in cultured stromal cells include aromatase activity, pregnancy-associated plasma protein-A, 51K secreted protein, prolactin and laminin . The hormonally regulated production of prolactin and laminin, both considered markers of decidualization, together with morphologic changes of stromal cells to decidual-like cells, strongly suggest that human endometrial stromal cells, when subjected to appropriate hormonal stimulation, are capable of differentiating into decidual cells in culture . Cultured stromal cells therefore offer a unique opportunity to examine the complex changes in gene expression associated with decidualization . In addition, in vitro decidualization may prove to be an effective diagnostic tool in certain cases of infertility . Finally, decidualization of cultured stromal cells represents a relevant end point for testing compounds of potential clinical importance, such as synthetic progestins or antifertility drugs.

Hum Cell, 1988 Jun, 1(2), 150 - 61
{Normal skin cells in vitro}; Yoshizato K et al.; Major cells in skin are epidermal cells in epidermis and fibroblasts in dermis . These cells can be isolated as a relatively pure population from the tissues using proteases and chelating agents . In this review we describe the way of culture where these two kinds of cells express normal function as they do in vivo . 1) It is important to consider the polarity of epidermal cell membranes in the transport of nutrients and metabolites when the cells are to be cultured in a healthy state in a long period . Epidermals cells expressed their normal polarities when cultured on a porous thin film of collagen and bathed on both sides (apical and basal) in culture media . 2) It is important to consider the interactions of fibroblasts with collagen when normal morphology and physiology are expected to be expressed in the cell in culture . Collagen affected the morphology of the cell and profoundly decreased the rate of DNA synthesis . We present a hypothesis which explains the fibronectin-independent interaction of fibroblasts with collagen . 3) It is important to consider the interactions between fibroblasts and epidermal cells when normal physiology of the skin as a whole is expected to be expressed in vitro, because exchange of information between them control their metabolic activities and functions . In this review, two examples for this exchange are presented: cell growth and collagenolysis.

Acta Trop, 1988 Jun, 45(2), 109 - 26
Differential growth requirements of several Leishmania spp . in chemically defined culture media; O'Daly JA et al.; 17 strains of Leishmania from 4 species: brasiliensis, mexicana, donovani and garnhami have been continually cultured at 26 degrees C, in the absence of proteins, in a medium containing salts, glucose, D-ribose, 2-deoxyribose, hemin, tricine, HEPES, 34 amino acids and intermediates of amino acid metabolism, 23 vitamins, 6 nucleotides and tetrahydrofolic acid . A wide variation in growth requirements was observed among leishmaniae which permitted the preparation of different minimum culture media for each Leishmania spp . Virulence of parasites was maintained after 30 passages in these chemically defined media . The requirements for differentiation to amastigotes also varied among the species as a function of the temperature of incubation and the protein content of the culture medium . Bovine serum albumin tryptic peptides substituted fetal bovine serum as growth factors at 30-34 degrees C.

Am J Pathol, 1988 Jun, 131(3), 569 - 77
Production of transforming growth factor-beta activity by Ki-1 positive lymphoma cells and analysis of its role in the regulation of Ki-1 positive lymphoma growth; Newcom SR et al.; The growth of activated human T lymphocytes in response to interleukin-2 (IL-2) is suppressed by transforming growth factor-beta (TGF-beta) . This study presents data that show a diminished response of two human lymphoma cell lines to physiologic regulation by TGF-beta . Cell line L-428 was derived from the malignant pleural effusion of a patient with far advanced nodular sclerosing Hodgkin's disease and has been shown to have clonal gene rearrangements characteristic of both B and T lymphocytes . Cell line Mac-1 was derived from the blood of a patient with clinically indolent cutaneous T-cell lymphoma . Both cell lines express the Hodgkin's disease associated antigen, Ki-1 . These Ki-1 positive lymphomas are shown to secrete TGF-beta into serum-free culture media . The addition of picogram quantities of exogenous TGF-beta to cell cultures of indolent Ki-1 lymphoma (Mac-1) suppresses IL-2-dependent mitosis; however, the suppression is less than 45% . This suppression correlates with a decrease in the number of IL-2 receptors . No inhibition of Ki-1 positive Hodgkin's cells (L-428) was observed, and proliferation dependent on polyclonal IL-2 was either not affected or was slightly potentiated by TGF-beta . Receptor analysis indicates the absence of IL-2 and TGF-beta receptors on L-428 cells . Thus, these Ki-1 lymphomas derived from activated lymphocytes appear to secrete TGF-beta activity but continue to proliferate because of defective suppression of IL-2 (and related lymphokine)-dependent DNA synthesis.

Exp Cell Res, 1988 Jun, 176(2), 336 - 43
Production of immunoreactive insulin-like growth factor I and response to exogenous IGF-I in small cell lung cancer cell lines; Jaques G et al.; Small cell lung cancer (SCLC) cell lines were examined for the presence of insulin-like growth factor I-related protein (IGF-I) in cell pellets and culture media . IGF-I immunoreactivity was detected in 11/14 pellets, ranging from 12 to 76 mIU/mg soluble protein . The IGF-I levels in the cell pellets showed a correlation to the corresponding culture media . IGF-I binding sites were found in all tested cell lines . The maximum binding (Bmax) ranged from 131 to 1230 fmol/mg protein and the dissociation constant (KD) from 0.89 to 5.21 nM . The incorporation of {3H}thymidine in the presence of recombinant human IGF-I resulted in a clearly increased DNA synthesis in two of seven cell lines . Thus, IGF-I may be an important growth factor in SCLC.

J Natl Cancer Inst, 1988 May 18, 80(6), 407 - 14
7,12-Dimethylbenz{a}anthracene adduct formation with Syrian hamster cheek pouch epithelial DNA: in vitro studies in organ explant culture; Lurie AG et al.; Studies examined the binding of radiolabeled 7,12-dimethylbenz{a}anthracene (DMBA) to epithelial DNA of hamster cheek pouch (HCP) maintained in organ explant culture . Adduct formation was studied as functions of {3H}DMBA dose, of the time after single {3H}DMBA applications, and of the route by which the DMBA was administered--either topically or in the culture media . Total DMBA-DNA adduct formation {total binding index (TBI)} was determined by DNA-bound 3H activity, and qualitative binding characteristics were further studied by high-pressure liquid chromatography . {3H}DMBA was applied either in the culture media at concentrations of 0.005-0.5 micrograms/ml or topically in mineral oil or ethanol in doses of 0.005-0.5 micrograms to each tissue fragment . Histopathologic changes in DMBA-treated HCP fragments included substantial aberrations in maturation of cornified and keratin layers and focal squamatization and dysplasia of the basal epithelium--considerable tissue necrosis was encountered in the high-DMBA-dose groups . Dose-response data were qualitatively similar among treatment types, with the greatest TBIs in topical ethanol groups and the lowest TBIs in culture medium groups . Kinetics of adduct formation and removal showed a rapid increase in TBIs to peak values at 24-72 hours followed by a biphasic decrease in TBIs, which leveled off at 7%-20% of peak values at 120-240 hours . Chromatographic analyses of selected samples at various times from all treatment groups showed three major peaks that are likely to be the same 1,2,3,4-tetrahydro-3,4-dihydroxy-1, 2-oxide-deoxyribonucleoside adducts observed in other rodent in vivo and cell culture systems . These results are consistent with those of other laboratories studying DMBA-DNA interactions and suggest that in vitro studies of DMBA-treated HCP explants are useful in studying the molecular nature of DMBA-DNA interactions in oral mucosal carcinogenesis.

J Biol Chem, 1988 May 15, 263(14), 6836 - 41
The membrane domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase confers endoplasmic reticulum localization and sterol-regulated degradation onto beta-galactosidase; Skalnik DG et al.; A hybrid gene has been constructed consisting of coding sequence for the membrane domain of the endoplasmic reticulum protein 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase linked to the coding sequence for the soluble enzyme Escherichia coli beta-galactosidase . Expression of the hybrid gene in transfected Chinese hamster ovary cells results in the production of a fusion protein (HMGal) which is localized in the endoplasmic reticulum . The fusion protein contains the high-mannose oligosaccharides characteristic of HMG-CoA reductase . Importantly the beta-galactosidase activity of HMGal decreases when low density lipoprotein is added to the culture media . Therefore, the membrane domain of HMG-CoA reductase is sufficient to determine both correct intracellular localization and sterol-regulation of degradation . Mutant fusion proteins which lack 64, 85, or 98 amino acid residues from within the membrane domain of HMG-CoA reductase are found to be localized in the endoplasmic reticulum and to retain beta-galactosidase activity . However, sterol-regulation of degradation is abolished.

Drug Alcohol Depend, 1988 May, 21(2), 137 - 9
Synergism of ethanol and clofibrate in the lauric acid hydroxylation; Pazo JA et al.; The effects of ethanol and clofibrate on lauric acid hydroxylation in isolated rat hepatocytes cultured for 72 h were studied . During culture the lauric acid hydroxylation activity decreases . When the hepatocytes were cultured for 72 h with the addition of 10(-5) M, 10(-4) M and 10(-3) M clofibrate, the lauric acid hydroxylation increased by 42%, 428% and 900%, respectively . The stimulation by ethanol was also significant . The addition of both ethanol and clofibrate to the culture media resulted in a potentiation of this induction.

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