J Reconstr Microsurg, 1995 Nov, 11(6), 447 - 53 Preservation of peripheral nerve grafts: a comparison of normal saline, HTK organ preservation solution, and DMEM Schwann cell culture medium; Lassner F et al.; The regeneration of peripheral nerve grafts was evaluated in a rat model, after pretreating the grafts with Schwann cell culture medium, HTK organ preservation solution, and normal saline, under cold ischemic conditions for different time periods . Following orthotopic replantation of the grafts into donor animals, the quality of regeneration was assessed after 6 weeks, compared to positive controls (autologous transplantation) and negative controls (acellular grafts) . The regenerative quality in the Schwann cell culture groups with ischemic periods of 32 and 72 hr was comparable to normal controls . Significantly minor regeneration was detected in specimens undergoing 14 and 120 hr of ischemia in the Schwann cell culture medium and in the HTK and normal saline groups, regardless of ischemic time . Among the conclusions was that controlled proliferation of Schwann cells seems to be a basic principle for preservation of peripheral nerve grafts. J Clin Microbiol, 1995 Nov, 33(11), 2839 - 41 Evaluation of Sanofi Diagnostics Pasteur Chlamydia Microplate EIA shortened assay and comparison with cell culture and Syva Chlamydia MicroTrak II EIA in high- and low-risk populations; Chan EL et al.; Seven hundred thirty-two female urogenital samples were collected for Chlamydia trachomatis testing by both the Sanofi Diagnostics Pasteur (Chaska, Minn.) Chlamydia Microplate EIA by the shortened protocol and the Syva (San Jose, Calif.) MicroTrak II EIA, and the results were compared with those obtained by cell culture . For the analysis of samples from female patients, the patients were divided into high- and low-risk categories . An additional 121 male urethral samples were collected and tested by the Sanofi Microplate EIA and cell culture; for the analysis of samples from male patients, the patients were divided into asymptomatic and symptomatic categories . All specimens positive by enzyme immunoassay (EIA) were confirmed by a blocking assay following the respective manufacturer's instructions . Specimens negative by EIA that fell within a gray zone 30% below the cutoff and negative cultures with one or more corresponding positive EIA results were tested further by cytocentrifugation and direct immunofluorescent assay . The overall sensitivity, specificity, positive predictive value, and negative predictive value for Syva versus culture were 94, 98.8, 85.5 and 99.6%, respectively . After resolution, the results were 94.5, 99.6, 94.5, and 99.6%, respectively . The parallel results for the Sanofi Microplate EIA versus culture were 94.0, 98.7, and 83.9, and 99.6%, respectively, and after being resolved, the results were 94.9, 100, 100, and 99.6%, respectively . In the small male population tested, the resolved results of the Sanofi Microplate EIA versus culture demonstrated sensitivity, specificity, positive predictive value, and negative predictive value of 100, 100, 100, and 100%, respectively . The present study demonstrated that the Sanofi Microplate EIA shortened protocol is highly sensitive and specific in comparison with cell culture and the Syva MicroTrak II EIA. J Gen Virol, 1995 Nov, 76 ( Pt 11), 2875 - 9 Varicella-zoster virus induces apoptosis in cell culture; Sadzot-Delvaux C et al.; Apoptosis is an active mechanism of cell death which can be initiated in response to various stimuli including virus infections . In this work, we demonstrate that lytic infection by varicella-zoster virus (VZV), a human herpesvirus, is characterized by nuclear fragmentation of DNA into oligonucleosomal fragments and by chromatin condensation . In vitro, VZV-induced cell death is actually mediated by apoptosis . The mechanisms developed by cells to protect themselves against apoptosis could be one of the parameters allowing the establishment of virus latency . In the case of VZV, which can remain latent in sensory ganglia, we have not yet identified a cellular or viral protein which could play this protective role, since the observed apoptosis mechanism seems to be independent from Bcl-2, the most frequently described inhibitor of apoptosis. Endocrinology, 1995 Nov, 136(11), 5028 - 33 Interferon-gamma increases intracellular calcium and inositol phosphates in primary human thyroid cell culture; Kung AW et al.; Interferon-gamma (IFN gamma) is believed to play a role in the pathogenesis of autoimmune thyroid disease, as it is known to exert diverse effects on thyroid metabolism . These include induction of human leukocyte antigen class II expression, inhibition of gene expression of thyroglobulin and thyroid peroxidase, as well as inhibition of cellular proliferation . However, the mechanism of action of IFN gamma in thyrocytes has not been clearly defined . We studied the action of IFN gamma on the production of inositol phosphates and intracellular Ca2+ mobilization in primary cultures of human thyrocytes using the fluorescent Ca2+ indicator fura-2 . IFN gamma increased the production of inositol mono-, bis-, and trisphosphates and caused a dose-dependent increase in intracellular Ca2+ ({Ca2+}i) at 37 C . Preincubation with 12-O-tetradecanoylphorbol-13-acetate, which activates protein kinase C, resulted in the abolition of the IFN gamma response, suggesting that protein kinase C was involved in a negative feedback loop resulting in inhibition of IFN gamma-induced {Ca2+}i rise . Prior release of intracellularly stored Ca2+ with thapsigargin, the microsomal Ca2+ pump inhibitor, also abolished the response of IFN gamma . Mobilization of {Ca2+}i resulted in Ca2+ entry across the plasma membrane, which could be blocked by La3+, the inorganic Ca2+ antagonist . The tyrosine protein kinase inhibitor, genistein, inhibited the production of inositol phosphates and the elevation of {Ca2+}i induced by IFN gamma, but had no effect on ATP, suggesting that tyrosine protein kinase is involved in the signaling transduction of IFN gamma . We conclude that the mobilization of intracellular Ca2+ and the production of inositol phosphates are two important signaling events for the action of IFN gamma in human thyrocytes. Mutat Res, 1995 Nov, 332(1-2), 1 - 8 Simultaneous evaluation of dexamethasone-induced apoptosis and micronuclei in rat primary spleen cell cultures; Krishna G et al.; Apoptosis or programmed cell death is a biological event that is biochemically and morphologically distinct from cellular necrosis . Nonetheless, its relationship has not been studied in terms of a cytogenetic endpoint such as micronucleus formation . In the present study, based on cytological observations, the incidence of dexamethasone-induced apoptotic cells was related to the frequency of micronucleated cells in vitro . Rat primary spleen cells were grown in 6-well plates with RPMI 1640 media using concanavalin A and lipopolysaccharide as mitogens . At culture initiation, the test agent dexamethasone (10, 20 or 40 microM) and a cytokinesis inhibitor cytochalasin B (3 micrograms/ml) were added . Cultures were harvested 18 h and 40 h later . Slides were prepared and stained with Diff-Quik stain . Frequencies of apoptotic cells and micronucleated binucleate cells were enumerated cytologically based on 500 cells per treatment from the same slides . The results showed a dose-dependent increase in the number of apoptotic cells in rat spleen cultures treated with dexamethasone . At 18 h, the percentages of apoptotic cells were 0.8, 1.6, 3.4 and 4.4 with 0, 10, 20 and 40 microM dexamethasone, respectively . The corresponding percentages of apoptotic cells at 40 h were: 2.8, 2.6, 5.6 and 10.4 . However, at the same concentrations of dexamethasone, the micronucleus frequency in binucleate cells remained relatively unchanged . The phenomenon of apoptosis induced by dexamethasone was confirmed biochemically based on a characteristic DNA 'ladder' pattern by gel electrophoresis . These data suggest that dexamethasone at the concentrations which induced apoptosis did not produce cytogenetic damage . Also, these findings indicate that micronucleus formation and nuclear changes leading to apoptosis are separate events and these endpoints may not be closely correlated for dexamethasone. J Gerontol A Biol Sci Med Sci, 1995 Nov, 50 Spec No, 142 - 4 Cell culture systems as tools for studying age-related changes in skeletal muscle; Peterson CA; Two aspects of muscle cell phenotype that may be affected by aging are amenable to study in vitro: proliferative potential and ability to differentiate . These processes in primary myoblast cultures as well as in established myoblast cell lines will be examined, and the usefulness of these in vitro systems in addressing fundamental molecular mechanisms of muscle aging will be discussed. Biochim Biophys Acta, 1995 Oct 19, 1269(1), 6 - 12 Metabolism of homocysteine, its relation to the other cellular thiols and its mechanism of cell damage in a cell culture line (human histiocytic cell line U-937); Hultberg B et al.; This study shows that the intracellular concentration of homocysteine in cultured cells is kept low due to an accumulation in the medium . The intracellular level of homocysteine was decreased when its precursor, methionine, was omitted from the culture medium . Intracellular glutathione and cysteine were lowered in cystine-deficient medium . Intracellular glutathione was also lowered when copper ions were added to the culture medium . It is evident from this study that the intracellular concentration of homocysteine was not influenced by the lowered level of glutathione and/or cysteine . High amounts of homocysteine added to the medium give rise to an increase of intracellular reduced homocysteine, which participates in the transsulfuration pathway and can replace cysteine in the synthesis of glutathione . The addition of relatively high amounts of reduced homocysteine (500 mumol/l) in the presence of copper ions (100 mumol/l) to the culture medium can be directly toxic to the cells, possibly due to oxygen radicals formed by thiol auto-oxidation . Whilst the level of homocysteine in this study using short-time cell culture experiment is much higher than the mild hyperhomocysteinemia thought to be atherogenic in humans, it is conceivable that over a longer time course these levels of homocysteine could be sufficient to induce endothelial dysfunction, eventually leading to atherosclerosis. J Bone Miner Res, 1995 Oct, 10(10), 1577 - 88 Structural and chemical characteristics and maturation of the calcium-phosphate crystals formed during the calcification of the organic matrix synthesized by chicken osteoblasts in cell culture; Rey C et al.; The calcium-phosphate (CA-P) crystals formed in the extracellular organic matrix synthesized by chicken osteoblasts in cell culture were examined after 30, 40, and 60 days of culture by a number of physical and chemical techniques including chemical analyses, X-ray diffraction, transmission electron microscopy of isolated crystals, and resolution-enhanced Fourier transform infrared spectroscopy . The data reveal that the solid inorganic calcium-phosphate phase consists of a very poorly crystalline apatite, having a low carbonate content and containing acid phosphate groups . The chemical and structural characteristics are generally similar to the apatite crystals found in young newly synthesized bone but there were small but significant differences found . The major significant differences noted were the rate at which maturational changes occurred in the crystals formed in cell culture compared with those noted in vivo and in synthetic carbonate apatite crystals equilibrated with the same cell culture medium, and the persistence of labile groups, especially HPO4(-2) ions during a relatively long period of incubation . Despite extensive chemical efforts to degrade the organic constituents and to disperse the individual crystals isolated from the organic matrix constituents, a large proportion of the crystals were found to be organized in both loosely and densely packed relatively large roughly spherical aggregates . A few of the aggregates were organized in the form of fibrils with the crystals oriented with their c-axes roughly parallel to the long axes of the crystal aggregate . With briefer periods of chemical treatment, larger aggregates of crystals were occasionally observed in which there was a distinct axial periodicity of approximately 70 nm . In such collagen-crystal fragments, the crystals were well-oriented with their c-axis roughly parallel to the long axes of the aggregate similar to the organization and relationships between crystals and collagen fibrils in native bone . Isolated crystals were in the shape of thin plates . At the end of 30 days of culture, many of the crystals were clearly larger than those observed in native chick bone, except for those in the very youngest (7- to 8-day-old) embryos . At the end of 40 and 60 days of culture, the crystal habit remained as thin plates but the crystals were predominantly smaller, similar to those found in older embryo and postnatal chicken bone . The marked tendency of the crystals to form relatively large aggregates that resist dispersion by techniques that readily disperse the crystals of bone, and the presence of a significant number of larger crystals has also been observed in studies of calcified cartilage . Resolution enhanced FTIR spectroscopy revealed the presence of a high concentration of labile phosphate groups, especially after 30 days of culture and just after the plateau of mineralization is reached. J Assist Reprod Genet, 1995 Oct, 12(9), 632 - 8 Ultrastructure of endometrial epithelial cells in a three-dimensional cell culture system for human implantation studies; Bentin-Ley U et al.; PURPOSE: A three-dimensional cell culture system imitating normal uterine endometrium has previously been established . To what degree do cultured epithelial cells retain their morphological characteristics as compared to in vivo material obtained simultaneously from the same tissue donor . RESULTS: We found a high degree of similarity between the in vivo and in vitro situations . The present culture system furthermore imitates the day-to-day morphology of the cycle . CONCLUSIONS: This indicates, that a correct timing of the biopsy tissue is important for future human implantation studies. Bone, 1995 Oct, 17(4 Suppl), 461S - 466S Canine bone marrow cell cultures infected with canine distemper virus: an in vitro model of Paget's disease; Mee AP et al.; We have previously shown that the canine paramyxovirus, canine distemper virus (CDV), is a possible aetiologic agent in Paget's disease of bone and in the canine bone disorder, metaphyseal osteopathy . More recently, we have examined the effects of CDV on the formation of multinucleated, tartrate resistant acid phosphatase positive, calcitonin receptor positive, osteoclast-like cells in cultures of canine bone marrow mononuclear cells, and shown that both in vitro and in vivo infection with CDV produced a dose dependent increase in the number and size of osteoclast-like cells . We have now extended these results to show that CDV infection induces interleukin-6 and c-Fos mRNA in these cells, similar to our recent findings in pagetic bone cells . These results further support the hypothesis that CDV might be involved in the aetiopathogenesis of Paget's disease and metaphyseal osteopathy and suggest that canine marrow culture systems will prove useful as an in vitro model to examine the disease processes in more detail. Mol Cell Probes, 1995 Oct, 9(5), 341 - 6 Evaluation of the effects of disinfectants on rotavirus RNA and infectivity by the polymerase chain reaction and cell-culture methods; Ojeh CK et al.; Rotaviruses have been linked to outbreaks of acute gastroenteritis of children in day-care centres and hospital paediatric wards . There is, therefore, the need for monitoring effective decontamination of such environments . We have evaluated the effects of seven different methods of disinfection/inactivation (four chemical and three physical) on rotavirus using the PCR and cell-culture methods . We observed that 6% H2O2, 2500 ppm chlorine, an ethano-phenolic disinfectant, u.v . irradiation and heat completely destroyed the infectivity of rotavirus as well as RNA amplifiable by PCR . On the other hand, treatment with 80% ethanol resulted in the loss of infectivity despite the fact that RNA was still amplifiable . Rotavirus subjected to drying over a 24 h period still retained amplifiable RNA but infectivity was reduced by 100-fold when compared to the control . This study demonstrated an agreement between PCR and cell-culture monitoring systems, however, PCR is a more rapid and sensitive assay. J Neurosci Res, 1995 Oct 1, 42(2), 252 - 8 Calbindin D28K-containing neurons, and not HSP70-expressing neurons, are more resistant to HIV-1 envelope (gp120) toxicity in cortical cell cultures; Diop AG et al.; HIV-1-associated cognitive/motor complex is one of the major neurological complications of AIDS and is associated with neuronal loss . Gp120, the HIV envelope protein, is toxic for neurons in cultures and produces a rise in intracytosolic calcium . This neurotoxicity is dose-dependent and time-dependent . We evaluated the selective gp120 toxicity in primary neuronal cultures for calbindin-free and calbindin-containing neurons with semi-quantitative immunocytochemistry using an anti-calbindin D28K monoclonal antibody . The number of immunolabelled neurons was inversely correlated to neuronal survival . In cultures exposed to gp120 (100 pM) for 24 hr the neuronal survival of initial platings was 19.7 +/- 2.1% and the percentage of neuronal survival was 84.6 +/- 4.9% in control cultures exposed to the vehicle . The corresponding percentages of immunolabelled neurons were 85.0 +/- 2.1% in treated cultures and 23.6 +/- 3.1% in control cultures (P < 0.001) . The expression of heat shock proteins by heating cell cultures did not protect neurons from gp120 toxicity . These results suggest that calbindin D2K28-containing neurons are more resistant to gp120-toxicity in this cell culture system. In Vitro Cell Dev Biol Anim, 1995 Oct, 31(9), 659 - 63 Intrinsic glycosylation potentials of insect cell cultures and insect larvae; Davis TR et al.; The glycosylation and subsequent processing of native and recombinant glycoproteins expressed in established insect cell lines and insect larvae were compared . The Spodoptera frugiperda (Sf21) and Trichoplusia ni (TN-368 and BTI-Tn-5B1-4) cell lines possessed several intrinsic glycoproteins that are modified with both N- and O-linked oligosaccharides . The N-linked oligosaccharides were identified as both the simple (high mannose) and complex (containing sialic acid) types . Similarly, the T . ni larvae also possessed intrinsic glycoproteins that were modified with O-linked and simple and complex N-linked oligosaccharides . Additionally, human placental, secreted alkaline phosphatase (SEAP) produced during replication of a recombinant baculovirus in T . ni larvae was modified with complex oligosaccharide having sialic acid linked alpha(2-6) to galactose. Exp Eye Res, 1995 Oct, 61(4), 487 - 93 Natural, high-mannose glycoproteins inhibit ROS binding and ingestion by RPE cell cultures; Lutz DA et al.; Previous studies have suggested that a mannose receptor mediates the phagocytic uptake of effete rod outer segments by retinal pigment epithelial cells . In the present study, the effect of adding a soluble ligand for the mannose receptor, horseradish peroxidase, was examined . Cultured retinal pigment epithelial cells from Long Evans rats were preincubated with various concentrations of horseradish peroxidase for 20 min followed by a challenge of FITC-labeled bovine rod outer segments for 3 h . Both counts of total rod outer segments (bound and ingested) and ingested rod outer segments were determined . Rod outer segment uptake was reduced, in a concentration-dependent fashion, by an average of 60% of control values when horseradish peroxidase was added to retinal pigment epithelial cultures . Similarly, total rod outer segment values were reduced to 50% of controls in the presence of at least a 10 micrograms ml-1 horseradish peroxidase concentration . Horseradish peroxidase inhibition of retinal pigment epithelial phagocytic capacity was reversible . Other high mannose glycoproteins, such as invertase, beta-glucoronidase, and ovalbumin, were equally effective in preventing rod outer segment ingestion by retinal pigment epithelial cells . These data further support the hypothesis that a mannose receptor on the retinal pigment epithelial apical surface facilitates phagocytosis of rod outer segments. Immun Infekt, 1995 Oct, 23(5), 158 - 60 {Effects of the oxidizing agents ozone and nitrogen dioxide on the lung: studies of a cell culture model}; Holz O et al.; In experimental exposure studies with ozone or nitrogen dioxide a change in lung function and unspecific airway sensitivity was observed . These effects were paralleled by an induction of cellular and biochemical changes . The exposure of isolated cells in vitro led to a decrease in vitality, an increase in permeability and the secretion of mediators very similar to the composition found in bronchoalveolar lavage after in vivo exposure . The cell culture model, therefore, allows the study of the mechanism of these oxidative air pollutants and allows further the understanding of the influence of different cell types on the observed effects in vivo. J Virol, 1995 Oct, 69(10), 5959 - 68 The dominant phosphoprotein pp65 (UL83) of human cytomegalovirus is dispensable for growth in cell culture; Schmolke S et al.; The phosphoprotein pp65 (ppUL83) of human cytomegalovirus (HCMV) is abundantly synthesized during lytic infection in cultured fibroblasts . As a major constituent of extracellular particles, it gains entry to infected cells immediately after adsorption and subsequently translocates to the cell nucleus . This efficient transport is mediated by unique nuclear localization signals . To study the function of pp65, a viral deletion mutant was constructed by replacing the pp65 gene with the bacterial neomycin phosphotransferase gene, driven by the simian virus 40 early promoter . The resulting virus, RVAd65, could be grown and selected on human fibroblasts without complementation . The deletion of the pp65 gene in RVAd65 was verified by using Southern blot and PCR analyses . The lack of expression from the gene was investigated by immunoblotting with pp65-specific monoclonal antibodies . Single-cycle growth analyses showed that RVAd65 grew to levels of infectivity comparable to those of the wild-type virus . Therefore, pp65 is nonessential for the growth of HCMV in human fibroblasts . Electron microscopy revealed no differences in the processes of virion morphogenesis, although the maturation appeared to be delayed . However, the kinetics of expression of the immediate-early genes UL122 and UL123, the early gene UL44, and the late gene UL32 were the same in RVAd65-infected cells as in wild-type virus-infected cells in immunoblot analyses . In vitro phosphorylation assays showed that some of the virion proteins were labelled to a markedly reduced extent by virion-associated kinases in RVAd65 compared with wild-type virus . We therefore conclude that although deletion of the pp65 gene does not abolish replication of HCMV, a recombinant virus lacking pp65 displays phenotypic alterations compared with wild-type virus during growth in cultured fibroblasts. Endocrinology, 1995 Oct, 136(10), 4572 - 81 Expression of the calcitonin receptor in bone marrow cell cultures and in bone: a specific marker of the differentiated osteoclast that is regulated by calcitonin; Lee SK et al.; We studied the temporal sequence of osteoclast (OC) differentiation from precursor cells in murine marrow cultures . Two markers of the OC phenotype, calcitonin (CT) receptor (CTR) and tartrate resistant acid phosphatase (TRAP), were assessed . Marrow cells from C57BL/6 mice were cultured for 3, 5, 7, and 9 days with or without 1,25-(OH)2vitamin D3 (10(-8) M) . In controls only small numbers of osteoclastic multinucleated cells 9MNCs) formed per well (< 15 per well) . In contrast, 1,25-(OH)2D3 strongly stimulated MNC formation (> 80 per well on day 7) . Messenger RNA (mRNA) for TRAP was detectable by reverse transcription-polymerase chain reaction amplification in both control and 1,25-(OH)2D3 treated groups at all times . However, TRAP mRNA was detectable in MNCs by the less sensitive in situ hybridization only on days 5, 7, and 9 and only in 1,25-(OH)2D3 treated cells . In control cultures, CTR mRNA was present on day 3 only in nonadherent cells and was not present in adherent cells (where MNCs formed) at any time point . In 1,25-(OH)2D3 treated cultures CTR mRNA was detectable in nonadherent cells on day 3 and in adherent cells on day 5 and thereafter . Peak levels of CTR mRNA were seen in adherent cells on day 7 (15-fold more than day 5 and 4-fold more than day 9) . CT (10(-7) M) treatment of 7 day cultures, which had been stimulated to express the osteoclastic phenotype, caused a marked decrease in CTR mRNA expression at 24 h . There was no effect of CT treatment on CTR mRNA expression at 3 h or on TRAP mRNA expression at 3 or 24 h . In neonatal mouse calvaria cultures, CTR mRNA expression was constitutively present and was markedly decreased by 48 h of CT treatment . Similarly, bone resorption in these cultures was inhibited at 24 h by CT treatment, but at 48 and 72 h there was escape from the inhibitory effects of CT on resorption . In the marrow cultures, MNCs were greater than 98% positive for {125I}-salmon calcitonin (sCT) binding and this binding was completely competed away by excess cold sCT (10(-7) M) . All primary isolated osteoclasts from 1- to 3-day-old mouse long bones exhibited {125I}-sCT binding and TRAP activity and were strongly positive for CTR and TRAP mRNA by in situ hybridization . Both MNCs that formed in bone marrow cultures and isolated primary osteoclasts formed resorption pits on bone slices.(ABSTRACT TRUNCATED AT 400 WORDS) Endocrinology, 1995 Oct, 136(10), 4254 - 60 Cortisol represses insulin-like growth factor II receptor transcription in skeletal cell cultures; Rydziel S et al.; Glucocorticoids have a number of effects on bone cell function, some of which might be mediated by changes in the synthesis or activity of insulin-like growth factors (IGFs) . Glucocorticoids inhibit IGF-I, but not IGF-II, synthesis in osteoblasts and decrease the expression of selected IGF-binding proteins . The effects of glucocorticoids on IGF-I and -II receptor messenger RNA (mRNA) expression in osteoblasts are not known, and changes in IGF-I or -II receptor levels could result in changes in IGF activity . We examined the effects of glucocorticoids on IGF-I and -II receptor mRNA expression in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells) . Cortisol at 1 microM for 2-48 h did not alter IGF-I receptor transcripts, as determined by Northern blot analysis and ribonuclease protection assay . In contrast, cortisol caused a time- and dose-dependent inhibition of IGF-II receptor mRNA levels . The effect was maximal at 0.1-1 microM for 24-48 h and was accompanied by a decrease in IGF-II receptor levels, as determined by affinity labeling, cross-linking and polyacrylamide gel electrophoresis, Western immunoblot, and Scatchard analysis . The effect of cortisol on IGF-II receptor transcripts was not dependent on de novo protein synthesis . Cortisol did not modify the IGF-II receptor mRNA half-life in transcriptionally arrested Ob cells and decreased the rate of IGF-II receptor RNA transcription in nuclear run-on assays . In conclusion, cortisol decreases transcription of the IGF-II receptor in Ob cell cultures, an effect that could mediate selected actions of glucocorticoids in bone. J Cell Sci, 1995 Oct, 108 ( Pt 10), 3171 - 80 Functional assessment of white and brown adipocyte development and energy metabolism in cell culture . Dissociation of terminal differentiation and thermogenesis in brown adipocytes; Klaus S et al.; We investigated the effect of insulin, triiodothyronine (T3) and dexamethasone (a synthetic glucocorticoid) on differentiation, lipid metabolism and thermogenesis of preadipocytes isolated from white fat (WAT) and brown fat (BAT) from the Siberian dwarf hamster (Phodopus sungorus) . Cell cultures from WAT and BAT were chronically treated with the above hormones alone or in any combination . After differentiation (day 8 or 9 of culture) we measured the following parameters: adipogenic index (number x size of adipocytes), protein content, lipolysis, cell respiration, and expression of the uncoupling protein UCP, which is unique to mitochondria of brown adipocytes . Insulin was the most important adipogenic factor for brown and white adipocytes and necessary for terminal differentiation, whereas dexamethasone alone completely inhibited differentiation . T3 had no effect on adipogenesis in WAT cultures, but further increased insulin stimulated adipogenesis in BAT cultures . Basal lipolysis was higher in WAT than in BAT cultures except when dexamethasone was present, which stimulated lipolysis in both culture types to the same extent . T3 had a pronounced dose dependent lipolytic effect on WAT cultures but very little effect on BAT cultures . Respiration rates were generally higher in differentiated adipocytes than in fibroblast like cells . T3 had no effect on thermogenesis in WAT cultures but increased thermogenesis in BAT cultures, and this was further elevated by insulin . UCP expression in BAT cultures could be detected by western blot in insulin treated, T3 treated and insulin+T3 treated cultures with highest expression in the latter . These results imply a possible dissociation of terminal differentiation and thermogenic function of brown adipocytes . In WAT cultures there was also a low level of UCP detectable in the insulin+T3 treated cultures . Immuno-fluorescence microscopy analysis revealed the presence of UCP in 10-15% of adipocytes from WAT cultures (in BAT cultures: 90%), indicating the presence of some brown preadipocytes in typical WAT deposits. Eur J Biochem, 1995 Oct 1, 233(1), 171 - 8 Prevention of cholesteryl ester accumulation in P388D1 macrophage-like cells by increased cellular vitamin E depends on species of extracellular cholesterol . Conventional heterologous non-human cell cultures are poor models of human atherosclerotic foam cell formation; Asmis R et al.; Since the cellular role of the antioxidative vitamins in the formation of foam cells has not yet been studied in detail, we investigated the effect of alpha-tocopherol and ascorbic acid loading of P388D1 macrophage-like cells on their cholesterol and cholesteryl ester levels and their response to the exposure to different lipoproteins . alpha-Tocopherol loading, but not ascorbic acid loading, of P388D1 cells strongly reduced their cellular cholesteryl ester/cholesterol ratio (the crucial indicator of foam cell formation) when fetal calf serum was the only extracellular source of cholesterol . Balance studies suggest that this effect of alpha-tocopherol was mainly due to a reduced uptake of fetal-calf-serum-derived cholesterol . alpha-Tocopherol loading, however, did not reduce the cholesteryl ester/cholesterol ratio when human unmodified low-density lipoprotein (LDL) was added to culture medium containing fetal calf serum . Thus, the uptake of fetal-calf-serum-derived cholesterol was competitively reduced by human LDL, the uptake of which remained unaffected by alpha-tocopherol . Similarly, alpha-tocopherol loading did not prevent cholesteryl ester formation induced by human LDL either oxidized with Cu2+, ultraviolet light or HOCl, or modified by acetylation, aggregation or by malondialdehyde treatment . The present experimental conditions lacked any pro-oxidative burden, since (a) ascorbic acid, either alone or combined with alpha-tocopherol, did not affect cellular cholesteryl ester levels, (b) foam cell formation was not a linear function of the degree of oxidative LDL modification, and (c) alpha-tocopherol lacked specific effects on oxidatively modified LDL . Thus, the reduction of cellular cholesteryl esters by alpha-tocopherol in the absence of human unmodified LDL was hardly due to common antioxidative properties of vitamin E . In conclusion, the present observation that a desirable alpha-tocopherol effect on the cholesteryl ester balance in mouse-tumor-derived P388D1 cells strongly depended on the species of extracellular cholesterol carrier, cautions against premature generalizations of conventional non-human cell culture data. Eur J Biochem, 1995 Oct 1, 233(1), 132 - 9 Purification and properties of codeinone reductase (NADPH) from Papaver somniferum cell cultures and differentiated plants; Lenz R et al.; Codeinone reductase (NADPH), which catalyzes the stereospecific reduction of (-)codeinone to (-)codeine, was detected and purified to electrophoretic homogeneity from a cytosolic fraction of Papaver somniferum L . cell cultures . The purification involved ammonium sulfate precipitation (40-80%), affinity chromatography (matrex red A), gel filtration (fractogel TSK HW 55S), affinity chromatography (fractogel TSK AF Blue), ion-exchange chromatography (DEAE-Sephacel) and native PAGE . The purified codeinone reductase was found to be a monomeric protein of 35 +/- 1 kDa that is highly substrate-specific, reducing only the C6 oxo group of codeinone and morphinone as well as a few analogues . The physiological forward reaction has a pH optimum at 7.0, the reverse reaction at 9.1 . The temperature optimum is at 40 degrees C and the isoelectric point (p1) at 4.4 . The apparent Km values (forward reaction) for codeinone and NADPH are 23 microM and 168 microM, respectively . Using capsule tissue of differentiated P . somniferum plants as an enzyme source, two codeinone reductase (NADPH) isoenzymes were detected and purified to homogeneity . These isoenzymes could not be separated for characterization and showed slightly different kinetic features (Km values: codeinone 9 microM; NADPH 81 microM) compared with the cell culture enzyme. J Clin Endocrinol Metab, 1995 Oct, 80(10), 3018 - 24 Interleukin-1-mediated stimulation of prostaglandin E production is without effect on plasminogen activator activity in human granulosa lutein cell cultures; Hurwitz A et al.; In continuation of earlier observations on the involvement of interleukin-1 (IL-1) in ovarian function, we examined the ability of IL-1 to modulate plasminogen activator (PA) activity and prostaglandin (PG) synthesis in human granulosa lutein cells (GLCs) . Toward this goal, GLCs were obtained from women undergoing in vitro fertilization, preincubated with 10% fetal calf serum for 48 h, and subsequently cultured for 48 h in serum-free media in the absence or presence of IL-1 beta (10 ng/mL) . Cellular PA activity was measured by plasminogen-dependent cleavage of the chromogenic substrate H-D-valyl-L-leucyl-L-lysine-p-nitroanilide (S-2251) . Prostaglandin E (PGE) levels were assayed by conventional RIA . Exposure of GLCs to IL-1 resulted in a 50% increase in PGE production, a 33% suppression of PA activity, and a 75% increase in the ability of the corresponding conditioned media to inhibit exogenous urokinase activity . The inhibitory capacity was attributable to an IL-1-mediated increase in PA inhibitor type-1 (PAI-1) production, inasmuch as urokinase inhibition could be abolished by the administration of a polyclonal antihuman PAI-1 immunoglobulin G . IL-1 treatment had no effect on plasmin or trypsin inhibition . Exposure of GLCs to IL-1 receptor antagonist abolished the ability of IL-1 to enhance PA inhibitory activity and PGE production, thereby establishing specific IL-1 receptor-mediated effects . The ability of IL-1 to suppress PA activity and to produce PAI-1 persisted in the presence of indomethacin, a potent inhibitor of PG synthesis . Likewise, transforming growth factor-beta 1 suppressed the ability of IL-1 to stimulate PGE production without affecting the IL-1-induced effects on the PA system . The present findings suggest a pluripotent response of GLCs to IL-1, characterized by the induction of PAI-1 and the suppression of PA occurring concurrent with, but independent of, PG production . These observations support the potential involvement of IL-1 in the regulation of human ovulatory processes. J Neuroimmunol, 1995 Oct, 62(1), 9 - 17 Modulatory effects of {Met5}-enkephalin on interleukin-1 beta secretion from microglia in mixed brain cell cultures; Das KP et al.; In the present study, functional interactions between {Met5}-enkephalin (ME), naloxone and lipopolysaccharide (LPS) on interleukin-1 beta (IL-1 beta) immunostaining and secretion have been assessed in mixed brain cell cultures from embryonic day 17 mice . Adding ME alone or together with LPS to the culture increased the release of IL-1 beta after 48 h in a concentration-dependent fashion . In situ hybridization studies showed that LPS, but not ME, increased the abundance of IL-1 beta mRNA . The enhanced release of IL-1 beta caused by ME or LPS was partially blocked by naloxone . LPS induced concentration-dependent morphological changes in microglia in mixed brain cell cultures, identified by a monoclonal antibody F4/80 which is specific for macrophages/microglia . Despite increasing IL-1 beta release into the media, ME (10(-8) M) did not induce morphological changes in microglia . Naloxone alone also had no effect on glial morphology; however, the LPS-induced morphological changes were blocked by naloxone . Our data indicate that both exogenous and endogenous opioids regulate IL-1 beta production by microglial cells in the mixed brain cell cultures. J Parasitol, 1995 Oct, 81(5), 821 - 4 Hammondia hammondi cysts in cell cultures; Riahi H et al.; In an attempt to obtain the continuous development of Hammondia hammondi in vitro, culture of the parasite was performed in 3 cell lines . Although organisms were present in culture fluids of feline kidney cells (CRFK) for as long as 3 mo, continuous culture was not possible . However, for the first time, cysts of H . hammondi were observed in cell culture from day 6 after inoculation of sporozoites . Ultrastructure of the H . hammondi cysts was similar to that observed for in vitro-obtained Toxoplasma gondii cysts . Feeding a cat with these in vitro-developed cysts resulted in oocyst shedding 5 days after ingestion. J Biotechnol, 1995 Sep 29, 42(2), 163 - 75 Specific growth rate as a parameter for tracing growth-limiting substances in animal cell cultures; Ljunggren J et al.; The specific growth rate (mu) reaches its maximum very early during culture (at 20 h), but declines again thereafter so that no exponential growth phase occurs in batch cultures of hybridoma cells . This growth rate limitation depends neither on exhaustion of any of the macro-nutrients, nor on inhibition by metabolic byproducts (Ljunggren and Haggstrom, 1994) . Intermittent additions of serum, after 20 and 40 h of culture, resulted in exponential growth throughout the growth phase . Insulin was found to be the main component responsible for the growth rate increasing effect . Intermittent additions of serum or insulin to a dual substrate (glucose and glutamine) limited fed batch culture increased the growth rate also here, and the results indicate the existence of a minimum growth rate (about 0.02 h-1) at a threshold glutamine level (0.005 mM) . Serum and insulin additions markedly enhanced the glucose consumption and lactate formation rates, a metabolic effect that was not coupled to the increase in mu . The reduced concentrations of glucose and glutamine in substrate limited fed batch cultures suppressed substrate consumption rates and byproduct formation (lactate, ammonium, alanine, other amino acids) even in the serum and insulin stimulated cultures and rendered the energy metabolism much more efficient than in batch culture . Further, the serum and insulin stimulated cells growing in substrate limited fed batch culture produced almost 4-times more antibodies, from the same amount of nutrients as supplied to the batch grown cells. Mol Cell Endocrinol, 1995 Sep 22, 113(2), 195 - 204 Somatostatin receptors in prostate tissues and derived cell cultures, and the in vitro growth inhibitory effect of BIM-23014 analog; Tatoud R et al.; We investigated somatostatin receptors (SSTRs) in surgical specimens of prostate cancer and benign prostate hyperplasia (BPH), a normal immortalized epithelial cell line (PNT1), epithelial cancer cell lines, and stromal cells in short-term culture derived from normal and BPH biopsies . Cross-linking studies with 125I-Tyr11-SRIF-14 (125I-SRIF) and the SRIF analog 125I-BIM-23104 identified one major 57-kDa band both in surgical specimens and in epithelial and stromal cells cultures . In membrane-enriched fractions and whole stromal cells from a normal prostate and from one BPH, a single type of SSTR was characterized (Kd = 6.10(-9) and 10(-8) M, respectively, Bmax = 1.6 pmol per mg of proteins) . mRNA for SSTR1 was detected in all epithelial and stromal cells tested except for PNT1, while SSTR2 mRNA was detected in one BPH stromal cell culture . BIM-23104 had no effect on the in vitro growth of the epithelial cells tested . Conversely, 10(-10) M BIM-23104 induced >50% growth inhibition of stromal cells after 6 days in culture . These results may have implications for therapeutic strategies using SRIF analogs in BPH and prostate cancer. Mol Cell Endocrinol, 1995 Sep 22, 113(2), 123 - 30 Regulation of alpha-subunit mRNA transcripts by pituitary adenylate cyclase-activating polypeptide (PACAP) in pituitary cell cultures and alpha T3-1 cells; Tsujii T et al.; Pituitary adenylate cyclase activating polypeptide (PACAP) increases glycoprotein hormone alpha-subunit mRNA levels suggesting a role for PACAP in maintaining the high levels of alpha-subunit protein characteristic of the pituitary . The present study used primary pituitary cell cultures and the alpha T3-1 pituitary cell line to investigate how PACAP affects alpha-subunit mRNA transcripts . Stimulation of cultured pituitary cells with 10 nM PACAP38, 10 nM GnRH, or the combination, for 24 h increased alpha-subunit mRNA levels 1.5-fold, whereas GnRH more effectively (P<0.01) stimulated alpha-subunit protein release than did PACAP38 (3.2- vs . 2.0-fold) . alpha-Subunit mRNA levels in alphaT3-1 cells were also increased by PACAP38 and by GnRH to maximum values at 12 h (P<0.05), and alpha-subunit protein secretion rose proportionately and in parallel with alpha-subunit mRNA levels . PACAP38 was a 100-fold more potent stimulator of alpha-subunit mRNA than was VIP, and a VIP-antagonist failed to block the stimulatory effect of PACAP38, suggesting an effect via type PACAP 1 receptors . Type I receptor mRNA transcripts were identified by Northern analysis in alphaT3-1 cells . Depletion of PCK activity by PMA failed to block the stimulatory effect of PACAP38, but prevented GnRH from increasing alpha-subunit mRNA levels and alpha-subunit secretion . PACAP38, like 8Br-cAMP and forskolin, stimulated (P<0.05) luciferase (LUC) activity in alphaT3-1 cells transfected with a plasmid containing the first 846 of 180 base pairs of the 5'-flanking region of the human alpha-subunit gene linked upstream to a LUC reporter gene . Finally, experiments using the transcription inhibitor DRB reveal that PACAP does not appreciably change alpha-subunit mRNA half-life . These findings are consistent with the proposal that PACAP contributes to the high levels of alpha-subunit protein characteristic of the pituitary by activating Type I receptors and stimulating alpha-subunit gene transcription in part by the cAMP/PKA pathway. Vopr Virusol, 1995 Sep-Oct, 40(5), 225 - 7 {Cross contamination of continuous cell cultures}; Iurkov SG; The possibility of air-droplet cell transmission during cell transfer was experimentally confirmed, this appearing to be the most probable route of cross contamination of cell cultures . The consequences of such contamination are assessed and measures reducing the risk of intercellular contamination proposed. Anal Biochem, 1995 Sep 1, 230(1), 149 - 53 Determination of gaseous and dissolved oxygen in a closed fat cell culture system; Bottcher H et al.; A novel method facilitates the determination of O2 content in the gas phase and in the culture medium of closed cell culture systems: Total O2 was swept out for 3 min by a stream of N2 under atmospheric pressure and carried to the measuring unit of a PBI Dansensor TIA-111-LV oxygen sensor, creating a peak-shaped signal, which was integrated over time . From the peak area, gas flow (determined by a Rotameter flow meter), and actual gas pressure and temperature, O2 content was calculated . The precision of the method was 1.1% (CV), recovery was 97.7% . O2 uptake was measured in white adipocytes, isolated by the collagenase method and cultured in a 3D matrix of agarose gel . As a basis of reference, DNA content of the cultures (9.5 pg/cell) was determined by a fluorimetric method . The viability of the cells was not affected by O2 determination, as shown by the maintenance of cellular adenine nucleotide content during the analytic procedure . The decline of O2 content in the cultures was constant over 72 h . The mean rate of cellular O2 consumption was 34.1 mumol/micrograms DNA.h. Steroids, 1995 Sep, 60(9), 656 - 61 Regulation of sex hormone-binding globulin production by isoflavonoids and patterns of isoflavonoid conjugation in HepG2 cell cultures; Loukovaara M et al.; The effect of the isoflavonoid phytoestrogens daidzein, equol, and genistein on sex hormone-binding globulin (SHBG) levels, SHBG mRNA transcript levels, and SHBG gene methylation was studied in HepG2 cell cultures by fluoroimmunometric SHBG assay and Northern and Southern hybridizations, respectively . The effect of 17 beta-estradiol on these parameters was studied as a control . The metabolism of isoflavonoids in HepG2 cells was determined by isotope dilution gas chromatography-mass spectrometry, after ion-exchange chromatography . Daidzein and equol increased SHBG levels in parallel intracellularly and extracellularly, whereas genistein increased SHBG levels only within the cells, resembling thus the effect of 17 beta-estradiol . The difference may originate from the fact that genistein has more hydroxyl groups than daidzein and equol . The regulation of SHBG production by phytoestrogens appears to occur at the post-transcriptional level . Firstly, daidzein, equol, or genistein did not have a clear effect on the steady-state SHBG mRNA levels . Secondly, no effect on SHBG gene methylation was observed by genistein . The findings applied also to 17 beta-estradiol . However, as the SHBG gene was more methylated in SHBG-negative MCF-7 cells than in SHBG-positive HepG2 cells, DNA methylation may play a role in the tissue-specific activation of this gene . The metabolism of isoflavonoids in HepG2 cells yielded mainly unconjugated and sulfated compounds . Similar metabolism in hepatocytes in vivo might retain their biological activity in tissues responsive to estrogens. Mol Mar Biol Biotechnol, 1995 Sep, 4(3), 193 - 9 ES-like cell cultures derived from early zebrafish embryos; Sun L et al.; Pluripotent embryonic stem (ES) cell cultures provide an efficient method for genome manipulation with many applications in marine biotechnology . To develop this technology we have been working to derive fish ES cell lines for in vitro studies of embryo cell growth and differentiation and for the generation of transgenic fish . Zebrafish embryonal cell cultures were derived from blastula-stage embryos in LDF medium supplemented with fetal bovine serum, trout serum, trout embryo extract, selenium, insulin, and leukemia inhibitory factor . Cultures derived under these conditions on feeder layers of zebrafish embryonic fibroblasts possessed a diploid karyotype and exhibited an ES-like morphology with elevated levels of alkaline phosphatase enzyme activity . Injection of primary cell cultures derived from embryos of transgenic fish carrying neo produced chimeric fish detected by polymerase chain reaction analysis . Embryo cells cultured on poly-D-lysine substrate in the presence of retinoic acid or Buffalo rat liver cell-conditioned medium (BRL-CM) and a reduced serum concentration differentiated into neuronal cell types exhibiting elevated levels of acetylcholinesterase enzyme activity and expression of neurofilament and glial fibrillary acidic protein. J Biol Chem, 1995 Sep 1, 270(35), 20599 - 607 Novel (Rp)-cAMPS analogs as tools for inhibition of cAMP-kinase in cell culture . Basal cAMP-kinase activity modulates interleukin-1 beta action; Gjertsen BT et al.; Novel (Rp)-cAMPS analogs differed widely in ability to antagonize cAMP activation of pure cAMP-dependent protein kinase I and II and to antagonize actions of cAMP on gene expression, shape change, apoptosis, DNA replication, and protein phosphorylation in intact cells . These differences were related to different abilities of the analogs to stabilize the holoenzyme form relative to the dissociated form of cAMP kinase type I and II . (Rp)-8-Br-cAMPS and (Rp)-8-Cl-cAMPS were the most potent cAMP antagonists for isolated type I kinase and for cells expressing mostly type I kinase, like IPC-81 leukemia cells, fibroblasts transfected with type I regulatory subunit (RI), and primary hepatocytes . It is proposed that (Rp)-8-Br-cAMPS or (Rp)-8-Cl-cAMPS should replace (Rp)-cAMPS as the first line cAMP antagonist, particularly for studies in cells expressing predominantly type I kinase . The phosphorylation of endogenous hepatocyte proteins was affected oppositely by (Rp)-8-Br-cAMPS and increased cAMP, indicating that (Rp)-8-Br-cAMPS inhibited basal cAMP-kinase activity . The inhibition of basal kinase activity was accompanied by enhanced DNA replication, an effect which could be reproduced by microinjected mutant cAMP-subresponsive RI . It is concluded that the basal cAMP-kinase activity exerts a tonic inhibition of hepatocyte replication . (Rp)-8-Br-cAMPS and microinjected RI also desensitized hepatocytes toward inhibition of DNA synthesis by interleukin-1 beta . This indicates that basal cAMP-kinase activity can have a permissive role for the action of another (interleukin-1 beta) signaling pathway. Klin Lab Diagn, 1995 Sep-Oct, (5), 52 - 4 {Optimizing conditions of cell culture use for virus indications by means of fluorescent probes}; Smelianskaia MV et al.; Cell cultures in a suspension on medium 199 with 1 x 10(6) cells per ml are recommended for virus indication by means of fluorescent probe method . When stored at 4 degrees C, the cells are fit for work for 5 days . The proposed conditions for the use of cell cultures are necessary for effective introduction of the fluorescent probe method in practical virological laboratories. J Eukaryot Microbiol, 1995 Sep-Oct, 42(5), 518 - 25 Encephalitozoon-like organisms in patients with alveolar hydatid disease: cell culture, ultrastructure, histoimmunochemical localization and seroprevalence; Furuya K et al.; We found Encephalitozoon-like organisms in an in vitro culture of a human liver lesion which was due to larval Echinococcus multilocularis . The organisms developed in the same fashion as an Encephalitozoon cuniculi . The spores that developed in parasitophorous vacuoles were 2.0-2.6 x 1.1-1.5 microns; each contained a single nucleus and 4-5 polar tubule coils, closely resembling E . cuniculi in its ultrastructure . Mature spores were collected from the supernatants by the use of Percoll centrifugation resulting in the banding of the spores on continuous gradients . We prepared three sorts of spores which were different in buoyant density in 0.15 M NaCl: 1.05-1.07 g/ml spores, 1.12 g/ml spores, and spores of over 1.14 g/ml . Polyclonal antibodies to a pool of each spore preparation were produced in a rabbit and applied to the detection of microsporidian antigen in situ . The histoimmunoperoxidase (HIP) procedure was used to detect the microsporidian antigen in echinococcal liver lesions from patients with alveolar hydatid disease (AHD) . Ten echinococcal liver lesions from different AHD patients were examined and four were found to be positive in the HIP test . The Percoll-separated spores were also used as an antigen to detect for antibodies in the sera from the patients with AHD by Western blotting . Antibodies were detected in 62 (52%) of the 119 AHD patients and in only 8 (5%) of the 159 normal healthy individuals. J Neurochem, 1995 Sep, 65(3), 1077 - 84 Properties of AMPA receptors expressed in rat cerebellar granule cell cultures: Ca2+ influx studies; Hack N et al.; Cultured cerebellar granule cells become vulnerable to excitatory amino acids, especially to NMDA and kainate, by 9 days in vitro . In the same time, the sensitivity of cells to (RS)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA), in terms of AMPA-induced toxicity or 45Ca2+ uptake, was very low . The low AMPA responsiveness was due to receptor desensitization, because agents known to block desensitization, cyclothiazide and the lectins concanavalin A and wheat germ agglutinin, rendered granule cells vulnerable to AMPA and produced a pronounced stimulation of 45Ca2+ accumulation . 45Ca2+ influx was induced specifically by AMPA-receptor stimulation, because it was blocked virtually completely by 2,3-dihydroxy-6-nitro-7-sulfamoylbenzoquinoxaline (NBQX) and the benzodiazepine GYKI 52466 (selective non-NMDA receptor antagonists) . Nevertheless, indirect routes activated by cellular responses to AMPA-receptor stimulation contributed significantly to the overall 45Ca2+ influx . These included Ca2+ uptake through NMDA-receptor channels, voltage-sensitive Ca2+ channels, and via Na+/Ca2+ exchange . However, nearly one-fifth of the total 45Ca2+ influx remained unaccounted for and this estimate was similar to 45Ca2+ influx observed under Na(+)-free conditions . This observation suggested that a significant proportion of the Ca2+ flux passes through the AMPA-receptor channel proper, a view supported by Co2+ uptake into nearly all granule cells on exposure to AMPA in the presence of cyclothiazide . Results are discussed in light of the reported AMPA receptor-subunit composition of cerebellar granule cells in vitro. J Bone Miner Res, 1995 Sep, 10(9), 1327 - 33 Phosphate transport in immortalized cell cultures from the renal proximal tubule of normal and Hyp mice: evidence that the HYP gene locus product is an extrarenal factor; Nesbitt T et al.; Whether renal phosphate wasting in X-linked hypophosphatemia (XLH) results from an intrinsic renal or humoral defect remains controversial . In studies of the murine homolog of XLH, harboring the Simian Virus 40 (SV40) large T antigen, we obviated the influence of renal cell heterogeneity and impressed memory by comparing Na(+)-phosphate cotransport in immortalized cells from the S1 segment of the proximal tubule . Cells from SV40 transgenic normal and Hyp mice exhibit characteristics of differentiated proximal tubule cells including gluconeogenesis and alkaline phosphatase activity . Surprisingly, however, we found two distinct populations of cells from the S1 proximal tubule of both normal and Hyp mice . In one, PTH treatment increases cAMP accumulation, while in the other both PTH and thyrocalcitonin enhance cAMP production . Kinetic parameters for Na(+)-phosphate cotransport were similar in both subpopulations of cells from normal (Km, 0.29 +/- 0.03 vs . 0.39 +/- 0.04 mM; Vmax, 4.6 +/- 0.6 vs . 5.2 +/- 0.4 nmol/mg/5 minutes) and Hyp mice (0.33 +/- 0.02 vs . 0.26 +/- 0.04; 6.0 +/- 0.7, 4.8 +/- 0.6) . More importantly, phosphate transport in S1 cells of either subpopulation from Hyp mice is no different than that of normals . These data indicate that renal proximal tubule cells from Hyp mice have intrinsically normal phosphate transport and support the hypothesis that a humoral abnormality underlies renal phosphate wasting in XLH. J Clin Microbiol, 1995 Sep, 33(9), 2454 - 7 Evaluation of an enterovirus group-specific anti-VP1 monoclonal antibody, 5-D8/1, in comparison with neutralization and PCR for rapid identification of enteroviruses in cell culture; Trabelsi A et al.; We evaluated the usefulness of a commercially available monoclonal antibody (MAb) directed against a group-specific epitope of the capsid protein VP1 of enteroviruses for the rapid identification of these viruses in cell culture . The MAb was assayed in an indirect immunofluorescence test with cultured cells infected by various serotypes of enterovirus; all 39 serotypes tested, including echoviruses 22 and 23, which are considered atypical enteroviruses, were reactive . The MAb was also tested with 61 strains recovered from clinical specimens inoculated into cell cultures in comparison with seroneutralization with intersecting pools of hyperimmune sera and PCR with primers from the 5' untranslated region of enteroviruses . There was total agreement between the results obtained with the MAb and those obtained by PCR, even for those strains of enteroviruses which were found to be untypeable with polyclonal antisera . These data demonstrate the usefulness of the MAb for rapid identification of enteroviruses in cell culture. Arch Biochem Biophys, 1995 Aug 20, 321(2), 397 - 404 Enzymes of B-ring-deoxy flavonoid biosynthesis in elicited cell cultures of "old man" cactus (Cephalocereus senilis); Liu Q et al.; Elicited cell cultures of the cactus Cephalocereus senilis produce a group of flavonoids with unsubstituted B-rings, including an aurone which represents a new class of phytoalexin . Preliminary enzymological studies indicated that the chalcone synthase (CHS) and chalcone isomerase (CHI) from cultures of C . senilis were active with cinnamoyl-CoA and 2',4',6'-trihydroxychalcone, respectively, probable intermediates for synthesis of flavonoids with unsubstituted B-rings . We now demonstrate that the cultures contain two isoforms of CHI, both of which are induced by elicitor treatment and are active with both 2',4,4',6'-tetrahydroxy- and 2',4',6'-trihydroxychalcone . (Hydroxy)-cinnamate:CoA ligase in the cactus cultures was active with cinnamic, 4-coumaric, caffeic, ferulic, and 4-methoxycinnamic acids, but not sinapic acid . A single form of CoA ligase, as resolved by chromatofocusing analysis, was active against both cinnamate and 4-coumarate . Cinnamic acid 4-hydroxylase (CA4H) activity was induced by elicitor treatment . Thus, elicited cultures contain the necessary enzymatic activities for synthesis of B-ring-hydroxy and -deoxy flavonoids . Synthesis of only the deoxy class in response to elicitation may result from some form of metabolic compartmentation through which the CA4H reaction is bypassed, leading to formation of cinnamoyl CoA which may then be incorporated into B-ring deoxy flavonoids via nondiscriminating CHS and CHI activities. Virology, 1995 Aug 20, 211(2), 377 - 84 Microevolution of type 3 Sabin strain of poliovirus in cell cultures and its implications for oral poliovirus vaccine quality control; Rezapkin GV et al.; Screening for sequence heterogeneities in Sabin Type 3 strains of attenuated poliovirus demonstrated mutations that consistently accumulate to significant levels following 10 passages in cultures of primary African green monkey kidney (AGMK) cells or continuous cultures of Vero cells . Fourteen newly identified mutations were quantified by mutant analysis by PCR and restriction enzyme cleavage in passages and in batches of commercial vaccines made in AGMK and Vero cells from the Sabin original (SO) seed virus and from a seed virus rederived by RNA plaque purification (RSO or "Pfizer" seed) . Nine of the 14 mutations were reproducibly observed in more than one series of passages . Although 5 other mutations were observed in only one set of passages each, their content gradually increased to a high percentage, suggesting that all the mutations that we found accumulated consistently . SO-derived samples accumulated more mutations than did RSO-derived ones, and the number of mutations and the rates of their accumulation were higher in Vero than in AGMK cells . While the rates of accumulation of most mutations were higher when passaging was performed at 37 degrees, a U-->C transition at nucleotide 5832 occurred faster at 34 degrees, the temperature used for vaccine production . Analysis of Type 3 oral poliovirus vaccine (OPV) monopools made by six manufacturers found only 5 of these newly identified mutations in vaccine batches (nucleotides 3956, 4935, 5357, 5788, and 5832) . Some of the mutations were found in trace amounts (less than 0.1%) while others were present at up to 1.8% levels . The pattern of these mutations was characteristic for the type of seed virus and the cell substrate but demonstrated no correlation with results of the monkey neurovirulence test . Therefore the only mutation occurring in Type 3 OPV which contributed to neurovirulence in monkeys was the previously described reversion at nucleotide 472 . Quantitation of reversion at nucleotide 472 can be utilized for assessment of acceptability of vaccine lots, while other mutations can be used for monitoring the consistency of vaccine production. Microsc Res Tech, 1995 Aug 15, 31(6), 507 - 18 Isolation, cell culture, and characterization of oviduct epithelial cells of the cow; Joshi MS; This report describes an easy method of isolation and cell culture of the epithelial cells of cow oviduct . Incubation of cow oviduct with 0.1 mg/ml collagenase in the lumen for 90 minutes helped to dislodge large numbers of ciliated and secretory epithelial cells . The isolated cells, when seeded on plastic, proliferated very quickly and became confluent in 8-10 days in 35 mm Petri dishes . The isolated ciliated cells which attached to the plastic dish lost their cilia after 4-5 days in culture . The cultured epithelial cells were keretin positive . The isolated bovine oviduct epithelial cells, when cultured on plastic precoated with 10 mg/ml matrigel, organized themselves into hollow tubes or spheres with microvilli directed towards the lumen . The epithelial cells seeded on 2 mg/ml matrigel became subconfluent in 15-20 days after seeding . The histoarchitecture of the secretory cells growing in vitro on matrigel resembled that of intact oviduct secretory epithelial cells . Occasional ciliated cells containing large number of mitochondria were observed in the monolayer cultured on 2 mg/ml matrigel substratum but possessed few cilia . The oviduct epithelial cells cultured on 2 mg/ml matrigel incorporated 35S-methionine linearly into protein up to 8 hours in the presence of estradiol or progesterone . The fluorograph of the newly synthesized proteins indicated the presence of an additional 60 kd protein in the cell extract of epithelial cells incubated with estradiol. Pediatr Allergy Immunol, 1995 Aug, 6(3), 170 - 4 Comparison of cytokine production in blood cell cultures of healthy children and adults; Elsasser-Beile U et al.; The production of the cytokines IL-1-alpha, IL-1-beta, IL-2, TNF-alpha, and INF-gamma was measured by a sensitive immunological assay in stimulated whole blood cell cultures from 52 healthy children (33 aged from 1 to 9 years and 19 aged between 10 and 17 years) and 67 healthy adults . When the higher absolute mononuclear cell counts in the peripheral blood samples of the children were taken into account, the relative production of all measured cytokines was lower in the cell cultures of the children than of the adults . In the group of the younger children (< 10 years) the differences were significant for all measured cytokines . In the group of older children (> or = 10 years) the values were higher than in the younger children but lower than in adults . The findings indicate that the cellular immunological competence is or can be reduced in children and adolescents, particularly young children below 10 years of age . There seems to be a gradual development of cytokine production during childhood. Biosci Rep, 1995 Aug, 15(4), 191 - 9 Hexachlorocyclohexanes affect the arachidonic acid release from phosphatidylinositol but not from other phospholipid classes in tubular cell cultures; Puente-Fraga JC et al.; Gamma- and delta-isomers of hexachlorocyclohexane caused marked decreases in the levels of radioactive phospholipids, and increases in the levels of {3H}arachidonate incorporated into free fatty acids in rat renal tubular cells . The increased radioactivity of free fatty acids arises from the decrease of {3H}arachidonate incorporated into phosphatidylinositol, but not into phosphatidylcholine, phosphatidylserine or phosphatidylethanolamine . This fact suggests that phosphatidylinositol can be broken down to the fatty acid from the sn-2 position and lysophospholipid by a phospholipase activity increased by hexachlorocyclohexanes . The observed specific toxicant action could be achieved in two ways: (a) operating upon a specific phospholipase A2 that acts on phosphatidylinositol, but not on other phospholipids as substrates and/or (b) involving substrate-phospholipase A2 interactions . Interestingly, the observed effect of the delta-isomer was more pronounced than that of the gamma-one. J Pharmacol Toxicol Methods, 1995 Aug, 33(4), 187 - 95 Toxic effects of anabolic-androgenic steroids in primary rat hepatic cell cultures; Welder AA et al.; Hepatic complications in athletes and bodybuilders after abusing anabolic-androgenic steroids (AAS) have been reported . Hepatic injury, including cholestasis, peliosis hepatis, hyperplasia, and tumors, have been attributed to abuse of the 17 alpha-alkylated AAS . Some of these pathological conditions have been reversed when individuals were converted to nonalkylated AAS regimens . The purpose of this study was to determine and compare the direct toxic effects of commonly abused AAS (both 17 alpha-alkylated and nonalkylated) in primary hepatic cell cultures . Primary cultures, established from 60-day-old Sprague-Dawley rats, were exposed to doses of 1 x 10(-8), 1 x 10(-6), and 1 x 10(-4)M 19-nortestosterone, fluoxymesterone, testosterone cypionate, stanozolol, danazol, oxymetholone, testosterone, estradiol, and methyltestosterone for 1, 4, and 24 hr . Lactate dehydrogenase (LDH) release, neutral red (NR) retention, and glutathione (GSH) depletion were evaluated to determine plasma membrane damage, cell viability, and possible oxidative injury, respectively . Those cultures exposed to the 17 alpha-alkylated AAS, methyltestosterone and stanozolol, at doses of 1 x 10(-4) M for 24 hr and the 17 alpha-alkylated AAS, oxymetholone, at 1 x 10(-4) M for 4 and 24 hr showed significant increased in LDH release and decreases in NR retention while there were no significant differences with the nonalkylated steroids (testosterone cypionate, 19-nortestosterone, testosterone, and estradiol) . GSH depletion was evaluated in cultures treated with 1 x 10(-8), 1 x 10(-6), and 1 x 10(-4) M concentrations of methyltestosterone, stanozolol, and oxymetholone for 1, 2, 4, and 6 hr . Cultures exposed to 1 x 10(-4) M oxymetholone were significantly depleted of GSH at 2, 4, and 6 hr; cultures exposed to 1 x 10(-4) M methyltestosterone were significantly depleted of GSH at 4 and 6 hr; and cultures exposed to stanozolol were not significantly depleted of GSH at any of the time periods tested . These data indicate that the 17 alpha-alkylated steroids (methyltestosterone, oxymetholone, and stanozolol) are directly toxic to hepatocytes, whereas the nonalkylated steroids (testosterone cypionate, 19-nortestosterone, testosterone, and estradiol) show no effects at similar doses . These data demonstrate a trend toward a structural-activity relationship to AAS-induced toxicity in primary cultures of rat hepatocytes. J Vet Med Sci, 1995 Aug, 57(4), 769 - 71 Rapid detection of mycoplasma contamination in cell cultures by enzymatic detection of polymerase chain reaction (PCR) products; Kobayashi H et al.; Enzymatic detection of polymerase chain reaction (ED-PCR) was applied for rapid and easy identification of mycoplasmas from contaminated cell culture . This method was based on the capture of amplified products via biotin-streptavidine affinity and the detection of an incorporated hapten in amplified products with enzyme-linked antibody . Primers corresponding to common sequence of Mollicutes in 16S ribosomal RNA dominated gene was used . Nineteen of twenty Mollicutes so far reported as cell contaminants appeared positive by ED-PCR, whereas remaining one, Acholeplasma axanthum, appeared negative . Samples from sixty-two cell culture were tested for contamination of mycoplasmas by means of ED-PCR, cultivation, and electronmicroscopy . The results of ED-PCR were the same as those of cultivating method . The time required for all the detection process in ED-PCR was about 5 hr for 20 samples . We suggest that ED-PCR can be used in the rapid detection of mycoplasms from cell culture. Eur J Endocrinol, 1995 Aug, 133(2), 251 - 4 Role for serotonin3 receptors in the control of adrenocorticotropic hormone release from rat pituitary cell cultures; Calogero AE et al.; Although several serotonin (5-HT) receptor types have been shown capable of stimulating the release of adrenocorticotropic hormone (ACTH) from the pituitary gland, relatively little is known about the role of the 5-HT3 receptor, a receptor that has generated a great deal of interest for its involvement in many behavioral and therapeutic effects . Hence, in this study, we tested the effects of the 5-HT3/4 receptor antagonist 3-tropanyl-indole-3-carboxylate (ICS 205-930) and the selective 5-HT3 receptor antagonist 3-tropanyl-3, 5-dichlorobenzoate (MDL 72222) on ACTH release stimulated by 5-HT from primary cultures of rat pituitary cells . Subsequently, we evaluated the effects of the selective 5-HT3 receptor agonist 1-(m-chlorophenyl)-biguanide (m-CPBG) on basal, corticotropin-releasing hormone (CRH)- and arginine vasopressin (AVP)-stimulated ACTH release . The maximal stimulatory effect of 5-HT (10(-9) mol/l) on ACTH release was antagonized by both ICS 205-930 and MDL 72222, suggesting that 5-HT stimulates basal ACTH release through activation of 5-HT3, receptors . Accordingly, m-CPBG stimulated basal ACTH release in a concentration-dependent fashion . In contrast to 5-HT, m-CPBG did not have any effect on CRH-stimulated ACTH release and inhibited AVP-stimulated ACTH release in a concentration-dependent manner . These data suggest that the 5-HT3 receptor is involved in the release of ACTH from the pituitary gland in vitro. Toxicol Appl Pharmacol, 1995 Aug, 133(2), 328 - 42 Anabolic-androgenic steroid-induced toxicity in primary neonatal rat myocardial cell cultures; Welder AA et al.; Recent literature reports of myocardial infarction in athletes who self-administer anabolic-androgenic steroid (AAS) and previous animal studies of the effects of AASs on the heart suggest that these drugs may be directly injurious to the myocardium . We have previously demonstrated that 100 microM testosterone cypionate (TC) inhibits all beating activity of primary neonatal rat myocardial cell cultures within 1 hr of exposure and causes significant LDH release by 4 hr of exposure, indicating a direct toxic effect of TC . The purpose of this investigation was to evaluate the effects of commonly abused AASs on primary neonatal rat myocardial cell cultures and to provide insight into early cellular changes that may lead to TC-induced toxicity . Significant LDH release was observed in 5-day-old primary myocardial cell cultures (obtained from 3-to-5-day-old Sprague-Dawley rats) exposed to 100 microM testosterone enanthate (TE), testosterone propionate (TP), and oxymetholone (O) for 4 and 24 hr and in cultures exposed to 100 microM testosterone (T) for 24 hr . Neutral red retention and MTT formazan production were significantly decreased in cell cultures exposed to 100 microM TE, TP, and O after only 4 hr of exposure, indicating a loss of viability and mitochondrial activity . However, there was no effect on viability of cell cultures exposed for 24 hr to 100 microM of a variety of other commonly abused AASs . Phase-contrast microscopy revealed complete disruption of the monolayer in cell cultures treated with 100 microM TE, TP, and O for 4 hr . Treatment of fura-2-loaded myocardial cell cultures with 100 microM TC produced no significant changes in calcium transients or baseline calcium levels for up to 13 min of exposure . These results indicate that O, T, TC, TE, and TP produce a direct toxic effect in heart cell cultures and that early (< 13 min) changes in calcium homeostasis are unlikely to participate in the mechanism of toxicity. Am J Clin Pathol, 1995 Aug, 104(2), 167 - 71 Quality control of imprint and tissue section DNA ploidy analysis in image analysis systems utilizing cell culture-based control materials; Ruby SG et al.; Currently, there are no standard controls available for quality control of DNA ploidy assays in image analysis systems . The authors have developed a system of controls based on cultured human cell lines that can mimic tissue sections and touch preparations . This allows for quality control for the entire process for DNA ploidy analysis from the initial cutting of the paraffin block or production of the touch preparation through fixation, staining, and interactive image analysis . The use of cell line controls rather than normal tissue is advantageous because of cell population homogeneity and the volume of uniform slides that are obtainable . This system, while providing an excellent means of controlling day-to-day performance of DNA ploidy analysis, may also be adapted for use as a means of multi-institutional proficiency testing. Proc Natl Acad Sci U S A, 1995 Aug 1, 92(16), 7162 - 6 Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-D-aspartate or nitric oxide/superoxide in cortical cell cultures; Bonfoco E et al.; N-Methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity may depend, in part, on the generation of nitric oxide (NO.) and superoxide anion (O2.-), which react to form peroxynitrite (OONO-) . This form of neurotoxicity is thought to contribute to a final common pathway of injury in a wide variety of acute and chronic neurologic disorders, including focal ischemia, trauma, epilepsy, Huntington disease, Alzheimer disease, amyotrophic lateral scelerosis, AIDS dementia, and other neurodegenerative diseases . Here, we report that exposure of cortical neurons to relatively short durations or low concentrations of NMDA, S-nitrosocysteine, or 3-morpholinosydnonimine, which generate low levels of peroxynitrite, induces a delayed form of neurotoxicity predominated by apoptotic features . Pretreatment with superoxide dismutase and catalase to scavenge O2.- partially prevents the apoptotic process triggered by S-nitrosocysteine or 3-morpholinosydnonimine . In contrast, intense exposure to high concentrations of NMDA or peroxynitrite induces necrotic cell damage characterized by acute swelling and lysis, which cannot be ameliorated by superoxide dismutase and catalase . Thus, depending on the intensity of the initial insult, NMDA or nitric oxide/superoxide can result in either apoptotic or necrotic neuronal cell damage. J Exp Med, 1995 Aug 1, 182(2), 575 - 9 Identification of Fc epsilon RIneg mast cells in mouse bone marrow cell cultures . Use of a monoclonal anti-p161 antibody; Kinzer CA et al.; A monoclonal hamster antibody (K-1) specific for a 161-kD mast cell surface glycoprotein was derived . p161 is expressed on normal and cultured mast cells and on some macrophages, but not on basophils or other hematopoietic cells . A population of Fc epsilon Rneg cells expressing p161 was found in short term cultures of bone marrow cells grown in interleukin (IL)-3 . These cells were purified and propagated for extended periods in IL-3 . They express c-kit and Fc gamma RII/III, contain alcian blue-positive granules and histamine, and secrete IL-3 in response to ionomycin treatment . Their morphology is consistent with that of mast cells . We propose that they represent Fc epsilon RIneg mast cells that can be detected and purified because of their p161 expression. Endocrinology, 1995 Aug, 136(8), 3648 - 56 Parathyroid hormone increases the number of tartrate-resistant acid phosphatase-positive cells through prostaglandin E2 synthesis in adherent cell culture of neonatal rat bones; Inoue H et al.; Tartrate-resistant acid phosphatase (TRAP) is expressed during one of the steps of osteoclast differentiation . The involvement of prostaglandin E2 (PGE2) synthesis in PTH-induced increases in TRAP-positive cell number was studied in an improved slowly adhering cell culture system, prepared by removing preexisting osteoclasts from disaggregated bone cells obtained from 6-day-old rats . The majority of the TRAP-positive cells that appeared were mononuclear . In 1-day culture, PTH (0.025-0.25 nM) and PGE2 (1 microM) increased the number of mono- and multinucleated TRAP-positive cells . Indomethacin (50 microM) inhibited PTH-induced increase, and the inhibition was significantly abolished by the addition of PGE2 . SC-19220, a specific antagonist for one class of PGE2 receptor, inhibited the increase induced by PTH and PGE2 . PTH significantly stimulated the production of PGE2 in the culture . Peroxides, which are produced as byproducts of PGE2 biosynthesis, were detected in the adherent cells using dichlorofluorescene and increased the number of TRAP-positive cells . Small resorption pits were recognized on the surfaces of bone slices on which slowly adhering cells had been incubated for 3 days with PTH . These findings showed that PTH, through its effects on PGE2 synthesis and the subsequent reactions, induced the step at which TRAP is expressed, possibly during the differentiation of osteoclasts. Diabetes, 1995 Aug, 44(8), 936 - 46 Insulin action and glucose metabolism in nondiabetic control and NIDDM subjects . Comparison using human skeletal muscle cell cultures; Henry RR et al.; Myoblasts from human skeletal muscle were isolated from needle biopsy samples of vastus lateralis and fused to differentiated multinucleated myotubes . Specific high-affinity insulin and insulin-like growth factor I (IGF-I) binding, glucose transporter proteins GLUT1 and GLUT4, glycogen synthase and pyruvate dehydrogenase proteins, and their specific mRNAs were identified in fused myotubes . Insulin and IGF-I stimulated 2-deoxyglucose uptake twofold with half-maximal stimulation by insulin at 0.98 +/- 0.12 nmol/l and maximal stimulation at 17.5 nmol/l . Acute insulin treatment (33 nmol/l) doubled glycogen synthase activity and glucose incorporation into glycogen while increasing pyruvate dehydrogenase approximately 30% . In cells cultured from NIDDM subjects, both basal (6.9 +/- 1.0 vs . 13.0 +/- 1.7 pmol.mg protein-1.min-1) and acute insulin-stimulated transport (13.5 +/- 2.0 vs . 22.4 +/- 1.3 pmol.mg protein-1.min-1) were significantly reduced compared with nondiabetic control subjects (both P < or = 0.005) . GLUT1 protein content of total membranes from NIDDM subjects was decreased compared with control subjects, while GLUT4 levels were similar between groups . A significant correlation (r = 0.65, P < or = 0.05) was present when maximal rates of insulin-stimulated glucose transport in cell culture from subjects were compared with their corresponding in vivo glucose disposal determined by hyperinsulinemic glucose clamp . In summary, differentiated human skeletal muscle cultures exhibit biochemical and molecular features of insulin-stimulated glucose transport and intracellular enzyme activity comparable with the in vivo situation . Defective insulin-stimulated glucose transport persists in muscle cultures from NIDDM subjects and resembles the reduced insulin-mediated glucose uptake present in vivo . We conclude that this technique provides a relevant cellular model to study insulin action and glucose metabolism in normal subjects and determine the mechanisms of insulin resistance in NIDDM. Neurochem Int, 1995 Aug, 27(2), 147 - 55 Melatonin biosynthesis in photoreceptor-enriched chick retinal cell cultures: role of cyclic AMP in the K(+)-evoked, Ca(2+)-dependent induction of serotonin N-acetyltransferase activity; Gan J et al.; The roles of cyclic AMP and calcium in the regulation of serotonin N-acetyltransferase (NAT) activity were studied in low density monolayer cultures of chick retinal photoreceptors and neurons . Photoreceptor-enriched retinal cell cultures were prepared from embryonic day 6 retinas and cultured for 6 days . NAT activity in these cultures could be induced by treatment with cyclic AMP protagonists, 8Br-cyclic AMP, forskolin, and 3-isobutyl-1-methylxanthine (IBMX), or by treatment with depolarizing concentrations of extracellular K+ . The stimulatory effect of K+, which involves Ca2+ influx through dihydropyridine-sensitive channels, was mediated at least in part by cyclic AMP, as indicated by the following observations . Depolarizing concentrations of K+ stimulated the formation of cyclic AMP, and the stimulatory effects of K+ on both cyclic AMP formation and on NAT activity were synergistically potentiated by the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) . MDL 12,330A, a putative adenylate cyclase inhibitor, inhibited K(+)-evoked cyclic AMP accumulation and induction of NAT activity over the identical concentration range . In contrast, MDL 12,300A failed to inhibit the induction of NAT elicited by 8Br-cyclic AMP . H-89, an inhibitor of cyclic AMP-dependent protein kinase, antagonized the induction of NAT activity by either forskolin or K+ with equal potency for both stimuli . These results suggest that cyclic AMP plays an essential role in the induction of NAT activity that occurs as a consequence of membrane depolarization . Cyclic AMP and Ca2+ may also interact at a step distal to adenylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS) J Hepatol, 1995 Aug, 23(2), 160 - 5 Parathyroid hormone-related peptide is expressed and rapidly inducible in human liver cell cultures that have a bile duct phenotype; Roskams T et al.; Parathyroid hormone-related peptide is the major factor responsible for hypercalcemia of malignancy . There is increasing evidence that parathyroid hormone-related peptide also plays an important role in the growth and differentiation of both neoplastic and non-neoplastic cells . Recently we found that reactive human bile ductules and cholangiocarcinomas, but not normal bile ducts, human hepatocytes nor hepatocellular carcinomas, express parathyroid hormone-related peptide and we speculated that parathyroid hormone-related peptide may function as a growth and differentiation factor for bile ductular epithelial cells . Using a specific polyclonal antibody for immunostaining and a digoxigenin-random prime-labeled probe for in situ hybridization assay, we found that only cell lines with a bile duct phenotype expressed parathyroid hormone-related peptide and its mRNA . HepG2 cells with hepatocellular phenotype (CK19-, CK7-, CK8+, CK18+, albumin+) do not express parathyroid hormone-related peptide . However, A16 (HepG2 derived cell line) expressing bile duct marker CK19, also expressed parathyroid hormone-related peptide, while hepatocyte markers CK8, CK18, CALLA and albumin were negative . In addition, the H1 cell line (adult human hepatocytes immortalized in our laboratory by SV40 DNA transfection, passaged at least 40 times and cultured for 13 months) expressed bile duct marker CK7 and parathyroid hormone-related peptide, while hepatocyte markers CK8, CK18, CALLA and albumin were negative . Previous studies demonstrated that parathyroid hormone-related peptide gene expression in keratinocytes can be modulated by serum, growth factors and cycloheximide although there is a species and cellular specificity.(ABSTRACT TRUNCATED AT 250 WORDS) Biochim Biophys Acta, 1995 Jul 13, 1257(2), 174 - 80 Effects of interactions of apolipoprotein A-II with apolipoproteins A-I or A-IV on {3H}cholesterol efflux and uptake in cell culture; Stein O et al.; Conflicting evidence has accumulated with years regarding the putative negative effect of apolipoprotein A-II on apo A-I mediated cholesterol efflux . In this study, this question was reexamined and in addition to the interaction of apo A-II with apo A-I, its possible effect on apo E and apo A-IV was investigated as well . Free cholesterol (FC) donors were the main components of atheroma, namely, mouse peritoneal macrophages (MP), bovine aortic smooth muscle (SMC) and fibroblasts labeled with {3H}FC . Acceptors of FC were dioleoylphosphatidylcholine (DOPC) liposomes containing apo A-I, rh-apo A-IV or rh-apo E alone or together with apo A-II . When {3H}FC labeled MP were incubated for 2 or 4 h with equimolar concentrations of apo A-I, A-II, A-IV or E, the lowest {3H}cholesterol efflux occurred with apo A-II . Exposure of {3H}FC MP to liposomes containing apo A-I/A-II at 1:2 M/M (keeping the total protein concentration at 50 micrograms/ml), resulted in a lower {3H}FC efflux as compared to apo A-I alone . However, when apo A-I or apo A-IV protein concentration was kept constant and supplemented with apo A-II, a lower {3H}FC efflux was found only at 1:3 M/M of apo A-I/A-II . Apo A-II added to apo E had no effect on FC efflux . With aortic SMC and fibroblasts, no inhibitory effect of addition of apo A-II to apo A-I or apo A-IV on cholesterol efflux was seen at apo A-I/A-II of 1:1 or 1:2 M/M . The uptake of macrophage derived {3H}FC by SMC or HepG2 cells was studied using the serum-free efflux media, containing PC liposomes + apolipoproteins, from 3H-labeled macrophages . The cellular uptake of {3H}FC was higher when apo A-II had been added to apo A-I or apo A-IV than when the apolipoproteins were added alone . In conclusion, apo A-II was found to be less effective in cholesterol efflux and to interfere with the action of A-I only when the cholesterol donors were macrophages and when the relative amount of apo A-I to apo A-II was low . This was not the case when SMC or fibroblasts served as cholesterol donors . In the presence of apo A-II, enhanced {3H}cholesterol delivery to cells was seen which could contribute to the proatherogenic activity of apo A-II. Brain Res, 1995 Jul 10, 685(1-2), 201 - 4 Eicosanoids are produced by microglia, not by astrocytes, in rat glial cell cultures; Matsuo M et al.; To determine principal sources of eicosanoid production in glial cells, we analyzed the metabolites of arachidonic acid in cultured rat glial cells by use of reversed-phase, high-performance liquid chromatography and an on-line radioisotope detector . Prostaglandin D2, leukotriene B4, leukotriene C4, and 5-hydroxyeicosatetraenoic acid were present in cultures in which microglia appeared on a monolayer astrocytes . None were detected in culture dishes that contained only astrocytes, although astrocytes have been believed to be a main source of eicosanoid production in brain. J Gen Virol, 1995 Jul, 76 ( Pt 7), 1571 - 81 Subpopulations of non-coding control region variants within a cell culture-passaged stock of BK virus: sequence comparisons and biological characteristics; Johnsen JI et al.; In the circular DNA genome of the human polyomavirus BK an approximately 400 bp non-coding control region (NCCR) separates the early and late genes . The NCCR contains the origin of replication as well as the promoter/enhancer with a mosaic of cis-acting elements involved in the regulation of both early and late transcription . The NCCR has been shown to be very heterogeneous between different BK virus (BKV) strains . This may affect the host cell permissivity and oncogenic potential of a given BKV strain . Our previous studies with BKT-1B, a continuous cell line established from a BKV (Gardner) -induced hamster fibrosarcoma, revealed that the BKV DNA is integrated in the host genome in multiple copies . The sequence of the integrated BKV NCCR was substantially different from (and even contained sequences not found in) that of the BKV (Gardner) strain supposedly used to establish the BKT-1B cell line . PCR amplification, cloning and subsequent sequencing revealed that the original BKV (Gardner) stock contained at least seven different subpopulations of viral genomes . None of them had a control region 'anatomy' which was identical to either the BKV (Gardner) strain, the variant found integrated in BKT-1B cells or any previously published NCCR . In order to study the biological significance of these new BKV NCCR variants we developed a simple cassette model allowing the NCCRs of the new variants to be cloned in an identical genomic background of BKV protein-coding sequences and performed transfection studies with the recombinant genomes in non-permissive rodent cells and in semi-permissive monkey cells . The results demonstrated that the NCCR variants conferred striking differences, in both transforming capacity and host cell permissivity, to the recombinant BKV genomes . Sequence comparisons suggested genetic explanations for these differences, as well as evolutionary relationships between BKV NCCRs. Ann Acad Med Singapore, 1995 Jul, 24(4), 523 - 7 A comparative study of the Abbott and Murex enzyme immunoassay, and cell culture in the detection of cervical chlamydial infection in female sex workers in Singapore; Chan RK et al.; Endocervical smears from 200 female sex workers were tested for Chlamydia trachomatis infection using the Abbott and Murex enzyme immunoassay (EIA) kits and the standard cell culture technique to compare the performance of these tests . The Abbott test had a sensitivity, specificity, predictive value of a positive result (PVP) and predictive value of a negative result (PVN) of 83.3%, 97.8%, 78.9% and 98.3% respectively . The corresponding figures for the Murex system were 77.8%, 98.4%, 82.4% and 97.8%; and of the standard culture test were 55.6%, 100% and 95.8% . EIA tests were more sensitive but slightly less specific than cell culture in the detection of Chlamydia trachomatis cervical infection in female sex workers in Singapore. Eur Cytokine Netw, 1995 Jul-Dec, 6(4), 225 - 30 Modulation of proinflammatory cytokine release in rheumatoid synovial membrane cell cultures . Comparison of monoclonal anti TNF-alpha antibody with the interleukin-1 receptor antagonist; Butler DM et al.; While there is an extensive literature on cytokine regulation in vivo using human cell lines or peripheral blood monocytes, very little is known about cytokine regulation within the multicellular environment of inflammatory sites in vivo . We have previously shown that in rheumatoid synovial membrane cultures, a complex, but pathophysiologically relevant mixture of cells, the addition of a neutralizing anti TNF-alpha antibody inhibits the production of IL-1 and GM-CSF, indicating the presence of a cytokine 'cascade' in this inflammatory tissue . In this paper we demonstrate that the interactivities between cytokines in rheumatoid arthritis also extends to other cytokines, such as IL-6 and IL-8, and that within the IL-1 family it is IL-1 beta in particular which is downregulated by neutralizing TNF-alpha activity . The cytokine interactions are unidirectional, in that neutralization of TNF-alpha reduced IL-1 beta, IL-6 and IL-8 production, whereas treatment of the rheumatoid synovial membrane cells with a neutralizing concentration of the IL-1 receptor antagonist (IL-1ra) reduced IL-6 and IL-8 production but not TNF-alpha production . These results suggest a rationale for the profound anti-inflammatory effects and consequent clinical benefit noted in RA patients treated recently in clinical trials with a chimeric anti-TNF-alpha antibody in vivo. Tsitol Genet, 1995 Jul-Aug, 29(4), 22 - 6 {The electron microscopic study of the cytoplast mitochondria of an ESK cell culture after the action of actinomycin D . Embryonic swine kidney}; Konstantinova SA et al.; The inhibitor of RNA synthesis actinomycin D substantially changed mitochondrial morphology and ultrastructure of cytoplasts in comparison with mitochondria of intact cells . These changes included an increase of matrix condensation, the number of cristae, and extension of intercristal and intracristal spaces . We suggest that mitochondria are characterized by significant independence from the nucleus that possibly controls a considerable part of mitochondrial RNA. Prostaglandins, 1995 Jul, 50(1), 19 - 32 Platelet activating factor (PAF) stimulates release of PGI2 from inflamed rabbit gallbladder cell cultures; Myers SI et al.; This study examines the hypothesis that PAF stimulates release of PGI2 from inflamed rabbit gallbladder explant cell cultures . New Zealand white rabbits underwent bile duct ligation for 72 h (72 h BDL), or sham operation, Sham and 72 h BDL gallbladder explants were placed in culture, and the cells grown to 75% confluence . The cells were exposed to increasing concentrations of PAF for 60 min . The media analyzed for eicosanoid release by EIA and the cells analyzed for cyclooxygenase and prostacyclin synthase content by immunoblot analysis . PAF increased release of 6-keto-PGF1 alpha from the 72 h BDL gallbladder cell cultures in a dose-related manner which was inhibited by indomethacin preincubation by 90% . The increased 72 h BDL cell release of 6-keto-PGF1 alpha was not associated with changes in the content of cyclooxygenase or prostacyclin synthase . PAF did not alter eicosanoid release from sham control cell cultures . These data suggest that PAF can only up-regulate endogenous 6-keto-PGF1 alpha release from the 72 h BDL cells that had been previously stimulated by inflammation . PAF may thus contribute to gallbladder distention and injury by chronic stimulation of inflamed gallbladder PGI2 release. J Cardiovasc Electrophysiol, 1995 Jul, 6(7), 551 - 68 Determination of impulse conduction characteristics at a microscopic scale in patterned growth heart cell cultures using multiple site optical recording of transmembrane voltage; Rohr S; It is well established that impulse propagation in cardiac tissue is determined by the interaction between active membrane properties and the passive electrical characteristics of the network formed by individual myocytes . In the past, the intricate microarchitecture of intact cardiac tissue and the limited spatial resolution of available recording techniques had rendered a systematic evaluation of the influence of the cellular microarchitecture on impulse propagation difficult . Recently, however, successful efforts have been undertaken to: (1) simplify the cellular arrangement by designing cardiac structures with defined two-dimensional geometries; and (2) measure impulse propagation in these preparations at the cellular/subcellular scale using optical techniques . This short review considers both of these developments, i.e., patterned growth of heart cells in culture and multiple site optical recording of transmembrane voltage (MSORTV), and summarizes first results obtained with the combination of both techniques. Endocrinology, 1995 Jul, 136(7), 3100 - 6 Combined magnetic fields increase insulin-like growth factor-II in TE-85 human osteosarcoma bone cell cultures; Fitzsimmons RJ et al.; In vitro exposure to low-energy, combined magnetic fields (CMF) increased the release of insulin-like growth factor (IGF)-II from human TE-85 osteosarcoma cells . Short-term CMF exposure of only 10 min increased IGF-II levels in conditioned medium 1 h post CMF exposure . IGF-II levels were measured with a radioreceptor assay using H-35 cells that contain abundant IGF-II but not IGF-I receptors . This assay also uses a recently validated BioGel P-10 acid gel filtration method to remove IGF binding protein before quantitation of either IGF-I or IGF-II . In addition to an increase in IGF-II levels, DNA synthesis, as an index of cell proliferation, was increased during the 24-h period post CMF exposure . A monoclonal antibody against IGF-II blocked the increase in cell proliferation following CMF exposure, whereas a control monoclonal antibody against osteocalcin did not attenuate the mitogenic action of CMF exposure . The effect of CMF exposure to increase both cell proliferation and IGF-II was cell-density dependent with greater stimulation by CMF observed at lower densities . Together, these data are consistent with the hypothesis that CMF exposure stimulates release/production of IGF-II from bone cells and that increased IGF-II then promotes an increase in cell proliferation. J Orthop Res, 1995 Jul, 13(4), 553 - 61 Microvessel endothelial cells and pericytes increase proliferation and repress osteoblast phenotypic markers in rat calvarial bone cell cultures; Jones AR et al.; To investigate the influence of microvessel cells on osteoblasts, we exposed osteoblast-enriched cultures of rat calvarial cells to cultured endothelial cells and pericytes using feeder-layer co-cultures, co-culture dish inserts, and conditioned media experiments . When co-cultured with growth-arrested feeder-layers of endothelial cells or pericytes for 10 days, bone cell cultures showed an increase in cell number and reduction in alkaline phosphatase activity . The response of bone cells to endothelial cells was nearly twice their response to pericytes . A similar response was demonstrated by exposure to microvessel cells in co-culture dish inserts and by exposure to media conditioned by microvessel cells . In long-term cultures of bone cells, the levels of osteocalcin and the number of mineralized nodules both were reduced by exposure to media conditioned by the microvessel cells . Transient exposure to conditioned media from the microvessel cell cultures for 3 days, during the period from initial plating to cell confluence, produced nearly the same effect on the cultures of bone cells as did continuous exposure to these conditioned media . The influence of isolated microvessel cells on osteoblast-enriched calvarial cells was found to be primarily mitogenic, mediated by soluble factors, independent of cell contact, and a cause of prolonged reduction in the expression of early and late markers of the osteoblast phenotype. Free Radic Biol Med, 1995 Jul, 19(1), 53 - 65 Antioxidant enzyme levels as a function of growth state in cell culture; Oberley TD et al.; Manganese superoxide dismutase (MnSOD) levels were monitored as a function of time in culture to determine whether these levels were altered at logarithmic growth versus when the cells exhibited density limitation of growth . For comparison, activities of the antioxidant enzymes copper, zinc superoxide dismutase (CuZnSOD), catalase, and glutathione peroxidase were also evaluated . Four cell lines were studied, two of which exhibited density limitation of growth and two of which did not . Each cell line showed a unique antioxidant enzyme profile . The two cell lines that showed density limitation of growth also demonstrated induction of MnSOD at the time when the cells stopped proliferating in culture, whereas the other two cell lines did not show induction of MnSOD . There was no strict correlation between density limitation of growth and activities of the other antioxidant enzymes . To determine whether SOD varied with various phases of the cell cycle, NIH/3T3 cells were synchronized using serum starvation, and then SOD activities were measured during quiescence (G0) and the phase of DNA synthesis (S-phase) . MnSOD was decreased during S-phase compared with G0, whereas CuZnSOD was increased during S-phase compared with G0, demonstrating alteration of SOD activities with varying phases of the cell cycle . This study suggests the possibility that increased MnSOD may correlate with decreased cell proliferation and suggests significant alterations in SOD activities during the cell cycle. Scand J Immunol, 1995 Jul, 42(1), 158 - 65 TNF inhibitors are produced spontaneously by rheumatoid and osteoarthritic synovial joint cell cultures: evidence of feedback control of TNF action; Brennan FM et al.; We have proposed the hypothesis that tumour necrosis factor alpha (TNF-alpha) has a pivotal role in the pathogenesis of rheumatoid arthritis, based on in vitro observations that in RA synovial joint cell cultures removal of TNF-alpha, inhibited other potentially pathogenic cytokines such as the equally proinflammatory cytokine interleukin 1 (IL-1) and the macrophage activating factor, GM-CSF . Here we describe that in both rheumatoid (RA) and osteoarthritic (OA) synovial cultures there is a homeostatic mechanism to regulate the activities of TNF-alpha . This concept is based on several observations . First in these synovial joint cell cultures the substantial discrepancy between the levels of bioactive and immunoreactive TNF-alpha indicates the presence of an inhibitor . Second, TNF binding proteins are produced spontaneously, which are the soluble variants of surface p75 and p55 TNF receptor . The amount of soluble TNF receptors (sTNF-R) produced varied between cultures; p75 sTNF-R was more abundant than p55 sTNF-R (as detected by ELISA), and both were produced at higher levels by RA synovial joint cells in culture, compared to OA cultures . These TNF binding proteins act as endogenous inhibitors of TNF-alpha, since blocking their activity in synovial joint cell culture supernatants with MoAb to p55 and p75 sTNF-R enhanced their cytotoxic activity in the TNF bioassay . The regulation of production of these sTNF-R in synovial joint cell cultures is important as the balance between TNF-alpha and sTNF-R production may determine the outcome of the inflammatory process. J Clin Endocrinol Metab, 1995 Jul, 80(7), 2077 - 81 Biochemical characteristics of human pituitary somatotropinomas with and without gsp mutations: in vitro cell culture studies; Adams EF et al.; Gsp oncogenes are present in about 40% of somatotropinomas . They result in excessive cAMP production and have been proposed to be the cause of increased GH secretion . We have used in vitro cell culture to compare the biochemical characteristics of somatotropinomas with and without gsp oncogenes (gsp-positive and gsp-negative tumors, respectively) . Of 30 somatotropinomas examined, 10 proved to be gsp positive, as determined by sequence analysis of DNA generated by the polymerase chain reaction . The somatostatin analog, octreotide, powerfully inhibited GH secretion by gsp-positive somatotropinomas, but had no effect on 8 of 13 gsp-negative tumors . Five of 20 gsp-negative and 4 of 10 gsp-positive tumors failed to respond to GHRH, whereas stimulatory effects ranging from 37-500% increases in GH secretion occurred in the remainder . However, strong stimulation (> 4-fold) occurred only in 5 of the gsp-negative tumors . The basal phosphatidylinositol turnover rate was elevated in about 25% of gsp-negative somatotropinomas . These results demonstrate similar and highly variable effects of GHRH on both types of somatotropinoma, whereas the absence of gsp oncogenes is often associated with resistance to octreotide . The phosphatidylinositol turnover data suggest that defects within this second messenger system may be present in a subset of somatotropinomas without gsp oncogenes. Clin Exp Metastasis, 1995 Jul, 13(4), 303 - 14 Differences in proliferation and invasion by normal, transformed and NF1 Schwann cell cultures are influenced by matrix metalloproteinase expression; Muir D; Loss of negative growth regulation and high invasive potential are neoplastic traits often associated with abnormal expression of matrix metalloproteinases (MMPs) . We previously found MMP-3 (stromelysin/transin) was secreted by quiescent rat Schwann cell cultures and expressed potent antiproliferative activity . In the present study we observed that human Schwann cells and cutaneous neurofibroma Schwann cell cultures secreted abundant MMP-3 and their proliferation was inhibited by autologous and rat Schwann cell conditioned media . Antiproliferative activities were depleted by immunoadsorption with anti-stromelysin antibodies . In contrast, plexiform neurofibroma cultures did not secrete MMP-3 and failed to respond to Schwann cell antiproliferative activities associated with MMP-3 . Quiescent Schwann cells constitutively secreted low levels of MMP-2 (gelatinase A) and showed a low invasion potential in filter-based assays of basement membrane invasion . Cyclic AMP elevation, which profoundly influences cell differentiation, increased the invasion potential of rat Schwann cells and caused a corresponding increase in secretion of MMP-2 . Schwann cells immortalized by protracted elevation of cAMP, as well as a schwannoma cell line (D6P2T), also rapidly invaded a reconstituted basement membrane and over-expressed MMP-2 . Similarly, neurofibroma Schwann cells were highly invasive and secreted up to 10-fold more MMP-2 than normal human Schwann cells . Additionally, only cutaneous neurofibroma Schwann cell cultures secreted MMP-9 (gelatinase B) and MMP-1 (interstitial collagenase) and also invaded native type I collagen barriers . Cultures of normal Schwann cells and plexiform neurofibroma tumor expressed little or no MMP-1 and did not invade type I collagen barriers . These results suggest a role for MMPs in the control of proliferation and invasion by Schwann cells and in the formation of peripheral nerve sheath tumors. Fundam Appl Toxicol, 1995 Jul, 26(2), 203 - 10 Evaluation of chick embryo neural retinal cell culture as a screen for developmental toxicants; Daston GP et al.; This paper describes a study to evaluate the concordance with in vivo results of an in vitro screen for developmental toxicants . The screen is a primary culture of chick embryo neural retina cells (CERC) which undergo processes of cell-cell recognition and interaction, growth, and differentiation over a 7-day culture period . Each of these developmentally significant events is measured separately as formation of multicellular aggregates, protein content, and glutamine synthetase activity, respectively . A total of 45 chemicals, 24 of which have been shown to be teratogenic at some dosage to mammalian embryos in utero, 7 of which are embryotoxic (but not teratogenic) in utero at high dosage, and 14 of which have not produced developmental toxicity in vivo, were evaluated in this assay by investigators who were blinded to the identity of the chemicals . Chemicals were tested up to concentrations that were frankly cytolethal, or up to a maximum of 5 mg/ml . Chemicals were present only during the first 24 hr of culture . The chemicals were selected to be representative of a variety of chemical classes (e.g., solvents, metals, food additives, anticonvulsants, antineoplastics) . In several cases, pairs of structurally similar compounds with different developmental toxic potencies (e.g., valproate and 2-en-valproate, formamide, and N,N-dimethylformamide) were tested . Of the 31 developmental toxicants, 25 affected at least one endpoint in the assay at concentrations which are achievable in vivo (i.e., below the systemic concentration at a lethal dose), yielding a false-negative rate of 19%.(ABSTRACT TRUNCATED AT 250 WORDS) J Recept Signal Transduct Res, 1995 Jul, 15(6), 801 - 9 Translocation of protein kinase C isozymes in rat pituitary lactotroph-enriched cell cultures by substance P: effects of sex and age; Mau SE et al.; It is generally accepted that the phospholipid and calcium-dependent enzyme protein kinase C (PKC) plays a significant role in secretion of hormones from anterior pituitary cells . The present study was undertaken to study age and sex-related changes in 1 . levels of immunoreactivity of PKC isozymes and 2 . distribution of immunoreactivity of PKC isozymes after stimulation with substance P (SP) in rat lactotroph-enriched cell cultures . The alpha, beta, delta and zeta isozymes were present in both sexes and at all ages . There was a sex-specific differential regulation of the different PKC isozymes as a function of sexual maturation . In male rats there was an up-regulation of the alpha isozyme throughout the sexual development, while the beta subtype showed a small, but significant decrease in immunoreactivity with increasing age . In female rats, on the other hand, the beta species was up-regulated with increasing age while the other subtypes remained constant . The concentration of the delta and zeta isozymes was unaffected of sex and age . Stimulation of lactotroph-enriched cell cultures with substance P (SP) resulted in translocation of the alpha and beta isozymes from the soluble to the particulate fraction while the delta and zeta species were left unchanged independently of age and sex . However, a decrease in responsiveness was observed in adult male rats, although a significant degree of translocation of alpha and beta species was still detected . On the basis of these results it is suggested that in lactotroph-enriched cell cultures basal levels of PKC subtype immunoreactivity and distribution of immunoreactivity of PKC isozymes after SP challenge might be regulated as a function of sex and age. Acta Otolaryngol, 1995 Jul, 115(4), 517 - 21 Laminin promotes differentiation, adhesion and proliferation of cell cultures derived from human acoustic nerve schwannoma; Baur AM et al.; The influence of laminin on cell cultures derived from unilateral acoustic nerve schwannomas was investigated . Cell cultures were initiated from 12 schwannomas, removed via the enlarged middle cranial fossa approach . Tumor tissue was dispersed by collagenase treatment and cells seeded in uncoated or laminin-coated culture dishes . Confluent cultures were immunocytochemically characterized with antibodies against S-100, CD 68, factor VIII-related antigen and type IV collagen . Cell adhesion in response to different doses of laminin was evaluated with an electronic cell counter . The effect of laminin on cell proliferation was assessed by measuring the incorporation of 5-bromo-2'-deoxy-uridine (BRDU) into cellular DNA . Cells cultured on laminin as substrate appeared more differentiated with long, fusiform, cytoplasmic processes . Cultured cells stained positive for S-100, not for factor VIII-related antigen or CD 68 . Only cells cultured on laminin deposited a dense extracellular network of type IV collagen . When laminin was added to the culture medium, cell attachment and proliferation was stimulated in a dose dependent manner . Maximal stimulation of both was observed with a laminin concentration of 50 micrograms/ml, which induced a nearly 2-fold increase in cell attachment and an approximately 66% increase in DNA content . Since laminin is a major component of the extracellular matrix in schwannomas, the possibility exists that laminin is also mitogenic for human neoplastic Schwann cells in situ. Diabetologia, 1995 Jul, 38(7), 764 - 71 Effects of tumour necrosis factor alpha (TNF alpha) on glucose transport and lipid metabolism of newly-differentiated human fat cells in cell culture; Hauner H et al.; Tumour necrosis factor alpha (TNF alpha) has been found to cause a delipidation of fat cells and a decrease of the adipose tissue mass . In the present study, we tried to elucidate some of the mechanisms responsible for this phenomenon by investigating the action of TNF alpha on specific pathways which are involved in lipid storage . Cultured