Cell cultures

J Bone Miner Res, 1996 Dec, 11(12), 1926 - 34
Lack of evidence for an increase in interleukin-6 expression in adult murine bone, bone marrow, and marrow stromal cell cultures after ovariectomy; Vargas SJ et al.; Interleukin-6 (IL-6) has been implicated as a mediator of postmenopausal bone loss . In vitro studies of bone and bone marrow cells have suggested that estrogen regulates bone turnover by controlling the production of IL-6, a potent stimulator of osteoclastogenesis and bone resorption . To investigate this hypothesis in an in vivo model, we examined the effect of ovariectomy or estrogen replacement on IL-6 mRNA and protein expression in adult mouse bone and bone marrow in vivo and in marrow stromal cell cultures . Eight-week-old CD-1 mice were sham-operated (SHAM), ovariectomized (OVX), or ovariectomized and subcutaneously implanted with 21-day slow-release pellets containing 10 micrograms of 17 beta-estradiol (O + E) . Placebo pellets were implanted in the SHAM and OVX mice . Uterine weights at 1, 2, or 3 weeks after surgery were significantly decreased (68-76%) in OVX animals compared with SHAM or O + E . In mice sacrificed at 1 or 3 weeks after surgery, we found by nonquantitative reverse transcribed polymerase chain reaction (RT-PCR), that SHAM, OVX, and O + E calvariae (CALV) constitutively expressed IL-6 mRNA . In contrast, IL-6 mRNA was either barely detectable or absent in the tibia (TIB) and bone marrow (BM) . In the mice sacrificed 3 weeks after surgery, we determined by quantitative RT-PCR that IL-6 mRNA in the CALV from the OVX and O + E groups were decreased by 81 and 92%, respectively, compared with SHAM . IL-6 protein levels in the flushed bone marrow (BMSups) were detectable and were not significantly different among the groups . In bone marrow cells that were cultured for 1 week, basal levels of IL-6 protein were low and did not differ significantly among the SHAM, OVX, or O + E groups sacrificed 1, 2, or 3 weeks after surgery . After the addition of hrIL-1 alpha, IL-6 protein levels increased 100- to 1300-fold over control . IL-6 levels in cells from animals sacrificed 2 weeks after surgery were significantly lower in the hrIL-1 alpha-stimulated OVX and O + E groups than in hrIL-1 alpha-stimulated SHAM cell cultures . In conclusion, in this model we could find no increase in IL-6 production with in vivo estrogen withdrawal in calvaria, long bones, bone marrow, or marrow stromal cell cultures . If increases in IL-6 expression are involved in the effects of estrogen withdrawal on bone, the magnitude of these changes are relatively small and below the limits of detection of the assays that we employed.

J Bone Miner Res, 1996 Dec, 11(12), 1889 - 96
Inositol trisphosphate receptor gene expression and hormonal regulation in osteoblast-like cell lines and primary osteoblastic cell cultures; Kirkwood KL et al.; The inositol trisphosphate receptor (IP3R) is an intracellular calcium channel that mediates the cellular actions of a wide variety of hormones, growth factors, and cytokines . In osteoblastic cell cultures, many bone resorbing hormones increase phosphoinositide turnover, inositol trisphosphate production, mobilization of intracellular calcium, and the secretion of osteoclast recruitment and activating factors . In this study, the effects of 17 beta-estradiol, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), phrobol ester, and serum on IP3R mRNA levels were evaluated in osteogenic-osteosarcoma cells and in primary osteoblastic cultures derived from neonatal rat calvaria . Type-specific reverse transcription polymerase chain reaction (RT-PCR) indicated that all cell types evaluated (G-292, U-2 OS, Saos-2, MC3T3-E1, UMR-106, and calvarial osteoblastic cells) express IP3R mRNA type I; G-292, U-2 OS, MC3T3-E1, and calvarial osteoblastic cells also express type II IP3R mRNA; and UMR-106 and the calvarial osteoblastic cells express type III IP3R mRNA . Northern blot and RT-PCR analyses of human G-292 osteosarcoma cells and rat calvarial osteoblastic cells showed that phorbol ester and serum increase IP3R mRNA levels, whereas 17 beta-estradiol and 1,25(OH)2D3 decrease these levels . In G-292 cells, the effect of 17 beta-estradiol was not due to accelerated IP3R mRNA degradation and required continued protein synthesis . The results show that multiple IP3R types are expressed in osteoblasts and osteoblastic osteosarcoma cells and that this expression is regulated by 17 beta-estradiol and other osteoporotic and antiosteoporotic hormones . These findings indicate that hormonal control of IP3R expression may be relevant in the chronic regulation of osteoblast secretory activity.

J Pharmacol Exp Ther, 1996 Dec, 279(3), 1520 - 6
Effects of silibinin and antioxidants on high glucose-induced alterations of fibronectin turnover in human mesangial cell cultures; Wenzel S et al.; To elucidate the primary mechanism of high glucose cytotoxicity, the cytoprotective properties of antioxidants against metabolical disorders were assessed in human mesangial cell (HMC) cultures . An 8-day incubation of HMC with high glucose concentration (30 mM) resulted in an extracellular accumulation of the matrixprotein fibronectin (FN), owing to both an expansion of the matrix-associated pericellular FN and a 60% increase of the soluble molecule in the culture medium . The high glucose-induced FN alterations were not due to osmotical effects, as assessed by an iso-osmotic mannitol control . Rather, they are mediated by oxygen-free radicals because the combined treatment of HMC with high glucose and either the antioxidative flavonoid silibinin (given as the water soluble derivative silibinin-C-2,3-dihydrogensuccinate disodium salt) or a radical scavenger cocktail totally prevented the extracellular FN accumulation . This is corroborated further by the determination of malondialdehyde, a product of lipid peroxidation . Incubation of HMC with high glucose resulted in an increase of malondialdehyde in cell homogenates which was completely counteracted by either silibinin or a radical scavenger cocktail . Silibinin alone had no effects on protein synthesis and culture growth . The data presented are compatible with oxidative stress induced by high glucose concentration in HMC cultures . The study further substantiates the proposed role of silibinin in the amelioration of glucose cytotoxicity in renal cells.

J Pharmacol Exp Ther, 1996 Dec, 279(3), 1268 - 73
The role of interferon-gamma in antiretroviral activity of methionine enkephalin and AZT in a murine cell culture; Sin JI et al.; The ability of spleen cells treated with methionine enkephalin (Met-ENK) in the presence of 3'-azido-3'-deoxythymidine (AZT) to produce cytokines and inhibit Friend leukemia virus (FLV) replication in Mus dunni cell cultures was investigated . In the presence of murine spleen cells, combination treatments using AZT plus Met-ENK or concanavalin A reduced FLV replication by 63% and 84%, respectively, as compared with 47% for AZT alone . When interleukin (IL)-2, IL-4 and interferon (IFN gamma) levels were measured in FLV-infected cell cultures, both AZT and Met-ENK treatments induced a higher production of IFN gamma and a slight increase in IL-2 and IL-4, as compared with either treatment alone . Subsequent treatment of FLV-infected cells with concanavalin A-stimulated cell supernatants, containing approximately 10 U/ml each of IFN gamma and IL-2, resulted in inhibition of viral replication . Thus, in the absence of spleen cells, IFN gamma was added to cell cultures to determine whether this cytokine contributed to combination antiviral effects . Results show that addition of IFN gamma alone results in a slight suppression of FLV expression, whereas treatment with both AZT and IFN gamma inhibits FLV replication significantly . Subsequently, addition of anti-IFN gamma antibody to cell cultures treated with Met-ENK blocked antiviral effects due to this neuropeptide . Thus anti-FLV effects of spleen cells treated with Met-ENK in combination with AZT are mediated to a large degree by IFN gamma.

Antiviral Res, 1996 Dec, 33(1), 21 - 31
Inhibition of bovine immunodeficiency virus by anti-HIV-1 compounds in a cell culture-based assay; Tobin GJ et al.; The bovine immunodeficiency virus (BIV) and human immunodeficiency virus types 1 and 2 (HIV-1 and -2) are members of the lentivirus genus of retroviruses . Although DNA sequences of these viruses have diverged considerably, the BIV genome organization, function of structural and regulatory genes, and replication cycle are very similar to that of HIV-1, making BIV a potentially useful model to study compounds with anti-HIV-1 activity . A cell culture-based antiviral assay was developed to test compounds for inhibition of BIV replication . The assay uses an embryonic rabbit epithelial (EREp) cell line that is highly sensitive to BIV infection and cytopathology . The 50% effective concentrations (EC50) at which the virus was inhibited in EREp cells were determined for 13 nucleoside analog, non-nucleoside, tumor-suppressive, or membrane-surface inhibitory compounds . The nucleoside analogs (3'-azido-2',3'-dideoxythymidine, 2',3'-dideoxyinosine and 2',3'-dideoxycytosine), surface-membrane inhibitors (dextran sulfate, hypericin, Chicago Sky Blue and quinobene), the nucleoside reductase inhibitor (hydroxyurea), and a tumor-suppressive phorbol ester (prostratin) inhibited BIV with EC50 values similar to those derived in HIV-1 lymphocyte (CD4+)-based assays . BIV was markedly more resistant to inhibition with HIV-1-specific non-nucleoside reverse transcriptase inhibitors (NNRTIs) (thiazolobenzimidazole, oxathiin carboxanilide and thiocarbamate) than was HIV-1, which parallels results with NNRTIs in HIV-2 assays.

J Cell Physiol, 1996 Dec, 169(3), 411 - 9
Effects of cell culture time and bone matrix exposure on calmodulin content and ATP-dependent cell membrane acid transport in avian osteoclasts and macrophages; Williams JP et al.; Osteoclasts mediate bone resorption by secretion at the site of bone attachment . This process depends on calmodulin concentrated at a specialized acid-secreting membrane . We hypothesized that increased calmodulin and bone attachment were required for acid secretion . We tested this by studying calmodulin, bone attachment, and membrane acid transport in osteoclasts and their precursor mononuclear cells . Osteoclasts and macrophages were isolated from medullary bone of hens; cell fractions were prepared after culturing cells with or without bone . Calmodulin was visualized by Western analysis; calmodulin mRNA was determined by Northern hybridization, and ATP-dependent membrane acid transport was assayed by acridine orange uptake . Calmodulin decreased in osteoclasts cultured without bone . Calmodulin in isolated macrophages was approximately 25% of osteoclast levels, but increased several fold by 5 days . Bone had no effect . Calmodulin mRNA was similar in osteoclasts with or without bone . However, only osteoclasts cultured with bone retained acid transport capacity . Macrophage calmodulin mRNA was not affected by bone, but increased three fold by day 5, paralleling protein production . Macrophages developed acid transport capacity at 3-5 days, but at lower levels than osteoclasts, and bone had no measurable effect . Chicken cells express 1.6 kb and inducible 1.9 kb calmodulin transcripts; in macrophages and osteoclasts, the 1.9 kb transcript predominated . We conclude that, following isolation, calmodulin levels decline in osteoclasts via a post-transcriptional mechanism . In cultured macrophages, by contrast, calmodulin mRNA, protein, and acid secretion increase with time independently of bone substrate, possibly reflecting differentiation in vitro . Increased calmodulin correlated with membrane acid transport capacity in both cell types . The macrophage findings indicate that stimuli other than bone influence acid transport capacity in this family of cells.

J Infect Dis, 1996 Dec, 174(6), 1324 - 7
Contamination of commercially available fetal bovine sera with bovine viral diarrhea virus genomes: implications for the study of hepatitis C virus in cell cultures; Yanagi M et al.; The establishment of cell cultures for hepatitis C virus (HCV) is important for its study as a human pathogen . However, in reported cell lines, HCV demonstrates low levels of replication detected primarily by reverse transcription-polymerase chain reaction (RT-PCR) assays . In attempts to culture HCV, an additional complication was observed . From mock-infected cultures, cDNA of appropriate size was obtained by RT-PCR with primers deduced from conserved domains of the 5' noncoding region of HCV . However, sequence analysis revealed that the cDNA was amplified from bovine viral diarrhea virus (BVDV) . All of 7 bovine sera tested were contaminated with BVDV . In conclusion, most commercially available bovine sera are contaminated with BVDV and, although there is no evidence that the virus is infectious, bovine sera should be screened for this virus by RT-PCR when used in conjunction with HCV or for the development or production of vaccine.

J Urol, 1996 Dec, 156(6), 2067 - 72
Ureteral cell cultures II: Collagen production and response to pharmacologic agents; Wolf JS Jr et al.; PURPOSE: To investigate the in vitro response of ureteral cells to potentially anti-fibrotic agents . MATERIALS AND METHODS: Cultured human uroepithelial cells, smooth muscle cells, and myofibroblasts were assayed for proliferation and production of collagen types I and III, with and without the presence of hydrocortisone, colchicine, retinol, verapamil, and D-penicillamine . RESULTS: Hydrocortisone stimulated the proliferation of all three cell types and reduced the type I and type III collagen production by myofibroblasts and smooth muscle cells, respectively . Verapamil enhanced the growth of uroepithelial cells and decreased collagen III production by both uroepithelial and smooth muscle cells . D-penicillamine increased the proliferation of uroepithelial and smooth muscle cells, and inhibited collagen type III production by all three cell types . CONCLUSIONS: In vitro evidence suggests that hydrocortisone, verapamil, and D-penicillamine have effects that could favorably alter the healing of endoscopic ureteral incisions.

J Biol Chem, 1996 Nov 22, 271(47), 29612 - 8
The bZIP transcription factor Nrl stimulates rhodopsin promoter activity in primary retinal cell cultures; Kumar R et al.; In vitro DNA binding assays and transient transfection analysis with monkey kidney cells have implicated Nrl, a member of the Maf-Nrl subfamily of bZIP transcription factors, and the Nrl response element (NRE) in the regulation of rhodopsin expression . We have now further explored the role of the NRE and surrounding promoter elements . Using the yeast one-hybrid screen with integrated NRE and flanking DNA as bait, the predominant clone obtained was bovine Nrl . Recovery of truncated clones in the screen demonstrated that the carboxyl-terminal half of Nrl, which contains the basic and leucine zipper domains, is sufficient for DNA binding . To functionally dissect the rhodopsin promoter, transient expression studies with primary chick retinal cell cultures were performed . Deletion and mutation analyses identified two positive regulatory sequences: one between -40 and -84 base pairs (bp) and another between -84 and -130 bp . Activity of the -40 to -84 region was shown to be largely due to the NRE . On co-transfection with an NRL expression vector, there were 3-5-fold increases in the activity of rhodopsin promoter constructs containing an intact NRE but little or no effect with rhodopsin promoters containing a mutated or deleted NRE . Nrl was more effective than the related bZIP proteins, c-Fos and c-Jun, in stimulating rhodopsin promoter activity . The -84- to -130-bp region acted synergistically with the NRE to enhance both the level of basal expression and the degree of Nrl-mediated trans-activation . These studies support Nrl as a regulator of rhodopsin expression in vivo, identify an additional regulatory region just upstream of the NRE, and demonstrate the utility of primary retinal cell cultures for characterizing both the cis-acting response elements and trans-acting factors that regulate photoreceptor gene expression.

J Clin Invest, 1996 Nov 15, 98(10), 2346 - 50
Human primary myoblast cell cultures from non-diabetic insulin resistant subjects retain defects in insulin action; Thompson DB et al.; Insulin resistance is a predictor of the development of noninsulin-dependent diabetes mellitus (NIDDM) in humans . It is unclear whether insulin resistance is a primary defect leading to NIDDM or the result of hyperinsulinemia and hyperglycemia . To determine if insulin resistance is the result of extrinsic factors such as hyperinsulinemia primary skeletal muscle cell cultures were established from muscle biopsies from Pima Indians with differing in vivo insulin sensitivities . These cell cultures expressed a variety of muscle-specific phenotypes including the proteins alpha-actinin and myosin, muscle-specific creatine kinase activity, and RNA encoding GLUT4, MYF5, MYOD1, and MYOGENIN . Labeled glucose was used to measure the insulin-stimulated conversion of glucose to glycogen in these cultures . The in vivo rates of insulin-stimulated glycogen production (insulin resistance) were correlated with in vitro measures of glycogen production (P = 0.007, r = 0.58) . This defect in insulin action is stable in a uniform culture environment and is retained over time . The retention of insulin resistance in myoblast derived cell cultures is consistent with the expression of an underlying biochemical defect in insulin resistant skeletal muscle.

Indian J Exp Biol, 1996 Nov, 34(11), 1169 - 71
Direct detection of bovine herpes virus-1 DNA from cell culture fluids using polymerase chain reaction; Sreenivasa BP et al.; A pair of oligomers of 20 and 23 bp were designed for amplifying a 381 bp sequence from glycoprotein IV gene of bovine herpesvirus 1 . The primer pairs were used for amplifying genomic DNA of BHV-1 directly from cell culture fluids under different experimental conditions such as, untreated cell culture fluid, thermal denaturation and proteinase K treatment in presence of detergent . The results reveal that direct thermal denaturation of cell culture fluid is sufficient to detect the virus by polymerase chain reaction.

Biofizika, 1996 Nov-Dec, 41(6), 1284 - 8
{The use of 1H-NMR spectroscopy for the study of cell cultures}; Iurkevich DI et al.; Production of lactate by the HSR-1, HSR-8, HET-SR fibroblasts have been investigated by 1H-NMR method . Were investigated both monolayer cell cultures and cells immobilized in collagen lattice . Represented data demonstrate the possibility of the NMR-spectroscopy to investigate growth's processes in the cell cultures.

Int J Dev Neurosci, 1996 Nov, 14(7-8), 823 - 39
Characterization of olfactory receptor neurons and other cell types in dissociated rat olfactory cell cultures; Pixley SK; In dissociated cell cultures, control over the cellular environment facilitates study of the differentiation of mature cellular phenotypes . Central to this approach is a rigorous characterization of the cells that reside in culture . Therefore, we have used a battery of cell type-specific antibody markers to identify the cell types present in dissociated cultures of olfactory mucosal cells (containing cells from both the epithelium and lamina propria) . To identify olfactory receptor neurons in the cultures, staining with antibodies against neuron-specific tubulin was compared to staining with antibodies to neuron-specific enolase, the neural cell adhesion molecule, N-CAM, and the adhesion molecule, LI . Staining of mature olfactory neurons in culture, with an antibody against the olfactory marker protein, was compared to staining with antibodies to carnosine . In contrast to tissue section staining, the overlap between carnosine and olfactory marker protein staining was not complete . Olfactory nerve glial cells were immunoreactive for the S100 beta protein and nestin, an intermediate filament found in early neuronal progenitor cells and Schwann cells . Antibodies to nestin did not label olfactory neurons or progenitor cells . An antibody to an oligodendrocyte-Schwann cell enzyme, 2',3'-cyclic nucleotide 3'-phosphodiesterase, did not label olfactory glia, but did label oligodendrocyte-like cells that appeared to be derived from the CNS glial feeder layer . An antibody against the heavy (200 kDa) neurofilament protein stained a minor subset of cells . The cultures also contained muscle cells, cartilage cells and macrophages (and/or microglia) . These results demonstrate that multiple cell types either maintain or re-establish differentiated, cell type-specific phenotypes in dissociated olfactory cell cultures.

J Math Biol, 1996 Nov, 35(1), 97 - 113
Modeling tendon morphogenesis in vivo based on cell density signaling in cell culture; Schwarz RI; A mathematical model of tendon morphogenesis is presented that is consistent with the dramatic transitions seen in this tissue as it progresses from rapid growth early in development to no growth in the adult . To accomplish this change, the embryonic chick tendon is hypercellular with each cell dedicating half of its protein production to procollagen but over time, as growth subsides, the tissue gradually becomes hypocellular with each cell producing only about 1% procollagen . Making this transition from the embryonic to the adult state, forming a roughly cylindrical tissue composed of approximately 90% collagen, and linking the correct muscle to the right bone, is a complex task . The proposed solution requires only two factors: an activator of growth and an inhibitor complex, composed of the activator and another molecule that modifies the activity of the activator . From a diverse set of cell culture observations, these two factors were deduced as the primary components of the mechanism that allows cells to signal their presence to their neighbors . Since cell density signaling is the principal regulator of both collagen synthesis and cell proliferation, its components should play the key role in tendon development . A mathematical model based on the changes in the concentrations of these factors with cell density correlates well with the transitions observed in vivo . Furthermore, the model predicts that in the maturing chicken there should be a high cell density region at the muscle tendon interface . Experimental observations of frozen sections of tendon from a 4 month old chicken confirm this prediction.

Acta Radiol, 1996 Nov, 37(6), 923 - 6
Toxicity of ethanol in low concentrations . Experimental evaluation in cell culture; Tapani E et al.; PURPOSE: To define the threshold ethanol concentration that is toxic to cultured cells . METHODS: Three malignant cell lines and freshly isolated normal rat hepatocytes were exposed to 0-50% (vol.) ethanol (concentrations used were 0, 5, 10, 15, 20, 25, 30, 40 and 50%) on tissue culture plates for 0.25-60 min (exposure times used were 0.25, 5, 10, 20, 30, 40 50 and 60 min) . Cytotoxicity was estimated by trypan blue exclusion test and from 3H-thymidine incorporation . RESULTS: All cells were killed by a 15-s exposure to 30-40% ethanol while a concentration as low as 15-20% gave a total response after 5-10-min exposures . After a one-hour exposure of F9 carcinoma cells and hepatocytes, a total or nearly total response was achieved with 10% ethanol . The cytotoxic effect was thus dependent both on the exposure time and on the concentration of ethanol . There were no significant differences in ethanol tolerance among the cell types . CONCLUSION: Ethanol seemed to kill cells in the cell culture effectively in much lower concentrations than those currently used in tumour ablation.

Trop Anim Health Prod, 1996 Nov, 28(4), 289 - 92
Theileria annulata schizonts found in calf kidney cell culture; Abd el Raouf Y et al.; Mononuclear cells infected with Theileria annulata schizonts were isolated at 4 different times from monolayer cultures of primary kidney cells derived from apparently healthy, 2 to 6 weeks old zebu calves . The presence of such an organism could interfere with the manufacturing procedure of rinderpest tissue culture vaccine if it is to be carried out in calf kidney cell culture.

Acta Otolaryngol, 1996 Nov, 116(6), 805 - 11
Establishment of primary cell culture from stria vascularis explants . Morphological and functional characterization; Kim HN et al.; To provide the prerequisite for long-term study of the inner ear related to structural and functional integrity, tissue of stria vascularis with spiral ligament was isolated from Wistar rat cochleas and cultured using the explant-culture technique . The following culture media were used: EMEM with Hepes buffer, hydrocortisone (400 ng/ml), transferrin (5 micrograms/ml) . triiodothyronine (10(-9) M), cholera toxin (10(-10) M), insulin (5 micrograms/ml), and epidermal growth factor (10 ng/ml) . To characterize the cells growing out from the explant, immunofluorescence with cytokeratin (cytokeratin 18) and ultrastructural examination with SEM and TEM were performed . The marginal cell function was investigated by expression of Na+, K(+)-ATPase antisera against beta 2 subunit of rat Na+, K(+)-ATPase and P-NPPase . We were able to maintain the cultured cells for 3 weeks or more . Monolayered marginal cells were observed beyond 14 days in vitro and the expression of cytokeratin 18 was especially enhanced . The cultured marginal cells were almost identical to in vivo cells both as regards ultrastructural features and Na+, K(+)-ATPase activity . The present results suggest that the primary explant culture technique is a reliable in vitro model of strial marginal cells . However, establishment of the cell line is needed for long-term study.

Ren Fail, 1996 Nov, 18(6), 867 - 82
Cytotoxicity, zinc protection, and stress protein induction in rat proximal tubule cells exposed to cadmium chloride in primary cell culture; Liu J et al.; Primary cell culture was utilized to study the relationships between stress protein induction by zinc in vivo and cadmium toxicity in vitro . Effects of cadmium on cell viability were evaluated by the alamar blue assay, in conjunction with the ultrastructural morphology of cells by transmission electron microscopy . The expression of stress protein gene products was evaluated by 35S two-dimensional gel electrophoresis . The results showed cytotoxicity of CdCl2 at and above 129 microM (14.55 micrograms cadmium/mL medium) following 4 h of exposure . Prior zinc administration (20 mg zinc/kg, s.c., two daily doses) in vivo significantly protected the cells in vitro as demonstrated by improved cell viability . The 35S labeling of proteins induced by CdCl2 exposure clearly demonstrated for the first time that gene product of the 70-kDa family was induced in cultured rat proximal tubule cells which are the target cells for cadmium toxicity in vivo . Zinc in vivo pretreatment of animals induced proteins in the 90-, 70-, and 38-kDa families, which may act together with metallothionein to protect cells against cadmium toxicity . The results also indicate that the protective effect of zinc remains after the cells have been put in culture and thus provides a system in which we can study the changes that occur as a result of zinc exposure that decreases cadmium toxicity.

Am J Clin Pathol, 1996 Nov, 106(5), 634 - 9
Quality control of proliferation marker (MIB-1) in image analysis systems utilizing cell culture-based control materials; Ruby SG et al.; Standard controls for quality control of cell growth (proliferation) assays in image analysis systems are not currently available . The authors have developed a system of controls, based on cultured and harvested human cell lines, that can mimic tissue sections . These controls help ensure quality control for the entire cell proliferation analysis process, from the initial cutting of the paraffin block through fixation, immunohistochemical staining, and interactive image analysis . The use of cell line controls is advantageous because of the greater cell population homogeneity and the volume of uniform slides that are obtainable, as opposed to the use of a heterogeneous tissue sample . This system provides an excellent means of evaluating the day-to-day performance of cell proliferation analysis and may also be adapted for use as a method of multi-institutional proficiency testing.

Toxicol Lett, 1996 Nov, 88(1-3), 35 - 7
Tumor necrosis factor alpha stimulates arachidonic acid metabolisms and mucus production in rat tracheal epithelial cell cultures; Nettesheim P et al.; Air-liquid interface (ALI) cultures of rat tracheal epithelial (RTE) cells were used to study the response of differentiated airway epithelial cells to the inflammatory cytokine, tumor necrosis factor alpha (TNF alpha) . We found that the cultures expressed low levels of TNF alpha receptors . TNF alpha stimulated the AA-cascade: cytoplasmic phospholipase A2 (cPLA2) and prostaglandin H synthase 2 (PGHS2) were upregulated; as a result prostaglandin E (PGE2) secretion was increased . Subsequent to the increase in PGE2 mucus secretion increased, suggesting that PGE2 may act as an autocrine regulator of mucus secretion.

J Bone Miner Res, 1996 Nov, 11(11), 1694 - 702
The mechanism of beta-glycerophosphate action in mineralizing chick limb-bud mesenchymal cell cultures; Boskey AL et al.; Differentiating chick limb-bud mesenchymal cells plated in micromass culture form a cartilage matrix that can be mineralized in the presence of 4 mM inorganic phosphate (Pi), and 1 mM calcium . Previous studies showed that when beta-glycerophosphate (beta GP) is used in place of Pi, the mineral crystals formed are larger and differ in distribution . The present study shows that the difference in distribution is not associated with alterations in cell proliferation, protein synthesis, or with collagen, proteoglycan core protein, or alkaline phosphatase gene expression . Cultures with 2.5, 5, and 10 mM beta GP did show different levels of alkaline phosphatase activity, and in the presence of low (0.3 mM) Ca had different Pi contents (4, 6 and 9 mM, respectively), indicating that the increase in CaxP product may in part be responsible for the altered pattern of mineralization . However, cultures with beta GP in which alkaline phosphatase activity was inhibited with levamisole still had an altered mineral distribution as revealed by Fourier transform-infrared (FT-IR) microspectroscopy . The presence of a casein kinase II-like activity in the mineralizing cultures, the ability of specific inhibitors of this enzyme to block mineralization, and the known ability of beta GP to block phosphoprotein phosphatase activity suggests that altered patterns of matrix protein phosphorylation may influence mineral deposition in these cultures.

Glia, 1996 Nov, 18(3), 211 - 23
Aged median eminence glial cell cultures promote survival and neurite outgrowth of cocultured neurons; Chauvet N et al.; We have recently shown that tanycytes, a particular type of glial cell that has morphological and biochemical similarities with radial glial cells, constitute a preferential support for the regeneration of lesioned neurohypophysial axons . The present study was designed to explore the possible neurotrophic role of tanycytes in vitro . Glial cells derived from the median eminence or from the cerebral cortex of 10-day-old rats were cultured for 4-7 weeks . At these times the majority of the cells identified in the median eminence cultures exhibited immunostaining patterns of tanycytes, as detected in the mediobasal hypothalamus of 10-day-old and adult rats, i.e., they were immunoreactive to vimentin (VIM), to DARPP-32 (a dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein), and to a lesser extent to glial fibrillary acidic protein (GFAP) antibodies . On the other hand, the majority of cells in cortex cultures showed immunostaining patterns of astrocytes, i.e., they were intensely immunoreactive to GFAP and VIM antibodies but negative to DARPP-32 . Cells obtained from the dissociation of 3-day-old rat mesencephalon, cortex, and hypothalamus were cocultured on these glial monolayers, and the number of surviving neurons and their neurite length were quantified after 8 days . Our data showed that, when compared with astrocytes, tanycytes greatly improved both survival (six-to ten-fold higher) and neurite outgrowth (two- to five-fold longer) of cocultured neurons whatever their origin . Experiments performed by coculturing neurons on millicell inserts placed above the glial monolayers showed that diffusible factors from median eminence glial cells slightly increased survival (1.7-fold higher) of cocultured neurons but had no significant effect on neurite outgrowth . These observations indicate: 1) that aged tanycytes have a capacity to support survival and neurite outgrowth for a variety of postnatal neurons; and 2) that this neurotrophic effect is exerted mainly by means of specific molecules bound to the tanycytic plasmalemma limiting membrane and/or to the extracellular matrix.

Am J Vet Res, 1996 Nov, 57(11), 1594 - 8
Attempted transmission of Ehrlichia canis by Rhipicephalus sanguineus after passage in cell culture; Mathew JS et al.; OBJECTIVES: To compare the transmissibility by the brown dog tick, Rhipicephalus sanguineus, of a recent isolate of Ehrlichia canis (Ebony) with that of another isolate (Oklahoma) that had been passaged in cell culture, and to assess the genetic similarity of the 2 isolates as reflected in the nucleotide (NT) sequence of 16S rDNA . ANIMALS: 13 healthy dogs of various ages and breeds . PROCEDURE: Larval and nymphal ticks were acquisition fed on acutely infected dogs, and, after molting, they were transmission fed as nymphs and adults, respectively, on Ehrlichia-naive dogs . All dogs were monitored daily by blood smear evaluation for evidence of parasitized leukocytes and by physical examination for clinical signs of ehrlichiosis . Serologic and hematologic values were measured weekly . Using a nested polymerase chain reaction, the 16S rDNA was amplified, and the NT sequence of the template DNA was determined . RESULTS: The Ebony isolate of E canis was successfully transmitted to dogs by nymphal and adult ticks . In contrast, no ticks that fed on dogs harboring the cell-cultured isolate (Oklahoma) transmitted it to dogs . On the basis of 16S rDNA sequence, the 2 isolates were 99.9% similar, with only 1 NT difference . CONCLUSIONS: These results reconfirm the vector potential of R sanguineus for E canis . Passage of the Oklahoma isolate of E canis in cell culture apparently adversely affected its transmissibility by ticks, raising the possibility that cell-cultured isolates of this rickettsia may lose their affinity for ticks . Determination of 16S rDNA sequence suggests minor strain variation within the species E canis.

Arch Dermatol, 1996 Nov, 132(11), 1323 - 9
Enhanced interaction of patients' lymphocytes with human dermal microvascular endothelial cell cultures in active Adamantiades-Behçet disease; Treudler R et al.; BACKGROUND AND DESIGN: To elucidate the role of lymphocyte/endothelial cell interactions in patients with Adamantiades-Behcet disease (ABD), we studied 16 patients of German and Turkish nationality (aged 18-57 years), all with active ABD, and 12 healthy volunteers (controls) of similar age and nationality . Peripheral blood lymphocytes (PBL) of patients were coincubated with human dermal microvascular endothelial cells (HDMEC) and human keratinocytes (HK) in vitro; interactions of PBL with HDMEC and HK were investigated using an established fluorometric assay . Interactions of patients' PBL with HDMEC, HK, or both were the main outcome measures . RESULTS: A significant increase of fluorescence with increasing PBL/HDMEC ratios was seen in patients and controls (P < .001); patients showed a significantly higher increase of fluorescence at higher PBL/HDMEC ratios (P < .05) . The PBL/HK coincubation did not show significant alterations compared with the basal fluorescence signals of HK monolayers alone . Peripheral blood lymphocyte and HDMEC fluorescence values that were more than 2 SDs of controls (defined as positive result of assay) were found in a significantly higher number of patients with 2 or more active symptoms at the time of investigation (83%) compared with patients with only 1 active symptom (10%) (P = .008) . Other clinical data did not correlate with the results of the PBL/HDMEC coincubation assay . CONCLUSIONS: Our results indicate enhanced in vitro interaction of PBL from patients with ABD with HDMEC, which was additionally shown to be a marker of the activity of the disease.

J Nutr, 1996 Nov, 126(11), 2831 - 42
7S globulin from soybean is metabolized in human cell cultures by a specific uptake and degradation system; Lovati MR et al.; We examined the biological fate of 7S globulin from soybean in a hepatoma cell line (Hep G2) and in human skin fibroblasts (HSF) to gain new insights into the 7S globulin cell process, the final effect of which is an enhanced expression of the LDL-receptor . The ability of 7S globulin to bind and to be internalized and degraded by both cell types was investigated under different experimental conditions . In all cases, specific uptake (binding + internalization) and degradation of 125I-7S globulin were curvilinear functions of substrate concentration at 37 degrees C . The two processes were saturated at around 80 mg/L, a concentration at which an up-regulation of LDL-receptor was previously reported . The specific uptake of 125I-7S globulin at 37 degrees C was a curvilinear function of time, and achieved equilibrium after 6 and 12 h in HSF and Hep G2 cells, respectively . Binding experiments, conducted at 4 degrees C in Hep G2 cells, showed a specific and saturable association of 7S globulin to the cell membrane . Linear Scatchard analysis demonstrated a single population of binding sites . The amount of 7S globulin bound at saturation (Bmax) was about 2.73 mg/L, with an apparent Kd of 21 micromol/L, assuming 175 kDa as the 7S globulin molecular weight . SDS-PAGE of Hep G2 membrane proteins incubated with 125I-7S globulin revealed a specific interaction of 7S globulin with a cell protein component with molecular weight between 14 and 21 kDa . Further studies are needed to ascertain whether this interaction is directly or indirectly related to the observed stimulation of the LDL-receptor.

J Cell Physiol, 1996 Nov, 169(2), 269 - 80
Differential growth factor responses of epithelial cell cultures derived from normal human prostate, benign prostatic hyperplasia, and primary prostate carcinoma; Chopra DP et al.; Because of a lack of information of the optimum nutritional requirements, epithelial cells derived from normal human prostate and prostate tumors have been difficult to propagate in vitro, which hinders research in prostate carcinogenesis . In an effort to establish optimum nutritional conditions and differences in growth characteristics of normal human prostate (NP), benign prostatic hyperplasia (BPH), and prostatic carcinoma (PCA), we have compared the effects of several growth factors on cell proliferation and elucidated growth properties of low passage epithelial cells derived from NP, BPH, and PCA of an African-American patient . Primary and low passage cultures were propagated in serum-free keratinocyte basal medium (KBM) supplemented with insulin (5 micrograms/ml), hydrocortisone (0.5 microgram/ml), epidermal growth factor (EGF, 10 ng/ml), bovine pituitary extract (BPE; 50 micrograms/ml), cholera toxin (10 ng/ml), and antibiotics . Almost all NP, BPH, and PCA cells were positive for cytokeratins and prostate-specific antigen (PSA) . The NP, BPH, and PCA cells were essentially diploid and lacked mutations in c-K-ras and c-Ha-ras oncogenes, and p53 tumor suppressor gene . However, they exhibited progressively accelerating growth parameters . The population doubling times of NP, BPH and PCA were 51 hr, 37 hr, and 29 hr, respectively; their saturation densities were 2.9 x 10(4)/cm2, 3.3 x 10(4)/cm2, and 7.2 x 10(4)/cm2, respectively . The NP and BPH cells required all of the growth factors in the medium, as deletion of any one of the above factors strongly inhibited their growth . The PCA cells, however, were independent of EGF and hydrocortisone . PC-3, an established human prostate cancer cell line, was independent of the growth factors tested . Fetal bovine serum (FBS) inhibited the growth of NP, BPH and PCA cells . In contrast, FBS stimulated the growth of the PC-3 cells in a concentration-dependent manner . These results indicate that in the absence of any apparent karyotype alterations and mutations in c-K-ras, c-Ha-ras and p53 genes, epithelial cells derived from NP, BPH, and PCA exhibit significant differences in their growth properties and responses to growth factors . These variations may represent early changes involved in prostate cancer, while gene mutations and cytogenetic alterations occur in advanced and/or metastatic tumors.

Br J Haematol, 1996 Nov, 95(2), 354 - 63
A role for paclitaxel in the combination chemotherapy of acute myeloblastic leukaemia: preclinical cell culture studies; Curtis JE et al.; Paclitaxel dose responses in culture have been investigated alone and in association with cytosine arabinoside (ARA-C) and all-trans retinoic acid (ATRA), with the objective of identifying a role for paclitaxel in the treatment of acute myeloblastic leukaemia (AML) . Initial studies were done to determine if paclitaxel dose responses of AML blast cell precursors were altered by regulatory compounds known to modify the dose responses of ARA-C . In contrast to ARA-C, paclitaxel dose responses were independent of cell culture method, the growth factors G-CSF and GM-CSF, and the ligands all-trans retinoic acid (ATRA) and hydrocortisone . Most blast cell populations were sensitive to paclitaxel; compared with normal marrow progenitors the dose responses were markedly heterogenous with some more, and others less, sensitive . Remission marrow progenitor paclitaxel responses resembled those of AML blasts in heterogeneity . The cell culture model tested the effect of pacliataxel and ATRA on the ARA-C dose responses of OCI/ AML-5; paclitaxel exposure was either before or after ARA-C to test for an effect of schedule; ATRA was added to the MEC cultures after paclitaxel and ARA-C . Repeat experiments were done to test three dose levels each of paclitaxel and ATRA . When paclitaxel was given after ARA-C, synergism was found for all but one of the dose combinations tested; only three examples of synergy were seen when paclitaxel preceded ARA-C . The studies justify trials combining ARA-C, paclitaxel and ATRA using a schedule suggested by the cell culture findings.

J Neuroimmunol, 1996 Nov, 70(2), 123 - 9
Reduction of the microglial cell number in rat primary glial cell cultures by exogenous addition of dibutyryl cyclic adenosine monophosphate; Dalmau I et al.; The present work examined the effects induced by dibutyryl cyclic adenosine monophosphate (dB-cAMP) on microglial cells in primary glial cell cultures from newborn rats . Microglial cells were identified by OX42 immunohistochemistry and nucleoside diphosphatase histochemistry . Double staining for astrocytes was carried out by combination with glial fibrillary acidic protein immunolabeling . Addition of 0.25 mM dB-cAMP to the cultures decreased the microglial cell number about sixfold . The findings suggest that the effect of dB-cAMP on the microglial cells might be either a direct action of dB-cAMP on the microglial cells or an indirect effect mediated by the astroglial cells.

J Biomed Mater Res, 1996 Nov, 32(3), 333 - 40
In vitro bone formation by rat marrow cell culture; Ohgushi H et al.; Fresh marrow cells were obtained from the femora Fischer rats and cultured in a medium containing 15% fetal calf serum (FCS) to leach confluent . After trypsinization, cells were subcultured at a cell density of 100 x 10(3)/35 mm well in the presence of FCS, 10 mM beta-glycerophosphate, 82 micrograms/mL ascorbic acid phosphate, and 10(-8)M dexamethasone (Dex) . Osteoblastic cells and microscopic mineralized nodules began to appear at about 1 week after the subculture, and at 2 weeks many macroscopic nodules that showed high alkaline phosphatase activity (ALP) and appearance of bone Gla protein (BGP) mRNA were evident . As demonstrated by in situ hybridization, the mRNA was manifested by cuboid-shaped cells (osteoblastic cells) . X-ray diffraction (XRD) and Fourier-transform infrared spectroscopy (FTIR) showed the mineralization of fine crystals of hydroxyapatite comparable to natural rat bone mineral . In contrast to these findings, subculture done under the same conditions except for the lack of Dex did not show mineralized nodules, nor did they show the osteoblastic phenotype expression . These analyses indicate that Dex-induced mineralization using rat bone marrow cell culture is an in vitro counterpart of bone formed in vivo . Such a culture is useful for investigating materials/ osteogenic cells interactions.

Endocrinology, 1996 Nov, 137(11), 4665 - 70
Autocrine down-regulation of collagenase-3 in rat bone cell cultures by insulin-like growth factors; Delany AM et al.; Insulin-like growth factors (IGF)-I and -II are presumed to act as autocrine regulators of bone formation . Recently, we demonstrated that IGF-I and -II inhibit bone collagen degradation and collagenase-3 synthesis in osteoblast cultures . Therefore, we tested the autocrine role of IGFs in the endogenous expression of collagenase-3 in cultures of osteoblast-enriched cells from 22-day fetal rat calvariae (Ob cells) . Steady-state messenger RNA (mRNA) levels were determined by Northern blot analysis and collagenase concentrations in the culture medium were determined by Western immunoblot . Basal level collagenase-3 transcripts decreased in Ob cell cultures, coinciding with an increase in IGF-I and -II protein levels . Removal of the conditioned medium modestly increased collagenase-3 mRNA levels and restored the ability of exogenously added IGF-I to repress collagenase-3 transcripts . IGF neutralizing antibodies and IGF binding proteins-2 and -3 in excess increased and sustained collagenase mRNA, heterogeneous nuclear RNA, and protease levels in Ob cell cultures . In conclusion, IGF-I and -II are autocrine repressors of collagenase-3 synthesis, and this effect may contribute to their actions on the maintenance of a normal bone collagen matrix.

Transplantation, 1996 Oct 27, 62(8), 1085 - 9
Comparison of adenovirus gene transfer to vascular endothelial cells in cell culture, organ culture, and in vivo; Merrick AF et al.; A replication-defective adenovirus 5 vector carrying the beta-galactosidase reporter gene was tested for its efficiency for gene delivery to vascular endothelial cells in various situations . Both porcine and human primary vascular endothelial cell cultures were very efficiently infected (>90%) at adenovirus concentrations of 10(10) pfu/ml or higher . Cultured rat fibroblasts and keratinocytes were even more readily infected, with >90% infection with adenovirus titers of 10(8) pfu/ml or higher . However, nondividing vascular endothelium in situ was very poorly transduced . Pieces of aorta from adult pigs, sheep, rabbit and rat, and pieces of human umbilical artery and vein were studied in organ culture . These showed only occasional positive vascular endothelial cells when exposed to the adenovirus vector at concentrations up to 5x10(11) pfu/ml . Kidney perfusion studies in rats and pigs gave similar results . The only exception to the above findings was in very young (3-4 day old) piglets, which showed excellent (>90%) infection of vascular endothelium with the adenovirus vector at titers of 10(10) pfu/ml . Our data suggest that adenovirus vectors will not be of value for gene delivery to uninjured vascular endothelium in situ, and are therefore unsuited for ex vivo genetic manipulation of vascular endothelium in organs for transplantation.

Biomed Tech (Berl), 1996 Oct, 41(10), 278 - 83
{A new osteoblast cell culture system for standardized testing of biomaterials}; Hendrich C et al.; For in vitro testing of new biomaterials cultured fibroblasts are employed . In the case of the agar diffusion test survival of cells is involved in the presence of the material to be tested . Further statements on the biological effects of a biomaterial require the use of cell cultures adapted to the tissue concerned and the underlying problem being investigated . In the present study, an osteoblast cell culture system with which implant surfaces in contact with bone can be tested as required by the relevant standards is described . Test bodies made of titanium, polystyrene or copper were used in the conventional agar diffusion test, and were also overgrown with fibroblasts or a cell line of foetal human osteoblasts . For the agar diffusion test, the test criterion was the extent of the inhibition area on staining with neutral red, while for the overgrowth, the mean cell diameter and the number of cells was employed . The phenotype of the osteoblast cell line was determined immunohistochemically by means of alkaline phosphatase or immunohistologically by means of collagen I and osteocalcin . Calcification was demonstrated using the v . Kossa stain . In the case of the osteoblasts, a differentiation of a collagen I and alkaline phosphatase-positive phenotype over an osteocalcin-positive phenotype to an increase in calcium deposition was shown . As in the case of the agar diffusion test, direct overgrowth also revealed no cytotoxic effect for titanium and polystyrene . In contrast, a cytotoxic effect consisting in a decrease in the number of cells and also a left shift in the size distribution was observed for copper . The standard deviations of the individual tests were less for overgrowth than for the agar diffusion test . The culture system for osteoblast cells thus meets the criteria of the EN/DIN 30993-5 in terms of the quality and accuracy of the results obtained . In addition to excluding direct cytotoxicity, this test system offers a new possibility of examining the influence of the material on cell growth . Consequently, it permits a repeatable examination of proliferation and differentiation of the osteoblasts on each material surface.

Trends Biotechnol, 1996 Oct, 14(10), 388 - 96
Hematopoietic cell culture therapies (Part II): Clinical aspects and applications; McAdams TA et al.; High-dose chemotherapy, followed by hematopoietic stem cell transplantation, holds significant promise for increasing the probability of long-term remission and possibly cure in a variety of cancers . Hematopoietic cell culture, or ex vivo expansion of hematopoietic cells, may play a significant role in reducing the danger and expense associated with the transplantation procedure . Phase I clinical trials have shown that ex vivo expanded cells have no significant toxicities, and some benefits . Ex vivo expansion of hematopoietic cells is likely to find other applications in gene therapy, tumor purging, production of dendritic cells for immunotherapy and the production of mature blood cells for transfusion therapies.

Int J Dev Neurosci, 1996 Oct, 14(6), 785 - 95
Neurotrophic effects of transferrin on embryonic chick brain and neural retinal cell cultures; Bruinink A et al.; The viability and differentiation promoting effects of various transferrins {iron-saturated (holo) and iron-depleted (apo) human and chick ovo (conalbumin)-transferrins, and bovine apo-transferrin} were studied, using serum-free, flat-sedimented cell cultures of embryonic chick brain and neural retina . The effects of transferrin (Tf) on the cell cultures depended on the type of Tf used and the parameter measured . Significant differences between brain and neural retina cultures in the effects of apo-ovoTf and iron {supplemented as ammonium-iron (III) citrate} were detected . Maximal levels of mitochondrial activity were observed in the presence of 2 mg/l apo-ovoTf in neural retina cell cultures . In brain cell cultures, 40 mg ovoTf/l were needed to achieve maximal levels . In brain, but not in neural, retina cell cultures ovoTf and optimal concentrations of Fe3+ exhibited similar effects on biochemical parameters of cell function and differentiation . Although, in the absence of ovoTf, neuronal outgrowth on areas not covered by glial cells was inhibited in both cell cultures, the differences were more prominent in neural retina cell cultures . Our data strongly suggest that Tf plays a key role in processes not connected directly with its iron transport capability.

Histochem J, 1996 Oct, 28(10), 671 - 80
S-100 protein subunits in bovine oviduct epithelium: in situ distribution and changes during primary cell culture; Walter I et al.; Cultures of bovine oviduct epithelial cells are widely used in co-culture systems to improve the results of in vitro fertilization . The aim of the present study was to evaluate the suitability of S-100 protein as a differentiation marker for bovine oviduct epithelial cells in vitro . The distribution of S-100 alpha and S-100 beta was examined immunohistochemically in bovine oviduct epithelium in situ and in primary cell cultures derived from it . Three segments of the Fallopian tube (isthmus, ampulla and fimbriae) were compared and analysed during different stages of the oestrus cycle (luteal phase and follicular phase) . Ciliated and non-ciliated cells of the epithelium reacted with anti-S-100 alpha, S-100a (alpha beta) and S-100 beta antibodies, except for isthmic non-ciliated cells, which did not bind anti-S-100 beta or anti-S-100a (alpha beta) . In addition, basal cells never showed immunoreactivity for S-100 . In confluent monolayers of cultured oviduct epithelial cells, disappearance of reactivity for S-100 paralleled morphological signs of dedifferentiation (loss of cilia, cytoplasmic vacuolization) . Free-floating oviduct epithelial cells, in contrast, retained morphological differentiation and still expressed S-100 antigen even after seven days in vitro . The immunohistochemical findings were confirmed by polyacrylamide gel electrophoresis and Western blotting . The results indicate that the presence of S-100 is closely connected to morphological differentiation and to the specific functional condition of bovine oviduct epithelial cells.

J Microsc, 1996 Oct, 184 ( Pt 1), 22 - 34
Use of primary cell cultures and intact isolated glandular epithelia for X-ray microanalysis; Hongpaisan J et al.; Changes in the elemental composition of cells during isolation of glandular epithelia were studied by electron probe X-ray microanalysis . Fine chopping of rat submandibular gland followed by enzymatic treatment for 15 min caused marked increases in Na and Cl and a decrease in K concentrations in acinar cells . After enzymatic treatment for 50 min, Na, Cl and K concentrations returned to close to the control level . Mechanical disaggregation of the acinar clumps following enzymatic treatment resulted again in minor increases in Na and Cl and a marked decrease in K concentration . Exposure of isolated acini to cholinergic stimulation in vitro resulted in secretion of Cl and K from the acinar cells . Dissection of the sweat gland from human skin caused a decrease in the K/Na ratio . Incubation of the gland for 30-45 min with collagenase gave rise to a gradual decrease in the K/Na ratio . After mechanical separation of the gland into the secretory coil and reabsorptive duct, a further reduction of the K/Na ratio was seen . However, the duct cells had a much lower K/Na ratio and higher Ca concentration than the coil cells . In primary cultures, the K/Na ratios of the coil and duct cells returned to the in situ level . The elemental composition of sweat gland cells incubated in collagenase-containing medium was no different from that in cells incubated in collagenase-free medium . In the intact collagenase-isolated tissue, Cl- secretion in the coil was elicited by carbachol but not by cAMP, whereas in the duct cells the reverse was the case . In primary cell cultures, Cl- efflux in both coil and duct cells could be elicited by both carbachol and cAMP . In conclusion, although changes in elemental composition of gland cells during the isolation procedure occur, physiological responses can be detected . When primary cell cultures are used, it should be borne in mind that cultured cells may have physiological properties different from those of the intact tissue.

J Dermatol Sci, 1996 Oct, 13(1), 25 - 9
Non-disulfided pro alpha 1(IV) chain in B16 melanoma cell culture; Tajima S et al.; We have previously reported that two species of pro alpha 1(IV) collagen chains, disulfided (500 kDa) and non-disulfided (180 kDa), were produced by B16 melanoma cells (J Biochem (1994) 116; 1039-1043) . The mechanism by which the non-disulfided pro alpha 1(IV) chain is produced was studied . No significant difference in prolyl hydroxylation in both polypeptides was observed . When the culture was treated with alpha, alpha 'dipyridyl', a potent inhibitor for hydroxylation of collagen, the secretion of the disulfided alpha 1(IV) chain was inhibited, but non-disulfided alpha 1(IV) chain secretion was not affected . Short pulse and pulse-chase experiments demonstrated that both chains appeared at the same time in the culture medium and the relative amounts of both chains in the medium were unaltered with increasing chase periods . These results indicate that the non-disulfided alpha 1(IV) chain is fully hydroxylated and that it's secretion is independent of it's hydroxylation level . A marked susceptibility of the 180 kDa alpha 1(IV) chain to pepsin at 4 degrees C suggests that the non-disulfided chain may be present in a denatured form.

J Parasitol, 1996 Oct, 82(5), 769 - 77
In vitro destruction of nerve cell cultures by Acanthamoeba spp.: a transmission and scanning electron microscopy study; Pettit DA et al.; Trophozoites of 4 species of Acanthamoeba were cytopathic for cultured rat B103 neuroblastoma cells . Cytopathogenicity was evaluated by a chromium release assay and by transmission and scanning electron microscopy . Acanthamoeba culbertsoni, Acanthamoeba castellanii, and Acanthamoeba polyphaga destroyed B103 target cells at 37 C as evidenced by the release of radiolabel . Acanthamoeba astronyxis did not produce cytopathology at 37 C but destroyed nerve cells at 25 C . Transmission and scanning electron microscopy of cocultures maintained at different time periods revealed that all species of Acanthamoeba exhibited long cylindrical structures, termed digipodia, which made contact with target cells . Following this effector cell-target cell contact, membrane blebbing on the nerve cells was observed . These events were followed either by lysis of target nerve cells or ingestion of the target cells via food-cups and their subsequent channeling into intracytoplasmic food vacuoles . Use of the TUNEL (TdT-mediated dUTP nick end labeling) technique indicated that approximately 40% of B103 cells incubated with A . culbertsoni, 20% of B103 cells cocultured with A . castellanii or with A . polyphaga, and less than 1% of B103 cells incubated with A . astronyxis at 37 C were apoptotic after 24 hr of coculture . Studies using electron microscopy indicated that Acanthamoeba trophozoites destroyed nerve cells both by cytolysis and by ingestion of whole nerve cells via food-cups.

Endocrinology, 1996 Oct, 137(10), 4451 - 9
Body temperature (37 C) specifically down-regulates the messenger ribonucleic acid for the major sperm surface antigen CD52 in epididymal cell culture; Pera I et al.; To study the role of scrotal vs . body temperature in epididymal function we established a simplified cell culture system from the dog epididymis in which the cells showed a pattern of gene expression similar to that in the human epididymis and retained many characteristics of epididymal epithelial cells . The cultured cells had an epithelial-type cytoskeleton, nuclear androgen receptor protein, and a striking temperature responsiveness . Exposure of the cells to a culture temperature of 37 C, compared to 33 C, had a fast and irreversibly suppressive effect on the levels of an abundant epididymal messenger RNA (mRNA), CE5, which represents the canine counterpart of the human CD52/HE5 mRNA, encoding a major glycosylphoshatidylinnositol (GPI)-anchored sperm membrane glycopeptide . The temperature effect on the mRNA was a direct and specific one, not mediated by temperature influences on the testis and not affecting other epididymal mRNAs . Exposure of the cells to 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (25 micrograms/ml culture medium) and cycloheximide (2 micrograms/ml) suggested that the steady state levels of CD52/CE5 mRNA may be controlled posttranscriptionally by changing the half-life of this specific mRNA in response to an extracellular temperature stimulus.

Endocrinology, 1996 Oct, 137(10), 4115 - 9
Expression and growth factor regulation of platelet-derived growth factor B transcripts in primary osteoblast cell cultures; Rydziel S et al.; Platelet-derived growth factor (PDGF), an important bone cell mitogen, exists as a homo- or heterodimer product of the PDGF-A and -B genes . Normal unstimulated cells of the osteoblast lineage express the PDGF-A gene, but it is not known whether they express the PDGF-B gene . We examined the expression of PDGF-B messenger RNA (mRNA) levels in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells) and determined whether they were modified by transforming growth factor-beta 1 (TGF beta 1), basic fibroblast growth factor (bFGF), insulin-like growth factor I (IGF-I), and PDGF-BB . Ob cells expressed PDGF-B transcripts of 3.5 kilo-bases, as determined by Northern blot analysis . Treatment of Ob cells with TGF beta 1 at 0.01-1.2 nM caused a dose-dependent increase in steady state PDGF-B mRNA, an effect that was initially observed after 2 h and was maximal after 6h . Cycloheximide induced PDGF-B transcripts and decreased the effect of TGF beta 1 . TGF beta 1 did not modify the half-life of PDGF-B mRNA in transcriptionally arrested Ob cells and increased the rate of PDGF-B gene transcription in nuclear run-on assays . In contrast, treatment with PDGF-BB at 3.3 nM, bFGF at 6 nM, or IGF-I at 100 nM for 2-24 h did not modify PDGF-B mRNA levels in Ob cells . In conclusion, normal Ob cells express the PDGF-B gene, and TGF beta 1 induces its transcription, whereas bFGF, IGF-I, and PDGF-BB do not enhance the levels of PDGF-B mRNA . PDGF-BB may act not only as a systemic but also as a local regulator of bone cell function.

Mol Gen Genet, 1996 Sep 25, 252(4), 465 - 9
Comparable processing of beta-lactoglobulin pre-mRNA in cell culture and transgenic mouse models; Donofrio G et al.; Eukaryotic pre-mRNAs undergo a variety of post-transcriptional modifications, including the removal of intronic sequences by splicing, leading to creation of a functional mRNA . We have compared the processing of transcripts generated from ovine beta-lactoglobulin gene constructs in stably transfected cells and in transgenic mice . In both the in vitro and in vivo model systems the removal of the middle two introns resulted in the inefficient splicing of the downstream, terminal intron . This intron-containing transcript was detected in the cytoplasmic RNA fraction . Thus, the initial in vitro analysis in cell lines of minigene constructs destined for expression in transgenic animals may provide a rapid and reliable indicator of the processing efficiency of the pre-mRNA produced by the construct in vivo . This is in contrast to the apparent limitations of in vitro systems in the analysis of transcription regulatory elements required for transgene expression.

Hear Res, 1996 Sep 15, 99(1-2), 71 - 8
Characterization and development of an inner ear type I fibrocyte cell culture; Gratton MA et al.; A method has been developed that allows successful maintenance of secondary cell cultures derived from explants of the cochlear lateral wall of young adult gerbils . The secondary cultures were characterized morphologically with light and transmission electron microscopy and immunocytochemically with protein markers specific to various lateral wall cell types . Structural studies revealed fusiform-shaped cells with a paucity of cytoplasm surrounding the nucleus and slender processes . The cells showed little evidence of intercellular contact even when confluent . The cultures were immunopositive for vimentin, carbonic anhydrase isozyme II, creatine kinase isozyme BB and smooth endoplasmic reticulum Ca-ATPase, but lacked reactivity for cytokeratins and Na,K-ATPase . The results indicate that the cultures are comprised of type I fibrocytes from the spiral ligament . These findings are the first to demonstrate that inner ear spiral ligament cells can be isolated and maintained in secondary culture while retaining many of their in vivo characteristics . Based upon their location and content of ion transport enzymes, type I fibrocytes are thought to be involved in the recycling of potassium from perilymph into the stria vascularis . The establishment of this cell line provides a means to analyze the role of spiral ligament fibrocytes in maintenance of inner ear homeostasis.

Virology, 1996 Sep 15, 223(2), 409 - 12
A hyperimmune serum against a synthetic peptide corresponding to the hypervariable region 1 of hepatitis C virus can prevent viral infection in cell cultures; Shimizu YK et al.; To investigate whether a principal neutralization epitope exists in hypervariable region 1 (HVR1) within the putative envelope of hepatitis C virus (HCV), we generated a hyperimmune rabbit serum against a synthetic peptide corresponding to HVR1 of HCV isolate H77 . The reactivity of the serum in the enzyme-linked immunosorbent assay was correlated with the 13 amino acids (position 398-410) in HVR1 . The serum prevented infection with H77 virus in cell cultures but did not prevent infection with H90 virus, a genetically divergent isolate from the same patient . The study demonstrated that neutralization of HCV was mediated, in part, by isolate-specific antibody recognizing HVR1.

Toxicology, 1996 Sep 2, 112(3), 219 - 26
Cadmium-induced production of superoxide anion and nitric oxide, DNA single strand breaks and lactate dehydrogenase leakage in J774A.1 cell cultures; Hassoun EA et al.; The involvement of reactive oxygen species in the toxicity of cadmium (Cd) has been proposed . We have, therefore, examined the effects of this cation on the production of superoxide anion and nitric oxide and DNA single strand breaks in J774A.1 macrophage cells in culture as well as the effects on lactate dehydrogenase (LDH) leakage and cell viability . Following a 48-h incubation, over 2-fold increases in superoxide anion and nitric oxide (NO) production were observed at a Cd concentration of approximately 0.60 microM, while a 50% decrease in viability was observed at this concentration . LDH leakage paralleled the superoxide anion and nitric oxide production . Concentration-dependent increases in DNA single strand breaks (SSB) were observed after incubation with Cd with a maximum increase occurring at a concentration of approximately 0.40 microM . The results indicate that Cd is toxic to the J774A.1 cell line, and support the hypothesis that the toxicity may be due at least in part to an oxidative stress induced by the production of reactive oxygen species following exposure to this cation.

J Mol Recognit . 1996 Sep-Dec;9(5-6):747.
Purification of an expressed insect transferrin from cell culture media using high-capacity Ni(2+)-dipicolylamine gel; Winzerling JJ et al.; Vertebrate transferrin is a well characterized iron transport protein . In contrast, little is known concerning the role of transferrin in insects . Yet, study of iron metabolism in insects could give insights into strategies for insect control, particularly for insects that transmit disease.

Toxicol Ind Health, 1996 Sep-Oct, 12(5), 683 - 96
An in vitro model for toxicological investigations of environmental neurotoxins in primary neuronal cell cultures; Schmuck G et al.; Currently, most neurotoxicological investigations are still conducted using various animal models (e.g . chickens, rodents) . In this report, alternative strategies of testing were examined to detect the neurotoxic potency of foreign compounds . Primary neuronal cell cultures from fetal rats are already an accepted model for mechanistic and pharmacological studies in drug research . Their suitability for neurotoxicological studies was examined by using industrial model compounds, which are well-known inductors of neuropathies: acrylamide, hexachlorophene, paraquat, n-hexane, and its neurotoxic metabolites acetylaceton and 2,5-hexandione . As a control compound, the nonneurotoxic solvent n-heptane was used . General cytotoxicity and the intracellular content of glial fibrillary acid protein, neuron-specific enolase, and neurofilaments were measured . n-Heptane induced an acute cytotoxicity and acrylamide and 2,5-hexandione produced a delayed cytotoxicity in primary neuronal cells, whereas the others showed no cytotoxic potency in the tested concentration range . These results were in agreement with the quantification of neurons by neuron-specific enolase . In contrast, with the exception of acetylaceton, glia cells were significantly affected by all neurotoxins at the later time . Signs of axonopathies were demonstrated for acrylamide, n-hexane and its metabolites, as well as for hexachlorophene and paraquat in vitro, by determining the intracellular neurofilament level . Therefore, the determination of cell-specific end points is necessary to detect the neurotoxic potency and quality of a compound, whereas the cytotoxicity assay limited the tested concentration range.

J Pathol, 1996 Sep, 180(1), 95 - 101
Cytokine production by cell cultures from bronchial subepithelial myofibroblasts; Zhang S et al.; Myofibroblasts have been previously described beneath the bronchial epithelium and were found to increase in number proportional to the accumulation of extracellular matrix in the bronchial lamina reticularis in asthma . The aim of this study was to assess further the contribution of these structural cells to allergic inflammation in the bronchial mucosa through their cytokine expression . Cell cultures were established from the lamina reticularis of human bronchial biopsies from asthmatic and non-asthmatic subjects . Cytokine secretion was measured by ELISA in supernatants of cultures with or without tumour necrosis factor-alpha (TNF-alpha) . The mRNA levels for granulocyte-macrophage colony-stimulating factor (GM-CSF) in the cultures were examined by ribonuclease protection assays (RPAs) . Bronchial myofibroblasts grown from bronchial biopsies were capable of producing GM-CSF, interleukin-6 (IL-6), interleukin-8 (IL-8), and stem cell factor (SCF) constitutively . The GM-CSF production by myofibroblasts was significantly increased in response to TNF-alpha simulation with a corresponding increase in GM-CSF mRNA expression . The enhancement of GM-CSF production by TNF-alpha in myofibroblasts was blocked by the inhibition of RNA synthesis . Prednisolone abolished the GM-CSF production . This study provides evidence for the role of bronchial myofibroblasts in the regulation of inflammatory cell recruitment and activation by interaction in the cytokine network in the bronchial mucosa.

Hum Reprod, 1996 Sep, 11(9), 1966 - 74
The influence of Vero cell culture on human embryo development and chorionic gonadotrophin production in vitro; Turner K et al.; Co-culture of human embryos with cell layers has generally shown that blastocyst formation rates are improved compared to routine culture in medium alone . In order to assess this further, we have additionally classified resulting blastocysts according to their morphology and secretion of human chorionic gonadotrophin (HCG) . A total of 70 supernumerary human embryos from 15 patients were divided equally and randomly between two culture conditions: (i) co-culture with Vero cells; and (ii) culture in our routine medium . Embryo development and morphology were recorded for up to 14 days in culture . The results showed that embryos on Vero cells had a significantly higher blastocyst formation rate (P < 0.02) by or on day 6 of development than those in routine culture medium alone (77 and 46% respectively) . For HCG analysis, the culture medium was changed in both culture systems on days 5, 7, 9, 12 and 14 of embryo development and analysed . Most embryos began to produce HCG between days 7 and 9, with HCG secretion being significantly higher from embryos on Vero cells between days 9 and 12 than from embryos in routine culture (P < 0.03) . The morphology of the blastocysts obtained was related to their ability to hatch and produce HCG but was not significantly better for one type of culture system than for the other.

Acta Otolaryngol, 1996 Sep, 116(5), 690 - 6
Auditory cortical neurons in vitro: cell culture and multichannel extracellular recording; Gopal KV et al.; Self organization, pattern generation, and pattern processing in local cortical circuits are difficult to study in vivo . The complexities of cortical circuits require simplified systems for study . We have developed a simplified model of auditory cortical neurons growing as monolayer networks in culture . Neurons dissociated from auditory cortex of 14-day mouse embryos were grown on photoetched microelectrode array containing 64 transparent indium-tin oxide electrodes . Cultures were maintained in incubators for up to 113 days . Neurons developed processes and made synaptic connections . All cultures were spontaneously active and exhibited complex temporal burst patterns . In a data set of 12 cultures, the number of active channels varied from culture to culture and ranged from 6-17 . Signal/noise ratios ranged from 3:1 to a maximum of 16:1 . No significant correlations were found between age of the culture and number of active channels, or signal/noise ratios . Spontaneous firing patterns recorded from various channels showed complex bursting patterns in all cultures . Within a culture, coordinated synchronous bursting were found among some channels, and independent bursting on others . Preliminary histological analysis of cultures using the Loots-modified Bodian stain showed neurons with axonal and dendritic profiles growing extensively on top of the glial carpet . Neuronal processes crossing the electrodes singly or in small groups were also observed . Pyramidal and non-pyramidal cells could be identified . In a pool of 2,093 neurons in a 49-day-old culture, the average size of the somata was found to be 16 microns, with a mode of 12 microns.

In Vivo, 1996 Sep-Oct, 10(5), 515 - 26
Cell culture observations of human postnatal thymic epithelium: an in vitro model for growth and humoral influence on intrathymic T lymphocyte maturation; Bodey B et al.; Sixteen postnatal human thymuses were obtained at the time of corrective cardiovascular surgery and maintained in vitro as separate cultures of thymocytes and reticulo-epithelial (RE) cells . The stages of differentiation of the thymocytes were investigated in situ with a library of 10 monoclonal antibodies (MoABs) directed against human lymphocyte differentiation antigens . Employing immunofluorescence staining and flow cytometric (FACS) analysis, in vitro immunophenotype (IP) changes were demonstrated, which appeared after use of a combination of mitogen (PHA), recombinant interleukin-2 (rIL-2) and autologous thymic RE cell culture supernatants . RE cell supernatant participated in increasing the expression of the IL-2 receptor (IL-2R) during combined stimulation with phytohaemagglutinin (PHA) and rIL-2 . Thymocyte proliferation was measured in 4 hour tritiated-thymidine (3H-TdR) incorporation (proliferation) assay . We were able to isolate the thymic nurse cells (TNC) with and without enzymatic tissue digestion . TNCs were separated from accompanying thymocytes and cultured . They grew as large, sometimes connected cells, but did not display the epithelial type of tissue organization . After in vitro culturing, the cytoskeleton of TNCs expressed high molecular weight cytokeratin and vimentin and intracytoplasmic tonofilaments, characteristic of epithelium . Whole thymic tissue pieces were cultured with and without previous trypsinization . The initial outgrowth of the cuboidal epithelial tissue layer occurred within 24-48 hours, and the RE cells remained functionally active for at least 15 days . RE cell supernatants were collected daily for two weeks and used in thymocyte differentiation experiments . The results indicated that thymic humoral factors contribute to a select, not fully understood differentiation pathway of thymocytes: a) more mature immunophenotype (IP) characterized by CD3 expression; b) de novo synthesis of interleukin-2 receptor (IL-2R); and; c) differentiation of the CD8+ subpopulation, identifying regulatory cells within the two major CD8+ and CD4+ subsets . Use of mitogenic (PHA) stimulation, after 5 days in vitro, resulted in a T helper (CD4+) oriented differentiation pathway of cortical thymocytes . At the same time, the cultured thymocytes expressed CD11 de novo, an early thymocyte differentiation antigen, and CD7, a marker not present on mature peripheral lymphocyte subsets (the IP changes demonstrated a dedifferentiation) . Our overall impression, following the studies with the proliferation assays, was that in our experimental in vitro model, thymic hormones did not contribute to the induction of generalized thymocyte proliferation.

Bone Marrow Transplant, 1996 Sep, 18 Suppl 1, S18 - 20
Detection of cancer cells in peripheral blood stem cells of women with breast cancer by RT-PCR and cell culture; Kruger WH et al.; The benefit of high-dose therapy and blood stem cell reinfusion for women with high-risk breast cancer is currently under investigation . Contaminations of autologous blood stem cells with cancer cells have been described . Cancer micrometastases may be detected by immunocytochemistry, culture techniques and cytokeratin-19 mRNA reverse transcriptase PCR . Women with breast cancer received adjuvant HD-CTM with peripheral blood stem cell (PBSC) support after surgical therapy and 4 cycles conventional chemotherapy . Peripheral blood stem cells were mobilised by G-CsF and harvested after the third or fourth cycle of standard therapy . Aliquots of PBSC-collections (10(7)-2*10(7) cells) were subjected to CK19-mRNA reverse transcriptase PCR . RNA was extracted by standard methods and reverse transcription was performed with MMV-RT . Integrity of RNA was checked by coamplification of housekeeping sequences . Aliquots of the RT-mix were subjected to PCR-amplification with outer and inner primer pairs, subsequently . A second aliquot of 2*10(7) cells was cultured over 42 days in liquid culture . Cytospins were prepared weekly from cultured cells and evaluated by light microscopy with or without prior immunocytochemistry . Ten leukaphereses from 6 women were available for PCR-analysis and cell culture . Six leukaphereses were negative for CK19-mRNA and for detection of cancer cells by culture technique, two samples were positive for CK19-mRNA and culturally enriched cells and two samples were positive for CK19-mRNA and negative for cultured cancer cells . No sample was positive for cultured cells and negative for CK19-mRNA . Overall, the results corresponded in 80% . Two sensitive techniques for the detection of cancer micrometastases were applied to aliquots from 10 leukaphereses of six breast cancer patients with corresponding results in 80% . PCR-mediated detection of cancer cells was confirmed by culture technique and light microscopy, however, further comparison of CK19-PCR with standard techniques like cell culture and immunocytochemistry is still necessary.

Keio J Med, 1996 Sep, 45(3), 200 - 6
Human brain microvessel endothelial cell culture as a model system to study vascular factors of ischemic brain; Nagy Z et al.; Cerebral ischemia is caused by reduced blood supply at the microcirculatory level . In the microvessels, the main elements of the reperfusion injury following brain ischemia are the transformation of endothelial cell-surface from anticoagulant to procoagulant property, leukocyte adhesion, sludge or clot formation . There is a paucity of information on how hemostatic factors, cytokines, lipoprotein(a) (Lp(a)) and endothelin-1 (ET-1), being responsible for ischemic/reperfusion injury, interact with human brain microvessel endothelium (HBEC) . There are no data furthermore about the expression of complement proteins of HBEC influenced by cytokines or fibrinolytic factors . Previously we established optimal conditions for culturing HBEC . Cell contraction induced by thrombin, plasmin, miniplasmin was recorded . The reassembly of F-actin was observed after thrombin treatment . ICAM-1 upregulation was measured following TNF-alpha, IL-1-alpha and thrombin incubation . Plasmin and miniplasmin downregulated the ICAM-1 in our cell culture system . Lp(a) modulated the thromboresistant cell-surface by reduction of t-PA and u-PA, but PAI-1 remained unchanged . Lp(a) modulated the ET-1 production by early increasing and late decreasing, in a bimodal manner . The increased secretion of ET-1 by cytokines (TNF-alpha, IL-1-alpha) was reduced in the presence of Lp(a) . Gradual increase of complement proteins (factor H, factor B, C4) was induced by cytokines . Plasmin and miniplasmin augmented a rapid increase of C4 . Some factors of complex relationship between regulators and modulators of endothelial adhesion molecules have been demonstrated in a human cell culture system prepared from brain microvessel endothelium . A unified concept of sequential events of ischemia/reperfusion in the brain has not yet developed.

Toxicon, 1996 Sep, 34(9), 1054 - 7
Neuroblastoma cell culture assay shows that Carcinoscorpius rotundicauda haemolymph neutralizes tetrodotoxin; Yeo DS et al.; Veratridine facilitates the influx of Na+ into Neuro-2A cells and this process is exacerbated by the presence of a Na+/K(+)-ATPase inhibitor, ouabain, leading to a reduction in cell viability . However, tetrodotoxin neutralizes the cytotoxic effects of veratridine, hence sustaining the viability of Neuro-2A cells . This neutralizing ability was negated when TTX was first reacted with cell-free haemolymph of Carcinoscorpius rotundicauda, resulting in reduced cell viability . These results therefore indicate a bona fide effect of the cell-free haemolymph against tetrodotoxin as demonstrated by in vitro cell culture technique.

J Neurophysiol, 1996 Sep, 76(3), 2111 - 4
Long-term depression of Aplysia sensorimotor synapses in cell culture: inductive role of a rise in postsynaptic calcium; Lin XY et al.; 1 . Activation of sensory neurons at 2 Hz for 15 min induces long-term depression (LTD) of isolated Aplysia sensorimotor synapses in cell culture . 2 . Prior infusion of the Ca2+ chelator 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) into the postsynaptic motor neuron blocks the induction of LTD, but not short-term synaptic depression . 3 . Invertebrate central synapses possess the capacity for LTD . This form of long-term synaptic plasticity may play an important role in learning in Aplysia.

J Neurosci Methods, 1996 Sep, 68(1), 49 - 53
Biochemical characteristics of a primary blood-brain barrier cell culture system as a function of the activity of the proteases used in tissue disaggregation; Ng KY et al.; Utilization of primary cultured brain capillary endothelial cells (BCECs) as an in vitro model of the blood-brain barrier (BBB) depends on the extent to which cultured BCECs retain the in vivo characteristics . Recently, we have reported that consistent isolation of BCECs that mimic the in vivo BBB depends on whether a specific ratio between the weight of the isolation enzyme (collagenase/dispase) and the weight of the capillaries present during the isolation is used . Since it is possible for the same weight of an enzyme to possess different activity levels, it is felt that activity rather than weight of an enzyme should be used in arriving at the above ratio . Therefore, using bovine brain as the source of BCECs, we have quantified the amount of collagenase/dispase needed for optimal isolation of BCECs and retention of their phenotypic properties in terms of collagenase/dispase activity per g of capillaries . Monolayers of bovine BCECs isolated at 0.15 or 0.30 units of collagenase and 2.06 or 4.12 units dispase per g of capillaries gave the best overall quality as judged by their permeability characteristics and the activities of angiotensin converting enzyme, alkaline phosphatase and gamma-glutamyl transpeptidase.

Eur Respir J, 1996 Sep, 9(9), 1839 - 46
Cell cultures from bronchial subepithelial myofibroblasts enhance eosinophil survival in vitro; Zhang S et al.; Mechanisms of eosinophil accumulation and activation in the bronchial mucosa are crucial for the pathogenesis of asthma . The location of specialized fibroblasts, myofibroblasts, beneath the bronchial basement membrane and their proximity to infiltrating eosinophils potentially enable the myofibroblasts to modulate eosinophil survival and function in asthma . The aim of this study was to investigate the effects of bronchial myofibroblasts on eosinophil survival in vitro . Eosinophils from human peripheral blood were exposed to cell cultures from bronchial myofibroblasts and to myofibroblast-conditioned media . Eosinophil viability was assessed and granulocyte/macrophage colony-stimulating factor (GM-CSF) production was examined in co-culture supernatants and as messenger ribonucleic acid (mRNA) in myofibroblasts . Eosinophil survival was significantly increased and eosinophil apoptosis was inhibited by co-culture with myofibroblasts . Conditioned medium from tumour necrosis factor-alpha (TNF-alpha)-stimulated myofibroblasts also prolonged eosinophil survival . This effect could be blocked by GM-CSF antibody . GM-CSF mRNA and secretion from myofibroblasts were increased in co-cultures and by eosinophil-conditioned medium . Addition of antibodies to TNF-alpha and interleukin-1 alpha (IL-1 alpha) to co-cultures resulted in significant reduction both in eosinophil survival and GM-CSF levels . Blocking of fibronectin in the co-cultures did not affect the eosinophil survival enhancing activity . Prednisolone inhibited the eosinophil survival enhancing activity of the co-cultures by suppression of GM-CSF production . Soluble eosinophil-derived cytokines are involved in the interaction of eosinophils with myofibroblasts, which results in a tumour necrosis factor-alpha/interleukin-1 alpha mediated release of granulocyte/macrophage colony-stimulating factor from myofibroblasts . Bronchial myofibroblasts can, thereby, contribute to allergic inflammation by granulocyte/macrophage colony-stimulating factor-mediated inhibition of eosinophil apoptosis.

Biotechnol Prog, 1996 Sep-Oct, 12(5), 700 - 2
Photoimmobilization of insulin onto polystyrene dishes for protein-free cell culture; Ito Y et al.; Photoreactive insulin was synthesized by coupling with azidobenzoic acid . The insulin derivative was immobilized onto the wells of polystyrene culture plates by photoir-radiation . The photoimmobilized insulin enhanced the growth of anchorage-dependent cells such as Chinese hamster ovary CHO-K1 and mouse fibroblast STO cells by more than native or azidophenyl-derivatized insulin . A small amount of photoimmobilized insulin (1-10% of the amount of the native or derivatized insulin) enhanced the growth of CHO-K1 and STO cells . In addition, the maximal mitogenic effect of the immobilized insulin was greater than that of native or derivatized insulin . Photoimmobilization could be a universal means of immobilizing growth factors onto the surface of materials devoid of chemical functional groups scaffolding growth factors and providing a new protein-free cell culture system or tissue engineering materials.

J Neuroendocrinol, 1996 Sep, 8(9), 687 - 93
Identification and characterization of angiotensinIV binding sites in rat neurone and astrocyte cell cultures; Greenland K et al.; This study demonstrates the existence of the putative receptor for the hexapeptide (3-8) fragment of angiotensin II (AngIV) on rat astrocytes and neurons grown in cell culture . Binding of 125I-AngIV was saturable and distinct from that of the AngII receptor subtypes . Equilibrium binding was attained in 15 min in astrocytes and 75 min in neurons at 22 degrees C . The bound peptide was confirmed by HPLC to be intact AngIV while the bound peptide was substantially degraded, even in the presence of peptidase inhibitors . Scatchard analysis of equilibrium binding was consistent with a two binding site model, revealing a high affinity and a low affinity binding site in both cell types . In neurons, the respective association constants (Ka) were 2.72 +/- 0.23 nM-1 and 727 +/- 354 nM-1, with associated receptor densities of 109.30 +/- 58.87 and 1723 +/- 1167 fmol/mg protein . Similar analyses in astrocytes gave Kas of 5.71 +/- 2.85 nM-1 and 277 +/- 205 nM-1, and respective densities of 191.1 +/- 90.1 and 1425 +/- 1250 fmol/mg protein . However, the quantitative reliability of these binding isotherms may be influenced by the degration of unbound peptide . Competitive binding analysis was used to determine the specificity of the receptor site, with the relative order of affinities being AngIV > AngIII > AngII(4-8), and no displacement by AngII, Iosartan and PD123319 in either neurons or astrocytes . Autoradiography with 125I-AngIV performed on neuronal cultures demonstrated that binding was confined to a subpopulation of the total cells . These data support the existence of a specific binding site for AngIV in both neurons and astrocytes, consistent with the properties of binding reported previously in the brain, and distinguish this site from the AngII receptor subtypes.

J Neurosci Res, 1996 Sep 1, 45(5), 631 - 6
Selective toxicity of the general anesthetic propofol for GABAergic neurons in rat brain cell cultures; Honegger P et al.; The intravenous, short-acting general anesthetic propofol was applied to three-dimensional (aggregating) cell cultures of fetal rat telencephalon . Both the clinically used formulation (Disoprivan, ICI Pharmaceuticals, Cheshire, England) and the pure form (2,6-diisopropylphenol) were tested at two different periods of brain development: immature brain cell cultures prior to synaptogenesis and at the time of intense synapses and myelin formation . At both time periods and for clinically relevant concentrations and time of exposure (i.e., concentrations > or = 2.0 micrograms/ml for 8 hr), propofol caused a significant decrease of glutamic acid decarboxylase activity . This effect persisted after removal of the drug, suggesting irreversible structural changes in GABAergic neurons . The gamma-aminobutyric acid type A (GABAA) blocking agents bicuculline and picrotoxin partially attenuated the neurotoxic effect of propofol in cultures treated at the more mature phase of development . This protective effect was not observed in the immature brain cells . The present data suggest that propofol may cause irreversible lesions to GABAergic neurons when given at a critical phase of brain development . In contrast, glial cells and myelin appeared resistant even to high doses of propofol.

Mol Mar Biol Biotechnol, 1996 Sep, 5(3), 167 - 74
Transient expression of luciferase reporter gene after lipofection in oyster (Crassostrea gigas) primary cell cultures; Boulo V et al.; Transient expression of the luciferase gene, under transcriptional control of several heterologous promoters, was obtained in heart primary cell cultures of the Pacific oyster, Crassostrea gigas . Drosophila heat shock protein 70 promoter (hsp70), cytomegalovirus, and simian virus early promoters, controlling the luciferase gene, were transfected into the cell cultures using liposomes . Two culture media were used to establish primary cell cultures and tested as transfection media . Parameters such as the quantity of DNA and the ratio of DNA to liposome were analyzed to define the best transfection conditions . In oysters, the Drosophila inducible hsp70 promoter behaved in a way similar to that observed in other animal species . Moreover, for this study, hsp70 was more efficient than the cytomegalovirus and simian virus promoters.

J Gen Virol, 1996 Sep, 77 ( Pt 9), 2277 - 85
Glycoprotein gH of pseudorabies virus is essential for penetration and propagation in cell culture and in the nervous system of mice; Babic N et al.; Glycoprotein H (gH) of pseudorabies virus (PrV) is a structural component of the virion and forms a complex with another glycoprotein, gL . For a detailed analysis of the function of PrV gH, we isolated a gH-deficient mutant on trans-complementing gH-expressing cells after insertion of a beta-galactosidase expression cassette into a partially deleted gH gene . The absence of gH did not affect primary or secondary attachment of PrV but the mutant was not infectious . The defect in infectivity could partially be overcome by experimentally induced membrane fusion using PEG, which suggests that gH was necessary for fusion between virion and cellular membranes . After intranasal inoculation into mice, the LD50 of complemented gH- PrV was more than four orders of magnitude higher than that of wild-type PrV . Infection of the respiratory epithelium was much less efficient with complemented gH- PrV as compared with rescued PrV, reflecting the lack of direct cell-to-cell spread . Complemented gH- PrV was able to penetrate into a few trigeminal and sympathetic first order neurons accessible from the nasal cavity, whereas transneuronal transfer in the second order neurons was not observed . In summary, gH is essential for entry and cell-to-cell spread in cell culture, and for propagation in the nervous system of mice . This substantiates the hypothesis that transneuronal spread in vivo and direct cell-to-cell spread in cell culture are governed by similar mechanisms.

J Gen Virol, 1996 Sep, 77 ( Pt 9), 2067 - 71
Cell culture isolation of piscine neuropathy nodavirus from juvenile sea bass, Dicentrarchus labrax; Frerichs GN et al.; A virus causing a vacuolating encephalopathy and retinopathy in juvenile sea bass, Dicentrarchus labrax, was isolated from brain tissue in a fish cell line (SSN-1) derived from striped snakehead, Channa striatus . The isometric, non-enveloped, 30 nm diameter virus particles were resistant to pH 2-9 and heating at 56 degrees C for 30 min . Infectious particles had a buoyant density of approximately 1.31 g/cm3 in CsCl . Two structural polypeptides of molecular mass 40 and 42 kDa were identified and the ssRNA consisted of two fragments of molecular mass 1.10 and 0.51 x 10(6) Da . From these characteristics the virus was identified as a nodavirus . Due to the broad range of susceptible fish hosts and the consistent neuropathology of the disease condition, the generic term piscine neuropathy nodavirus (PNN) is proposed for this infectious agent.

Endocrinology, 1996 Sep, 137(9), 3802 - 7
Corticosterone selectively increases follicle-stimulating hormone beta-subunit messenger ribonucleic acid in primary anterior pituitary cell culture without affecting its half-life; Kilen SM et al.; We demonstrated previously that glucocorticoids differentially affect the levels of the two pituitary gonadotropins, LH and FSH, both in vivo and in vitro . In vivo, the effect of glucocorticoids is GnRH independent, indicating a direct action on the gonadotrope, and it leads to selective up-regulation of the pituitary content of FSH and FSH beta-subunit messenger RNA (mRNA) . The objective of the present study was to confirm the direct action of corticosterone (B) on FSH beta-subunit mRNA in primary anterior pituitary cell culture and to assess whether the selective B-induced rise in FSH beta mRNA is mediated through altered stability of the FSH beta transcript . Anterior pituitary glands collected from randomly cycling female rats were dissociated with trypsin . Cells were incubated at 37 C for 48 h and subsequently exposed to vehicle or B (1.7 microM) for an additional 42 h . At the end of the incubation, media were sampled for FSH and LH, cells were lysed, and total RNA was isolated for Northern blot analysis . Exposure to B for 42 h caused direct and selective upregulation of FSH release, FSH content, and FSH beta mRNA; decreased alpha-subunit mRNA; and had no significant effect on LH release, LH content, or LH beta mRNA . To evaluate the mRNA stability of the three subunits, cells were exposed to the transcription blocker actinomycin D (act D; 5 micrograms/ml) for an additional 6 h . The combined 6-h treatment with B and act D slightly, but significantly, suppressed alpha-subunit mRNA and did not change LH beta mRNA, confirming a long half-life of the two gonadotropin subunit mRNAs . In contrast, FSH beta mRNA was significantly suppressed by act D to the same level in vehicle- and B-treated cells . The posttranscriptional decay rate was examined by sampling at 0, 1, 2, 3, and 6 h during the 6-h act D treatment period . Decay curves for FSH beta mRNA were parallel in vehicle- and B-treated cells, indicating that B did not alter FSH beta mRNA stability . We conclude that the selective B-induced rise in FSH beta mRNA is mediated at the level of transcription rather than mRNA stabilization.

J Urol, 1996 Sep, 156(3), 1198 - 203
Ureteral cell cultures . I . Characterization and cellular interactions; Wolf JS Jr et al.; PURPOSE: To further understand the biology of ureteral cells, we studied the growth characteristics and in vitro cellular interactions of human ureteral uroepithelial cells, smooth muscle cells and myofibroblasts . MATERIALS AND METHODS: The proliferation, morphology and immunohistochemical characteristics of human ureteral cells grown in vitro were evaluated under varying conditions . RESULTS: The growth and morphology of ureteral cells were dependent upon media characteristics, especially the calcium concentration and presence of epidermal growth factor and bovine pituitary extract . Cells demonstrated specific stimulatory interactions via both soluble and insoluble factors . Most important, uroepithelial cells and smooth muscle cells displayed reciprocal enhancement of growth . CONCLUSIONS: The favorable interactions between ureteral cell types in vitro have implications for future work involving these cells.

Cell Immunol, 1996 Aug 25, 172(1), 77 - 83
The effects of the HIV-1 envelope protein gp120 on the production of nitric oxide and proinflammatory cytokines in mixed glial cell cultures; Kong LY et al.; Although the neurotoxicity induced by the HIV envelope protein, gp120, has been demonstrated to require the presence of glial cells (microglia/astrocytes), the mechanisms for the gp120-induced neurotoxicity are not well understood . Moreover, the neurotoxic potencies of gp120s obtained from various HIV isolates are different . Since nitric oxide (NO) and proinflammatory cytokines (TNF-alpha, IL-1, IL-6) produced by glial cells have been involved in the neuropathogenesis of various diseases, this study examined the effects of gp120 obtained from two strains, HIV-1IIIB and HIV-1SF2, of the HIV-1 virus on the production of NO, TNF-alpha, IL-1 alpha, IL-1 beta, and IL-6 in murine primary mixed glial cell cultures . The glial cells exposed to HIV-1IIIB gp120 released NO, TNF-alpha, and IL-6 in a dose-dependent manner, whereas IL-1 alpha and IL-1 beta were undetectable . The cells exposed to HIV-1SF2 gp120 increased the release of IL-6 only . The gp120-induced effects were significantly enhanced by priming glial cells with IFN-gamma . To investigate the cellular sources and mechanisms of the gp120-induced IL-6 production, in situ hybridization with mRNA for IL-6 was performed in HIV-1IIIB gp120- or HIV-1SF2 gp120-stimulated microgliaenriched or astrocyte-enriched cultures . HIV-1IIIB gp120 or HIV-1SF2 gp120 induced the expression of IL-6 mRNA in both microglia-enriched and astrocyte-enriched cultures, indicating that both microglia and astrocytes produce IL-6, and that the transcriptional regulation is involved in the gp120-induced IL-6 production . Taken together, these results demonstrate that the production of NO, TNF-alpha, IL-1, or IL-6 from glial cells is differentially regulated by HIV-1IIIB gp120 and HIV-1SF2 gp120 . These results may provide insights into the roles of NO and proinflammatory cytokines in the neurotoxicity of gp120s and the neuropathology of different strains of HIV-1 viruses.

Brain Res, 1996 Aug 19, 730(1-2), 67 - 74
Dexamethasone and forskolin synergistically increase {Met5}enkephalin accumulation in mixed brain cell cultures; McMillian MK et al.; Possible synergistic effects of the glucocorticoid dexamethasone (DEX, 10(-7) M) and the adenylate cyclase agonist forskolin (FSK, 10(-5) M) on {Met5}enkephalin (ME) accumulation were examined in enriched rat glial cultures and in mixed neuronal/glial cultures . In enriched glial cultures, DEX and FSK each stimulated the accumulation of ME 2-3-fold over basal media levels, but there was little additional stimulation when these agonists were combined . In contrast, mixed neuronal/glial cultures showed only weak responses to DEX or FSK alone, but the combination of these agonists produced a pronounced synergistic effect on media ME accumulation (6-10-fold over basal levels) . The DEX effect was mediated via a classical glucocorticoid receptor, since DEX was potent (acting over a concentration range of 10(-11)-10(-7) M), mimicked by corticosterone (10(-6) M), and blocked by the glucocorticoid receptor antagonist RU486 . There was a pronounced time lag (2 days) for the synergistic effects of DEX + FSK to develop . In situ hybridization and immunocytochemical studies suggested that astrocytes were the major source for the increased ME production in all mixed neuronal/glial cultures examined . Creating a mixed culture by plating fetal neurons onto confluent, enriched P7 glial cultures inhibited accumulation of ME in the media . DEX + FSK, but neither agonist alone, overcame this neuronal inhibition and increased accumulation of media ME to levels identical to levels in stimulated enriched glial cultures . The net effect was a 6-fold increase in ME accumulation in the mixed neuronal/glial cultures relative to a 2.5-fold increase in the enriched glial cultures . Neuronal inhibition of basal glial ME production could explain the similar synergistic effects of DEX + FSK observed in all mixed neuronal/glial cultures examined, and may be important in suppressing ME production by astrocytes in the brain.

J Immunol, 1996 Aug 15, 157(4), 1415 - 21
Inhibition of transcription factor Stat1 activity in mononuclear cell cultures and T cells by the cyclic AMP signaling pathway; Ivashkiv LB et al.; Activation of T cells results in a cascade of gene activation and subsequent proliferation and differentiation into effector phenotypes . The regulation of transcription factors belonging to the signal transducer and activator of transcription (STAT) family was analyzed in PHA-activated mononuclear cells and in purified T cells activated by cross-linking cell surface CD3 . Cell activation resulted in a delayed induction of STAT DNA-binding activity, which was sustained for several days, was composed predominantly of Stat1 and Stat3, and was blocked by cycloheximide and actinomycin D . Increased Stat1 and Stat3 mRNA and protein levels were detected, respectively 4 and 24 h after activation . Stimulation of the cAMP signal transduction pathway, which skews cytokine production toward a Th2 pattern, resulted in the preferential suppression of Stat1 activity . cAMP inhibited the induction of expression of IL-2 receptor components, but did not inhibit IL-4 receptor alpha-chain and CD69 expression or the induction of activator protein 1 transcription factors . cAMP signaling inhibited Stat1 at several different levels, including suppression of DNA binding and down-regulation of Stat1 protein and mRNA levels . Our results demonstrate the regulation of STAT activity by a signaling pathway that regulates the T cell functional phenotype and is distinct from the cytokine-activated Janus kinase-STAT signaling pathway.

J Immunol Methods, 1996 Aug 14, 194(2), 191 - 9
The protein hydrolysate, Primatone RL, is a cost-effective multiple growth promoter of mammalian cell culture in serum-containing and serum-free media and displays anti-apoptosis properties; Schlaeger EJ; The tryptic meat digest Primatone RL is a low-cost medium supplement of a complex nature which serves as a source of amino acids, oligopeptides, iron salts, some lipids and other trace low molecular weight substances . Its addition to mammalian and insect cell culture media significantly improves the cell growth properties of many cell lines . In this work the growth promoting effects of Primatone RL are described in more detail using different mouse hybridomas, a mouse myeloma cell line, and human promyelocytic leukemia HL-60 cells . The positive effects on cell growth induced by Primatone were observed in the presence of serum but were even more pronounced in serum-free culture . In addition the adaptation time from high serum to low (1%) or serum-free growth in the presence of Primatone is also significantly reduced . Primatone RL, when added to HL and DHI medium, improves cell growth under low serum or serum-free conditions by increasing the maximum cell numbers and in particular the viability of the culture . The observed decrease in cell death (apoptosis) induction leads to a significant improvement in antibody (recombinant protein) production by increasing the volumetric yields during long-term batch culture . The so-called anti-apoptotic effects of Primatone RL for mouse hybridomas, which is concentration dependent, is not fully understood.

Proc Natl Acad Sci U S A, 1996 Aug 6, 93(16), 8584 - 9
Perturbations in maturation of secretory proteins and their association with endoplasmic reticulum chaperones in a cell culture model for epithelial ischemia; Kuznetsov G et al.; The effects of ischemia on the maturation of secretory proteins are not well understood . Among several events that occur during ischemia-reperfusion are a rapid and extensive decrease in ATP levels and an alteration of cellular oxidative state . Since the normal folding and assembly of secretory proteins are mediated by endoplasmic reticulum (ER) molecular chaperones, the function of which depends on ATP and maintenance of an appropriate redox environment, ischemia might be expected to perturb folding of secretory proteins . In this study, whole animal and cultured cell models for the epithelial ischemic state were used to examine this possibility . After acute kidney ischemia, marked increases in the mRNA levels of the ER chaperones glucose-regulated protein (grp)78/immunoglobulin-binding protein (BiP), grp94, and ER protein (ERp)72 were noted . Likewise, when cellular ATP was depleted to less than 10% of control with antimycin A, mRNA levels of BiP, ERp72, and grp94 were increased in kidney and thyroid epithelial cell culture models . Since the signal for the up-regulation of these stress proteins is believed to be the accumulation of misfolded/misassembled secretory proteins in the ER, their induction after ischemia in vivo and antimycin treatment of cultured cells suggests that maturation of secretory proteins in the ER lumen might indeed be perturbed . To analyze the effects of antimycin A on the maturation of secretory proteins, we studied the fate of thyroglobulin (Tg), a large oligomeric secretory glycoprotein, the folding and assembly of which seems to require a variety of ER chaperones . Treatment of cultured thyroid epithelial cells with antimycin A greatly inhibited ( > 90%) the secretion of Tg . Sucrose density gradient analysis revealed that in antimycin A-treated cells Tg associates into large macromolecular complexes which, by immunofluorescence, appeared to localize to the ER . Furthermore, coimmunoprecipitation studies after antimycin A treatment demonstrated that Tg stably associates with BiP, grp94, and ERp72 . Together, our results suggest that a key cellular lesion in ischemia is the misfolding of secretory proteins as they transit the ER, and this leads not only to increased expression of ER chaperones but also to their stable association with and the subsequent retention of at least some misfolded secretory proteins.

Cytokine, 1996 Aug, 8(8), 622 - 30
Inflammatory cytokines induce intercellular adhesion molecule-1 (ICAM-1) mRNA synthesis and protein secretion by human retinal pigment epithelial cell cultures; Nagineni CN et al.; Retinal inflammatory diseases in man are associated with an upregulation in the expression of intercellular adhesion molecule-1 (ICAM-1) in cells within the retina and with an increase in soluble ICAM-1 within the vitreous . These studies suggest that this protein may contribute to immunopathological processes within the eye . The effects of inflammatory mediators on the regulation of the expression and secretion of ICAM-1 by human retinal pigment epithelial cell cultures (HRPE) were investigated in order to identify the possible source of soluble ICAM-1 and the conditions which enhance its production . Immunofluorescence studies on TNF-alpha and/or IFN-gamma treated HRPE cells demonstrated cellular expression of ICAM-1 which was predominantly localized to intercellular junctions . Moreover, treatment of HRPE for 24 h with tumour necrosis factor alpha (TNF-alpha) (10 ng/ml), interferon gamma (IFN-gamma) (500 u/ml), interleukin 1 alpha (IL-1 alpha) (10 ng/ml) and IL-1 beta (10 ng/ml) results in the secretion of ICAM-1, ranging from 9 to 13 ng per 10(6) cells . IFN-gamma acts synergistically with (TNF-alpha) and IL-1 in the secretion of ICAM-1 by HRPE . Only 1.75 ng of soluble ICAM-1 was detected in untreated HRPE cells . In contrast, lipopolysaccharide (LPS), IL-6, IFN-alpha or TGF-beta did not exhibit any influence on ICAM-1 secretion by these cells . Northern blot analysis reveals an increased expression of ICAM-1 mRNA in HRPE stimulated with IFN-gamma, TNF-alpha or IL-1 for 24 h . In untreated cells, ICAM-1 mRNA is not detectable . There is a progressive increase in ICAM-1 mRNA levels in cytokine-treated HRPE, that reaches steady state by 12 h . Furthermore, a close correlation is noted between ICAM-1 mRNA levels and the secretion of ICAM-1 protein, suggesting regulation at the level of gene transcription . ICAM-1 secretion by RPE might actively participate in the immune reactions in the retina, by recruiting and activating lymphocytes, and contribute to the immunopathological processes in inflammatory diseases.

J Dairy Sci, 1996 Aug, 79(8), 1353 - 60
Cell culture system for studying bovine neutrophil diapedesis; Smits E et al.; Neutrophils are the major defense against bacterial infection in the bovine mammary gland . Neutrophils migrate from blood into the lumen of the gland in response to inflammatory stimuli . This study describes the development of a system of cell culture that can be used to study neutrophil diapedesis through secretory and ductal mammary epithelial barriers . The culture system consists of successive layers of collagen, fibroblasts, collagen, and a confluent monolayer of secretory or ductal epithelial cells layered on a porous membrane . Confluence was determined by electrical resistance and trypan blue diffusion . Neutrophil diapedesis occurred from the basal to the apical surface of the monolayers . Purified complement C5a, fetal bovine serum that had been activated by zymosan, and fetal bovine serum that had been activated by Escherichia coli induced neutrophil diapedesis . Neutrophil diapedesis was greater across ductal cell monolayers . Blood neutrophils from five cows differed in their ability to migrate through the multilayered culture system in response to C5a . Monoclonal antibodies to C5a blocked diapedesis induced by purified C5a but had no effect on diapedesis induced by fetal bovine serum that had been activated by zymosan or by fetal bovine serum that had been activated by E . coli endotoxin, indicating that factors other than C5a were chemotactic for neutrophils . Monomeric IgG2, immune complexes, and E . coli endotoxin did not induce neutrophil diapedesis.

Prenat Diagn, 1996 Aug, 16(8), 749 - 54
Trisomy 16 mosaicism in amniotic fluid cell cultures; Tantravahi U et al.; Trisomy 16 mosaicism was found in amniotic fluid cells in a patient undergoing amniocentesis because of elevated second-trimester maternal serum alpha-fetoprotein (MSAFP) (2.80 MOM), a markedly elevated human chorionic gonadotropin level (hCG) (12.02 MOM), and a Down syndrome risk of 1:55 . Ultrasound evaluation of the fetus indicated the presence of an atrial septal defect and clinodactyly . Cytogenetic analyses of various fetal tissues using fluorescence in situ hybridization (FISH) failed to detect substantial numbers of trisomy 16 cells; however, trisomy 16 mosaicism was identified in placental tissue . Molecular genetic analysis at five different loci {four analysed by polymerase chain reaction (PCR) and one by Southern blot analysis} failed to show any evidence for uniparental disomy . Although trisomy 16 cells could not be clearly demonstrated in the fetus, the presence of a clinically significant proportion of aneuploid cells early in development could not be excluded and it therefore cannot be assumed that a 'confined placental mosaicism' existed . The markedly elevated hCG and elevated MSAFP levels are consistent with abnormal placental function in trisomy 16 mosaicism . Serial ultrasound evaluation (to detect any late-onset growth retardation) and fetal echocardiography may be indicated for patients with extraordinarily high levels of hCG, especially if MSAFP is also elevated.

J Vet Med Sci, 1996 Aug, 58(8), 805 - 7
Serological property and replication in cell cultures of porcine reproductive and respiratory syndrome viruses isolated in Japan; Shibata I et al.; The serological property and replication in swine alveolar macrophages (SAM) and MARC-145 cell cultures of 35 porcine reproductive and respiratory syndrome (PRRS) viruses isolated in SAM were investigated . All the isolates were reacted almost equally with antisera against three Japanese isolates including EDRD-1 strain (American type) in immunoperoxidase monolayer assay (IPMA), but weakly or did not react with antiserum against Lelystad virus (European type) indicating that the Japanese isolates are more closely related to an American type virus . Twenty two of 35 isolates replicated with CPE both in SAM and MARC-145 cells, whereas remaining 13 isolates did not show CPE in MARC-145 cells . The antigenicity of the isolates did not relate to the virus origin and the replication in the cell cultures.

Int J Dev Biol, 1996 Aug, 40(4), 813 - 6
Can insights into urodele limb regeneration be achieved with cell cultures and retroviruses?
Tsonis PA, Del Rio-Tsonis K, Wallace JL, Burns JC, Hofmann MC, Millan JL, Washabaugh CH.
Recent advances in the field of amphibian limb regeneration have provided insights into its cellular and molecular events . This review summarizes the development of cell lines from limb tissues and their application to the study of transdifferentiation and limb regeneration . In addition, the availability of suitable retroviral vectors for salamanders is discussed for it has opened new avenues for experimentation at the molecular level.

Br J Surg, 1996 Aug, 83(8), 1148 - 51
Prognostic value of monolayer culture patterning in primary cell culture of oesophageal cancer; Shimada Y et al.; The growth of primary cell cultures of oesophageal cancer was compared with the clinical outcome of patients from whom the cancers were taken . Ninety-three patients underwent curative resection, with no operative deaths, and were divided into three groups according to the monolayer culture pattern of the primary cell culture: culture from 43 patients (46 per cent) grew no malignant cells (group 1), 21 (23 per cent) produced monolayer epithelial growth (group 2) and the rest (from 29 patients) established cell lines (group 3) . The 5-year survival rate of patients in group 2 (29 per cent) and group 3 (23 per cent) was significantly lower (P < 0.005) than that of those in group 1 (51 per cent) . Monolayer epithelial growth potential is a significant prognostic factor in patients with oesophageal cancer.

Virology, 1996 Aug 1, 222(1), 21 - 30
Restrictive type of replication of ovine/caprine lentiviruses in ovine fibroblast cell cultures; Chebloune Y et al.; Caprine arthritis-encephalitis virus (CAEV) is a natural lentivirus pathogen of goats . CAEV, like all members of the ovine/ caprine lentivirus family, has an in vivo tropism for cells of the monocyte/macrophage cell lineage and activation of viral gene expression is observed only following differentiation of monocytes to macrophages . In addition to cells of the monocyte/ macrophage lineage, CAEV and the closely related maedi visna virus of sheep (MVV) can also replicate productively in fibro-epithelial cells derived from synovial membrane of goats (GSM) . However, these viruses varied greatly in their ability to replicate in fibroblasts . We studied the biological and biochemical properties of CAEV and maedi-visna virus (MVV) of sheep following inoculation into the three ovine/caprine cell types . Our data showed no substantial differences in virus titers, viral protein biosynthesis, or processing of the viral proteins between CAEV and MVV following inoculation into primary macrophages and GSM cells . However, unlike MVV, CAEV failed to replicate productively in ovine fibroblasts (sheep choroid plexus cells) . This correlated with a specific but abnormal proteolytic cleavage of the envelope glycoprotein of the virus . This abnormal proteolytic cleavage represents a novel type of host cell restriction of lentivirus replication.

Jpn J Cancer Res, 1996 Aug, 87(8), 805 - 15
Simultaneous measurement of unscheduled and replicating DNA synthesis by means of a new cell culture insert DNA retention method: rapid induction of replicating DNA synthesis in response to genotoxic carcinogens; Okumura A et al.; In order to measure simultaneously replicating DNA synthesis (RDS) and unscheduled DNA synthesis (UDS) in rat hepatocytes responding to exposure to carcinogens, a new method, namely the "cell culture insert DNA retention (CDR)" method, was developed . All CDR procedures for cell culture, digestion of cytoplasm and retention of DNA were performed on membranes attached to cell culture containers . Four subgroups of primary cultures of hepatocytes prepared from rats were exposed to a genotoxic or non-genotoxic carcinogen with or without 10 mM hydroxyurea and incubated for 4 h with 10 microCi/ml {3H}thymidine . The membranes were then processed for both liquid scintillation and autoradiography . Among seven tested chemicals, three genotoxic agents, 3,2'-dimethyl-4-aminobiphenyl, 2-acetylaminofluorene and diethylnitrosamine, and two non-genotoxic carcinogens, nafenopin and phenobarbital, induced RDS within 4 h after the exposure, indicating that these carcinogenic agents induce cell proliferation is non-proliferating rat hepatocytes prior to the emergence of genotoxic changes . Several indices were devised to characterize the genotoxicity of the tested chemicals . The induction patterns obtained showed a wide variation in the individual characteristics of carcinogen-induced genotoxicity and mitogenicity in the early phase of initiation . This is the first report of simultaneous measurement, by using a combination of autoradiography and liquid scintillation, of UDS and RDS induced in rat hepatocytes . The described CDR approach will be useful for risk assessment and characterization of carcinogenic and tumor-promoting agents.

Planta Med, 1996 Aug, 62(4), 359 - 60
Production of methyl 3-oxoartemisinate from methyl artemisinate by suspension cell culture of Mentha piperita; Kim SU et al.; Methyl artemisinate was fed to the suspension cell culture of Mentha piperita . The biotransformation product was isolated and identified as a novel compound, methyl 3-oxoartemisinate . The Mentha cells were apparently capable of extensively oxidizing at the allylic C-3 position, to give rise to an oxo group . The conversion of the fed methyl ester of the acid reached a maximum in 48 h with 5.5% conversion . The physicochemical data of the oxo compound are presented.

Ann Thorac Surg, 1996 Aug, 62(2), 526 - 32
Assessment of endothelial preservation in human cell cultures; Eberl T et al.; BACKGROUND . Impairment of microcirculation due to endothelial cell damage must be considered a limiting factor in organ preservation . The present study aims at a quantitative assessment of preservation-induced injury in cultured human endothelial cells . METHODS . Monolayer cultures of human umbilical vein endothelial cells were exposed to cold (40 degrees C) hypoxic storage in University of Wisconsin solution, histidine-tryptophane-ketoglutarate solution, Euro-Collins solution, and saline solution . Cellular integrity was evaluated by viable cell count, ultrastructural analysis, and prostacyclin release after 24, 48, and 72 hours of storage and subsequent 6 hours of reincubation in culture medium at 37 degrees C . Expression of intercellular adhesion molecule-1 was investigated after 6, 12, and 24 hours of cold preservation and after 6 hours of rewarming . RESULTS . Cellular viability was best maintained with University of Wisconsin and histidine-tryptophane-ketoglutarate solutions with no significant reduction of cell count up to 72 hours; Euro-Collins solution and saline solution caused a significant decline in cell numbers after 24 hours (p < 0.05) . Morphology was best preserved by University of Wisconsin solution . Prostacyclin values were elevated after 24 hours in Euro-Collins solution and saline solution, after 48 hours in histidine-tryptophane-ketoglutarate, Euro-Collins, and saline solutions, and after 72 hours in Euro-Collins solution (p < 0.05, compared with University of Wisconsin solution) . ICAM expression was weak after cold storage (24 hours) in University of Wisconsin solution, moderate after incubation in histidine-tryptophane-ketoglutarate and Euro-Collins solutions and intensive after storage in saline solution . In contrast, rewarming caused intensive expression of intercellular adhesion molecule-1 in all experimental groups as compared with controls, which showed baseline expression at any time . CONCLUSIONS . From our results we conclude that in this model cellular integrity is best protected by University of Wisconsin solution, increased prostacyclin release is consistent with morphologic alterations and intercellular adhesion molecule-1 expression is clearly up-regulated in endothelial cells under reperfusion conditions after cold hypoxic storage.

J Parasitol, 1996 Aug, 82(4), 638 - 40
Efficacy of serine protease inhibitors against Cryptosporidium parvum infection in a bovine fallopian tube epithelial cell culture system; Forney JR et al.; The anticryptosporidial potential of the protease inhibitors alpha-1-antitrypsin (AAT), antipain, aprotinin, leupeptin, methoxysuccinyl-ala-ala-pro-valine chloromethylketone (MAAPVCK), soybean trypsin inhibitor (SBTI), and phenylmethylsulfonyl fluoride (PMSF) was evaluated in a bovine fallopian tube epithelial (BFTE) cell culture system . Protease inhibitor concentrations of 5, 10, 50, 100, and 500 micrograms/ ml (PMSF at 1, 2, and 3 mM) in RPMI medium were mixed with Cryptosporidium parvum oocysts and used to inoculate BFTE cell monolayers . At 24 hr postinoculation (candlejar/37 C), cells were rinsed with RPMI medium, fixed in methanol, and stained with Giemsa . Parasites were enumerated in cell monolayers by brightfield microscopy . The mean number of parasites counted in each protease inhibitor treatment group was expressed as a percentage of the mean number of parasites counted in an infection control group . Leupeptin and SBTI reduced parasite numbers to 40-50% of the control mean at 500 micrograms/ml: AAT, antipain, and aprotinin reduced parasite numbers to 10-15% at the same concentration . PMSF reduced parasite numbers to 40% of the control mean at 3 mM . MAAPVCK did not significantly inhibit cryptosporidial infection . These findings suggest that a protease component of C . parvum may be essential for host cell infection.

Ann N Y Acad Sci, 1996 Jul 23, 791, 24 - 34
Amino acid and protein depletion in medium of cell cultures infected with Cowdria ruminantium; Neitz AW et al.; Cowdria ruminantium (Rickettsiales) causes heartwater in ruminants of Africa, and some islands off Africa and in the Caribbean Sea . The in vitro culture method for the organism devised in 1985, which provided for the first time a means for production of adequate quantities of live organisms and their products, is erratic and requires improvement . We studied depletion of amino acids (AAs) and major proteins in culture medium taken daily from infected and uninfected ovine and bovine vascular endothelial cell cultures . AAs of these samples were analyzed by Pico Tag reversed phase HPLC precolumn derivatization, and major proteins determined by capillary electrophoresis using a 57 cm x 75 microns fused silica tube at high pH . In both ovine and bovine cell cultures, significant depletion of arginine and glutamine occurred over a 5-day observation period regardless of whether they were infected or uninfected . This indicates that supplementation of nutrient media with these AAs might improve conditions for growth of the organism . Both AAs are essential for survival of cultured cells, and probably for the rickettsia (although the metabolism of C . ruminantium is poorly understood) . Concentrations of several AAs increased in infected cultures, implying de novo synthesis and/or proteolysis on the part of the organism . In fact, several protein fractions did decrease in culture medium throughout the course of infection, while increasing or remaining unchanged in uninfected control cultures . Proteolytic activity by C . ruminantium may be essential for nitrogen metabolism by the organism . It is suggested that studies such as these will facilitate the development of a specific medium for optimal in vitro growth of the heartwater organism, and may also lead to an understanding of the metabolic stratagem of C . ruminantium . This knowledge, in turn, could reveal the mechanism for pathogenesis of heartwater, with implications for control.

J Biochem Biophys Methods, 1996 Jul 10, 32(3), 191 - 4
A new microcatheter enables microcalorimetric assessment of thermogenesis after stimulation in cell cultures; Bottcher H et al.; A new, inexpensive technique facilitates the observation of long-lasting effects (> 2 h) of thermogenic agents on cell cultures . A special microcatheter enables addition of hormones or pharmacologic agents to biological samples during microcalorimetric analysis using a ThermoMetric ThermalActivityMonitor without removing the sample ampoules from the measuring position . Technical modification of the calorimeter is not required . Baseline stability with the connected catheter is +/- 0.2 microW over 10 h . The error of the method of catecholamine induced thermogenesis in white adipocytes is 13.4% (C.V.).

Biotechnol Prog, 1996 Jul-Aug, 12(4), 457 - 65
Metabolic inhibitors, elicitors, and precursors as tools for probing yield limitation in taxane production by Taxus chinensis cell cultures; Srinivasan V et al.; Inhibition of biosynthetic enzymes and translation and translocation processes, elicitation, and precursor feeding were used to probe biosynthetic pathway compartmentation, substrate-product relationships, and yield limitation of the diterpenoid taxanes in cell cultures of Taxus chinensis (PRO1-95) . The results suggest the following: (i) the source of isopentenyl pyrophosphate in taxane production is likely plastidic rather than cytoplasmic; (ii) baccatin III may not be a direct precusor of Taxol (Taxol is a registered trademark of Bristol-Myers Squibb for paclitaxel); (iii) baccatin III appears to have cytoplasmic and plastidic biosynthetic components, while Taxol production is essentially plastidic; and (iv) arachidonic acid specifically stimulates Taxol production but does not have a significant effect on baccatin III yield . Semiempirical mathematical models were used to describe these results and predict potential yield-limiting steps . Model simulations suggest that, under current operating conditions, Taxol production in Taxus chinensis (PRO1-95) cultures is limited by the ability of the cells to convert phenylalanine to phenylisoserine rather than by the branch-point acyl transferase . This result is supported by the lack of improvement of Taxol yield by feeding phenylalanine or benzoylglycine . The methods described in this article, while specifically expanding our knowledge of taxane production in PRO1-95 cultures, could be generally useful in investigating complex aspects of secondary metabolic pathways in plant cell cultures, especially when details of the pathway and compartmentation are sparse.

J Androl, 1996 Jul-Aug, 17(4), 375 - 81
Enzymatic digestion of bradykinin by rat Sertoli cell cultures; Monsees TK et al.; Sertoli cells play a key role in spermatogenesis . To study the involvement of the kallikrein-kinin system in the testis, the pattern of bradykinin-inactivating kininases in rat Sertoli cells was investigated . Exogenous bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) is cleaved at Pro7-Phe8, Phe5-Ser6, and Gly4-Phe5, as demonstrated by high performance liquid chromatography analysis . Degradation of bradykinin was strongly inhibited by phosphoramidon and thiorphan, which are specific inhibitors of neutral metalloendopeptidase-24.11 . The kininase type II-specific inhibitors, captopril and enalapril, were only partially effective in preventing peptidolysis . This indicates that the main kininases responsible for rapid bradykinin inactivation are neutral metalloendopeptidase and, to a lesser extent, kininase type II . Neutral metalloendopeptidase and kininase type II were shown to be located on Sertoli cell membranes . A low degree of bradykinin degradation was detected by simultaneous inhibition of neutral metalloendopeptidase-24.11 and kininase II, pointing out the involvement of further peptidases with minor activities . This remaining activity is probably not due to the action of kininase type I or cysteine proteases, as shown by specific inhibitors . The data presented indicate the occurrence of membrane-bound kininases, which are an important part of the kallikrein-kinin system, in rat Sertoli cell cultures.

J Neurosci Methods, 1996 Jul, 67(1), 53 - 6
A modified catalase assay suitable for a plate reader and for the analysis of brain cell cultures; Cohen G et al.; The enzyme catalase is present in relatively small amounts in neural tissue . A standard spectrophotometric assay for catalase has been modified to make it suitable both for automated assay with a plate reader and for the analysis of neural cell cultures . Catalase activity is determined by measuring residual hydrogen peroxide after incubation with the enzyme . Ferrous ions and thiocyanate are used for the spectrophotometric determination of hydrogen peroxide . The stable visible absorption of ferrithiocyanate can be measured either with a plate reader or a spectrophotometer . The method assays catalase activity from single wells of a tissue culture plate containing 2-3 mg of embryonic rat mesencephalon.

Anal Cell Pathol, 1996 Jul, 11(2), 127 - 36
Comparative flow cytometric analysis of DNA-bound PCNA and DNA content as estimators of S-phase cells in cell cultures; Bustamante AS et al.; Flow cytometric estimations of S-phase cells were carried out on cultures from three different cell lines and in frozen aliquots . A PCNA-extraction protocol was applied . Measurements of the S fraction estimated from bivariate PCNA/DNA analysis after detergent extraction of DNA non-bound PCNA were compared with those obtained from total DNA histograms (Vindelov and Christensen's technique, methanol-fixed whole cells and PCNA-extracted nuclei) . No significant differences between methods, or between fresh and frozen specimens, were found in the measurements of the percentage of S-phase cells . Nevertheless, nuclei yield following PCNA extraction was highly variable, ranging from 63% to 10% (mean: 26%) . In some cases, the extraction was not complete and samples had to be discarded . Usually, boundaries between S-phase events and G0/G1 or G2/M subpopulations were not clearly defined . Because of these shortcomings, and the fact that is more costly and time consuming, the estimation of the S-phase fraction by means of bivariate DNA-bound PCNA/total DNA flow cytometric studies does not seem to surpass that obtained from standard DNA cell cycle analyses.

Pharm Res, 1996 Jul, 13(7), 978 - 88
In-vitro cell culture models of the nasal epithelium: a comparative histochemical investigation of their suitability for drug transport studies; Werner U et al.; PURPOSE: To evaluate different in-vitro cell culture models for their suitability to study drug transport through cell monolayers . METHODS: Bovine turbinate cells (BT; ATCC CRL 1390), human nasal septum tumor cells (RPMI, 2650; ATCC CCL 30), and primary cell cultures of human nasal epithelium were characterized morphologically and histochemically by their lectin binding properties . The development of tight junctions in culture was monitored by actin staining and transepithelial electrical resistance measurements . RESULTS: The binding pattern of thin-sections of excised human nasal respiratory epithelium was characterized using a pannel of fluorescently-labelled lectins . Mucus in goblet cells was stained by PNA, WGA and SBA, demonstrating the presence of terminal N-acetylglucosamine, N-acetylgalactosamine and galactose residues respectively in the mucus of human nasal cells . Ciliated cells revealed binding sites for N-acetylglucosamine, stained by WGA, whereas Con A, characteristic for mannose moieties, labelled the apical cytoplasm of epithelial cells . Binding sites for DBA were not present in this tissue . Comparing three different cell culture models: BT, RPMI 2650, and human nasal cells in primary culture using three lectins (PNA, WGA, Con A) as well as intracellular actin staining and transepithelial electrical resistance measurements we found, that only human nasal epithelial cells in primary culture showed differentiated epithelial cells, ciliated nasal cells and mucus producing goblet cells, which developed confluent cell monolayers with tight junctions . CONCLUSIONS: Of the in-vitro cell culture models studied, only human nasal cells in primary culture appears to be suitable for drug transport studies.

Am J Reprod Immunol, 1996 Jul, 36(1), 58 - 63
The role of estradiol, progesterone, and transforming growth factor on human endometrioma cell culture; Badawy SZ et al.; The present study demonstrates several aspects of endometrioma cells in culture, namely, 1) cell growth, proliferation, and morphology, 2) effect of cell culture on estrogen and progesterone receptor concentration, 3) effect of estradiol, progesterone, and transforming growth factor . The tissue sample was obtained from ovarian endometriomas that were removed via laparotomy or laparoscopy . The tissue sample was digested with collagenase . After washing, the tissue was cultured in endothelial cell culture medium . Cell count was done by flow cytometry . Receptor study was done by immunohistochemistry . The results demonstrated the growth and proliferation of endometrioma cell in culture medium . Electron microscopy showed stroma-like cells . The cells lost their estrogen and progesterone receptors . Estradiol and progesterone added to these cultures did not affect the rate of growth and proliferation of the cells . Transforming growth factor significantly increased the rate of growth and proliferation of these cells.

J Appl Physiol, 1996 Jul, 81(1), 164 - 71
Effects of spaceflight and recovery on rat humeri and vertebrae: histological and cell culture studies; Zerath E et al.; Skeletal changes associated with spaceflight in the rat have been well documented, but few data are available on bone tissue and bone cell metabolism after subsequent on-Earth recovery . We therefore investigated the effects of microgravity and subsequent recovery on trabecular bone morphology and cellular activities in rat humeri and thoracic vertebrae and compared histomorphometric parameters in caudal vertebrae with the behavior of vertebral osteoblastic cells in culture . We report here that humeral weight showed normal growth during the experiment but was unaffected by spaceflight or recovery from spaceflight . However, the 14-day spaceflight resulted in inhibition of static indexes of bone formation in humeral proximal metaphyses and thoracic vertebral bodies . This was associated with a decrease in bone volume in humeral metaphyses . After 14 days of on-Earth recovery, osteoblastic and osteoid surfaces returned toward normal and bone volume was normalized in humeri, whereas the static bone formation parameters were not restored in thoracic vertebrae . In addition, histological indexes of bone formation and osteoblastic cell growth in vitro were not affected by spaceflight in caudal vertebrae . This study shows that rat humeri and thoracic and caudal vertebrae exhibit different patterns of response to spaceflight and subsequent on-Earth recovery, which could be due, at least in part, to the different loading pattern of these bones, and also to differences in bone turnover rate.

Obes Res, 1996 Jul, 4(4), 357 - 66
Influence of thyroxine in vivo on preadipocyte development and insulin-like growth factor-I and IGF binding protein secretion in fetal stromal vascular cell cultures; Chen NX et al.; Studies were conducted to determine the influence of thyroxine (T4) in vivo on preadipocyte development and insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) secretion in stromal-vascular (S-V) cultures . Fetal pigs were hypophysectomized (hypox) at 70 days of gestation, implanted with T4 pellets, and fetuses from the dam at 75 days of gestation . In a second experiment, hypox and T4 implantation were performed at 75 days and fetal pigs removed at 95 days of gestation . Primary cultures of stromal vascular (S-V) cells derived from fetal adipose tissue were established . Cultures were stained for morphological analysis and conditioned media were collected for IGF-1 determination by radioimmunoassay (RIA) and IGFBP analysis by Western blotting . After only 5 days of T4 treatment, fat cell cluster number and size and lipid deposition in cultures were significantly increased compared to cultures from untreated hypox fetuses . Fetal hypox reduced IGF-I secretion by preadipocytes at both ages and T4 treatment completely normalized IGF-I secretion (p < 0.05) . Four IGFBPs (BP-1, BP-2, BP-3 & BP-4) detected in S-V cultures derived from 95-day fetuses were decreased in concentration by hypox by 44 +/- 9.4%, 32 +/- 9.7%, 42 +/- 12% and 53 +/- 6.9% . In cultures derived from T4 treated hypox fetuses, the levels of these four IGFBPs were increased by 187 +/- 25%, 239 +/- 38%, 190 +/- 5% and 347 +/- 43% over control values, respectively . In cultures from 75-day fetuses, only IGFBP-2 (major one) and BP-1 (minor one) were detected and their secretion was also decreased by hypox and elevated by T4 treatment (190 +/- 49.5%, 156 +/- 30%, respectively, of controls) . The results provide direct evidence that T4 has a major influence on fetal preadipocyte development . T4 stimulated production of IGF-I and IGFBP in fetal S-V cultures, which in turn, may have mediated the capability of T4 to enhance fetal adipose tissue development.

J Pharm Sci, 1996 Jul, 85(7), 767 - 72
Uptake characteristics of loracarbef and cephalexin in the Caco-2 cell culture model: effects of the proton gradient and possible presence of a distinctive second component; Hu M et al.; The mechanisms of apical (AP) uptake of cephalexin (CEPH) and loracarbef (LOR) in the absence or presence of an (extemally imposed) proton gradient were determined using well-stirred diffusion chambers that minimize the effects of the unstirred water layer . The results indicated that, compared to AP uptake in the presence of an imposed proton gradient, AP uptake in the absence of an imposed proton gradient had higher K(m) values and lower Jmax values . Furthermore, when inhibition studies were performed in the absence of a proton gradient, only natural peptides were effective, whereas the peptide analogs (e.g., enalapril) were not . In addition to the effects of concentration and competitive inhibitors, the results also indicated that (1) the AP uptake of both drugs was decreased more than 60% by FCCP, regardless of whether the proton gradient was present or absent; (2) effects of protein kinase C promoter were dependent upon the presence of a proton gradient; and (3) AP uptake in the presence of an imposed proton gradient was not affected by feeding restriction, whereas AP uptake in the absence of an imposed proton gradient was . These results showed for the first time that two substrates with similar AP uptake characteristics in the presence of an imposed proton gradient may not share those characteristics in the absence of an imposed proton gradient . Taken together, these results suggest that the AP uptake component that functions in the absence of an imposed proton gradient is distinctly different from the one that functions in the presence of an imposed proton gradient . Data generated from the present study and those in the literature lend support to the hypothesis that this distinctive component represents the second binding site on the AP peptide transporter . However, an alternative hypothesis that there are two AP peptide transporters remains to be disapproved.

Cell Tissue Res, 1996 Jul, 285(1), 171 - 6
Keratin 19 in the adult human prostate: tissue and cell culture studies; Peehl DM et al.; Keratin 19 is found primarily in simple epithelia . In mammary epithelia, keratin 19 was localized to a subset of luminal cells, suggesting that keratin 19-negative cells may be the proliferative compartment of the secretory cell lineage . The structural and functional similarities of prostate and breast led us to examine keratin 19 expression in the prostate . Immunohistochemical studies revealed that keratin 19 expression was heterogeneous and frequently occurred in basal as well as in luminal cells of normal, dysplastic, and benign hyperplastic tissues . Keratin 19 was observed in cancer, but usually in a minority of cells . This was in dramatic contrast to invasive breast cancers, which are reportedly uniformly positive for keratin 19 . Prostatic epithelial cells cultured from tissues of all histologies expressed keratin 19 . In summary, keratin 19 does not obviously correlate with any epithelial cell lineage or phenotype in the prostate.

Eur J Immunol, 1996 Jul, 26(7), 1565 - 70
Early message expression of interleukin-4 and interferon-gamma, but not of interleukin-2 and interleukin-10, reflects later polarization of primary CD4+ T cell cultures; Gollob KJ et al.; The CD4+ T cell subpopulation identified by the Mel-14high phenotype represents naive T cells that develop into T helper 1 (Th1) cells upon stimulation with anti-CD3 and interleukin (IL)-2; however, the addition of IL-4 induces the development of high IL-4, IL-5 and IL-10-secreting cells (IL-4 SC) . Here, we investigate the timing of cytokine gene expression in purified naive, Mel-14high CD4+ T cells after stimulation under conditions that induce one of three dramatically different populations following secondary stimulation: anti-CD3 + IL-2 (Th1), anti-CD3 + IL-2 and IL-4 (IL-4 SC) and anti-CD3 + anti-interferon (IFN)-gamma/anti-IL-4 (null population) . The skewing toward IL-4 SC development induced by IL-4 in the primary stimulation was clearly visible by 40 h . The message for IL-4 was absent at 20 h after stimulation, but was induced 30-100-fold by 40 h and was accompanied by a decrease in IFN-gamma message . The message for IL-5 was undetectable . The development of Mel-14high T cells into a Th1 population follows kinetics similar to those of the development of IL-4 SC in that IFN-gamma gene transcription was undetectable at 20 h and induced 100-fold by 40 h post-stimulation . This induction was inhibited by the inclusion of anti-IFN-gamma/anti-IL-4 during the primary stimulation . Finally, IL-10 message is present in both cultures that developed into IL-4 SC and those that developed into Th1 populations, in contrast to the differentiated cultures where there is a segregation between IL-10 and IFN-gamma expression . Undetectable levels of message for both IL-2 and IFN-gamma were seen in the null cultures, which expressed both IL-2 and IL-10 message . T cells identified by the Mel-14low phenotype develop into a population that secretes high levels of all cytokines tested, and this phenotype was revealed within 20 h after stimulation as determined by message analysis . These results demonstrate an early correlation of gene transcription for IL-4 and IFN-gamma, with the development of IL-4 SC and Th1 cells, and a lack of a correlation at these early time points for expression of IL-2, IL-5 and IL-10.

Kaohsiung J Med Sci, 1996 Jul, 12(7), 394 - 9
Human prolactinomas in cell culture: structure-function correlation and indication of early relapse; Wang CJ et al.; Cell cultures of 7 micro- and 16 macroprolactinomas obtained from twenty-three patients (10 males and 13 females) by transsphenoidal adenomectomies were found to be capable of secreting hormones in vitro . Prolactin levels in the culture media were measured during the culture periods and showed that the high PRL levels in culture media may indicate early relapse . Morphological features of both micro- and macroprolactinomas in vitro resembled those of the original tumors . Most macroprolactinomas harbored densely-granulated cells, while most microprolactinomas sparsely-granulated cells . The cell culture techniques combined with morphological studies of prolactinomas allow further investigation of structure-function correlation in human prolactinomas.

J Med Entomol, 1996 Jul, 33(4), 656 - 64
Establishment of the tick (Acari:Ixodidae)-borne cattle pathogen Anaplasma marginale (Rickettsiales:Anaplasmataceae) in tick cell culture; Munderloh UG et al.; Anaplasma marginale is a tick-borne rickettsia that causes bovine anaplasmosis worldwide . Despite its importance, A . marginale has thus far not been established in a continuous culture system . We have propagated A . marginale continuously for the 1st time in a tick cell line derived from the black-legged tick, Ixodes scapularis Say, using infected bovine blood as the inoculum . Erythrocytic stages invaded the tick cells and multiplied in membrane-lined vacuoles to form colonies typical of those observed in naturally infected ticks as demonstrated by light and electron microscopy . The rickettsiae have been passaged serially for 3 yr and have been cryopreserved in liquid nitrogen . Antigens present in A . marginale from tick cell culture were recognized by bovine immune serum against the blood stages of A . marginale . A . marginale grown in this tick cell line was infective for calves, and male ticks fed on the calves transmitted A . marginale to a susceptible calf . The ability to culture A . marginale removes a major impediment to the study of Anaplasma biology in vitro, and will enhance development of vaccines and diagnostic tests.

Phytochemistry, 1996 Jul, 42(4), 1039 - 46
Betacyanins from plants and cell cultures of Phytolacca americana; Schliemann W et al.; Betacyanins from cell cultures of Phytolacca americana were characterized and compared with those of the stems and ripening fruits of the plant . Whereas in fruits prebetanin (betanin 6'-O-sulphate) and its isoform predominate, in the stem and cell cultures feruloylated derivatives occur as the major components . These were rigorously identified by various spectroscopic techniques (DAD-HPLC, NMR, LC-MS and electrospray MS-MS) and carbohydrate analyses as betanidin 5-O-{(5"-O-E-feruloyl)-2'-O-beta-D-apiofuranosyl} -beta-D-glucopyranoside, a new betacyanin of higher plants, and betanidin 5-O-(6'-O-E-feruloyl)-beta-D-glucopyranoside (lampranthin II), together with their isoforms.

Phytochemistry, 1996 Jul, 42(4), 1031 - 4
Aliphatic and aromatic glycosides from the cell cultures of Lycopersicon esculentum; De Rosa S et al.; The beta-D-glucoside, gentiobioside and 6-O-alpha-L-arabinosyl-beta-D-glucoside of benzyl alcohol, androsin and a new simple aliphatic glycoside, isopentylgentiobioside, have been found in the cell cultures of Lycopersicon esculentum . Their structures were elucidated from chemical and spectroscopic evidence.

Br J Cancer, 1996 Jul, 74(1), 73 - 7
Drug sensitivity testing for clinical samples from oesophageal cancer using adhesive tumour cell culture system; Terashima M et al.; A total of 83 specimens of surgically resected tumours from 78 patients with oesophageal cancer were assayed for drug sensitivity using an adhesive tumour cell culture system (LifeTrac CSA assay) . Seventyone of 83 specimens had a sufficient number of cells to permit growth in culture and 57 of 71 (80%) were evaluable for drug response . Cells (3 x 10(3) ml-1 well-1) were cultured for 14 days and exposed to drugs on days 3-8 . Growing cells were confirmed as cancer cells by immunohistochemical staining . IC90 values against several anti-cancer drugs were determined and population distributions of IC90 for each drug served as the basis for judging sensitivity . The 10th percentiles of IC90 (microgram ml-1) for CDDP, 5-FU, DOX, CPM, MTX, VP16, IFOS, VDS, BLM and CDDP + 5-FU were 0.3, 0.16, 0.005, 0.9, 0.006, 0.09, 0.8, 0.006, 0.04 and 0.15 + 0.09 respectively . The population distribution of IC90 against each drug showed a specific pattern that was very similar among histopathological gradings and stages of the disease . This system appeared to be a clinically applicable drug sensitivity test for human oesophageal cancer.

Neurosci Lett, 1996 Jun 28, 211(3), 151 - 4
Promotion of synthetic amyloid beta-peptide fibrillization by cell culture media and cessation of fibrillization by serum; Wegiel J et al.; Dulbecco's modified Eagle's medium promotes aggregation and fibrillization of the synthetic amyloid beta 1-40 and beta 1-42 peptides more than RPMI and OPTI media . Fibrillization in all of these media is faster than in phosphate-buffered saline and Tris buffer . Normal and heat-inactivated fetal bovine and human serum abolish amyloid fibril formation in buffers and cell culture media . Fibrillar amyloid formed during 2-day-long incubation in cell culture media and buffers is defibrillized by 1-day-long treatment with human and bovine serum . This study indicates that amyloid beta fibrillogenesis in cell culture should be studied in serum-free media or in media with a low concentration of serum.

J Biotechnol, 1996 Jun 27, 47(2-3), 203 - 14
The C5 Unit: a semi-automatic cell culture device suitable for experiments under microgravity; Vens C et al.; This paper presents data on a novel, semi-automatic cell culturing device called 'C5 Unit' (connectable circuit cell culture chamber) which was developed and adapted to the quality and size criteria set by the characteristics of the ESA Biorack . The suitability of the hardware for culturing cells under microgravity conditions was demonstrated by successful culture of primary mouse cells from neonatal cerebellum and testis aboard the Space Shuttle during the IML-2 mission.

Toxicology, 1996 Jun 17, 110(1-3), 27 - 37
The antimalarials quinacrine and chloroquine induce weak lysosomal storage of sulphated glycosaminoglycans in cell culture and in vivo; Lullmann-Rauch R et al.; The antimalarial agents quinacrine and chloroquine are well known as potent inducers of lysosomal storage of polar lipids (lipidosis) in cell culture and in vivo . In previous experiments on cultured fibroblasts, chloroquine was shown to additionally cause weak lysosomal storage of sulphated glycosaminoglycans (GAGs) thus inducing mucopolysaccharidosis (MPS) . In the present study, quinacrine was investigated for this ability, because we wished to know whether or not the acridine ring system in quinacrine would enhance the MPS-inducing potency as compared to chloroquine carrying an isoquinoline ring system . Tilorone (2,7-bis{2-(diethylamino)ethoxy}fluoren-9-one) known as a potent inducer of MPS served as reference compound . The compounds were compared at a concentration (3 microM) which did not enhance the secretion of the lysosomal enzyme beta-hexosaminidase (E.C . 3.2.1.52), since this would be an indication of unspecific drug effects upon the endosomal/lysosomal compartments of the cell . Additionally the liver of quinacrine- and chloroquine-treated rats was examined with the question whether the lysosomal GAG storage induced by either drug in cell culture had an equivalent in intact organisms . Both, in cell culture and in vivo, quinacrine was found to be a more potent inducer of lysosomal GAG storage than was chloroquine . The results suggest that the acridine ring system favours this drug side effect as compared with the bicyclic isoquinoline ring system . On the other hand, quinacrine was significantly less potent than tilorone and the Symmetrically substituted acridine derivative 3,6-bis{2-(diethylamino)ethoxy}acridine investigated previously . This suggests that the asymmetric structure of the quinacrine molecule reduces the potency as compared to the symmetrically substituted bisbasic compounds with planary tricyclic ring systems such as tilorone and congeners.

J Clin Invest, 1996 Jun 15, 97(12), 2833 - 41
Paradoxical effects of a synthetic metalloproteinase inhibitor that blocks both p55 and p75 TNF receptor shedding and TNF alpha processing in RA synovial membrane cell cultures; Williams LM et al.; We have previously hypothesized that the pro-inflammatory cytokine TNF alpha has a pivotal role in the pathogenesis of rheumatoid arthritis (RA) . It mediates its effects by cross-linking surface p55 TNF receptors (TNF-R), which can be proteolytically cleaved to yield soluble fragments . Upon binding TNF alpha soluble TNF-R (sTNF-R) can inhibit its function . We investigated the enzymatic nature of the proteases involved in TNF-R cleavage, and found that this process is blocked by a synthetic inhibitor of matrix metallo-proteinase activity (MMP), BB-2275 . Inhibition of TNF-R cleavage was observed in a number of different cell types, as detected by retention of surface bound TNF receptor and by less sTNF-R released into the cell supernatant . The augmentation of surface TNF-R expression was of biological relevance as TNF alpha-mediated necrosis of human KYM.1D4 rhabdosarcoma cells was enhanced approximately 15-fold in the presence of BB-2275 . The addition of BB-2275 to rheumatoid synovial membrane cell cultures totally inhibited MMP activity and also significantly reduced the levels of soluble TNF alpha (P < 0.006), p55 sTNF-R (P < 0.006), and p75 sTNF-R (P < 0.004) . Paradoxically, despite the reduction in soluble TNF alpha levels, the production of IL-1 beta, IL-6, and IL-8, cytokines whose production was previously demonstrated to be inhibited by the addition of neutralizing anti-TNF alpha antibody were not down-regulated by BB-2275 . These results raise the interesting possibility that a close relationship exits between the enzyme(s) which process membrane-bound TNF alpha, and those involved in surface TNF-R cleavage . Furthermore our observations suggest that hydroxamate inhibitors of MMP activity which block TNF alpha secretion and TNF-R cleavage may not modulate down-stream effects of TNA alpha, and as such suggest that the precise specificity of these compounds will be highly relevant to their clinical efficacy in inflammatory diseases.

Biochem J, 1996 Jun 15, 316 ( Pt 3), 953 - 8
Processing of chromogranins in chromaffin cell culture: effects of reserpine and alpha-methyl-p-tyrosine; Wolkersdorfer M et al.; Bovine chromaffin cell cultures were treated with either reserpine or alpha-methyl-p-tyrosine for up to 10 days . Afterwards the cells were harvested and the degree of proteolytic processing of secretogranin II, chromogranin A and chromogranin B was determined by immunoblotting and HPLC followed by RIA . There was a significant increase in the proteolysis of all three chromogranins after 4-6 days in the presence of reserpine . The small peptides formed in the presence of reserpine in vitro are also produced in vivo . A similar effect was observed with alpha-methyl-p-tyrosine, an inhibitor of tyrosine hydroxylase, but the response took up to 10 days to develop . Both drugs decreased catecholamine levels but reserpine was more effective, reaching a high degree of depletion after 4 days . In addition, experiments in vitro indicate that low millimolar amounts of either adrenaline (IC50 5.2 mM) or noradrenaline (IC50 2.4 mM) can significantly impair the proteolytic activity of recombinant murine prohormone convertase 1 when assayed with synthetic fluorogenic and/or peptidyl substrates . We conclude that a lowering of catecholamine levels in chromaffin granules leads to a concomitant increase in proteolytic processing of all secretory peptides . Apparently within chromaffin granules the endoproteases are inhibited by catecholamines and thus their removal leads to increased proteolysis.

Anal Biochem, 1996 Jun 15, 238(1), 72 - 5
Assay for tyrosine hydroxylation activity of tyrosinase from betalain-forming plants and cell cultures; Steiner U et al.; In our studies on tyrosinase-catalyzed tyrosine hydroxylation, possibly involved in betalain biosynthesis, we have evaluated different assays for the detection and quantification of the enzymatic product Dopa with respect to sensitivity, simplicity, and suitability for automatization . A tyrosinase assay including reversed-phase high-performance liquid chromatography with isocratic elution and fluorescence detection has been developed (native fluorescence of Dopa; excitation at 281 nm, emission at 314 nm) . This improved assay was sensitive (detection limit: 2 pmol Dopa) and showed a wide linear range of Dopa detection (10 pmol-20 nmol Dopa) . The method proved to be suitable for high-performance liquid chromatography with an autosampler and has been applied for measuring tyrosinase activity of cell cultures and different tissues of Portulaca grandiflora.

J Endod, 1996 Jun, 22(6), 284 - 9
Toxicity of camphorated phenol and camphorated parachlorophenol in dental pulp cell culture; Soekanto A et al.; The toxicity of phenol, parachlorophenol, camphorated phenol, camphorated parachlorophenol, and camphor was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay on established rat dental pulp cells (RPC-C2A) . RPC-C2A cells at the confluent stage were incubated for 24 h in an experimental medium containing each compound at different concentrations . All tested drugs showed cytotoxicity in the MTT assay in a concentration-dependent manner . It is believed that camphor is a vehicle, and it reduces the toxicity of phenol and parachlorophenol . However, camphor itself showed cytotoxicity, and the addition of camphor increased the toxicity of phenol and parachlorophenol, reconfirming the cytotoxicity of these classical antiseptics.

Eur J Cell Biol, 1996 Jun, 70(2), 106 - 16
Proliferating and differentiating Schwann cell cultures from embryonic chick sciatic nerve maintained for months in vitro without antimitotics or growth factors; Weiss B et al.; In general, the available methods for culturing Schwann cells require specific antibodies and/or the addition of antimitotics to suppress fibroblasts, plus various factors to support their growth . Moreover, the maximal culture period of Schwann cells normally is limited to a few weeks . Here, three easy novel methods to culture Schwann cells from embryonic chick sciatic nerve are presented, that require no growth factors or agents elevating intracellular cAMP . In contrast to the conventional antimitotic treatment with cytosine arabinoside, we use D-valine to suppress fibroblasts . Our modified medium C leads within a few days to highly enriched Schwann cell cultures (culture I) . Passage into a serum-reduced medium D allows for differentiating longterm cultures (culture II) . In cultures I and II, the rate of cell division is low . However, after passage into serum-containing SC-medium, proliferation increases within one week to high levels (culture III) . Cultures II and III can be grown for several months, during which time spontaneous immortalization can occur . The high purity of the cultures of about 95% is assessed using glia-specific antibodies for S-100 antigen, HNK-1 epitope, glia fibrillary acidic protein (GFAP), galactocerebroside (Gal C) and 3A7 . These culture procedures are easy to perform and are suitable for differentiation, proliferation and coculturing experiments.

Glia, 1996 Jun, 17(2), 94 - 102
Gradual inhibition of inducible nitric oxide synthase but not of interleukin-1 beta production in rat microglial cells of endotoxin-treated mixed glial cell cultures; Vincent VA et al.; In cultures of purified microglial cells and astrocytes from newborn rats, the immunocytochemical localization of interleukin-1 beta (IL-1 beta) and inducible nitric oxide synthase (iNOS) using recently developed antibodies, as well as the release of IL-1 beta and nitric oxide (NO), was studied following exposure of the cells to endotoxin {lipopolysaccharide (LPS)} . In the absence of LPS, IL-1 beta- and iNOS-immunoreactive microglial cells and IL-1 beta or NO release were not observed, whereas in the presence of the endotoxin, the production of NO and IL-1 beta by microglial cells dramatically exceeded their synthesis and release by astrocytes . Interestingly, microglial cells cultured for 4-8 days in the presence of astrocytes appeared to lose their ability to produce iNOS, whereas the release of IL-1 beta remained unaltered . Moreover, endotoxin-stimulated microglial cells appeared to regain their ability to synthesize iNOS following their separation from astrocytes . These data show that microglia are primarily responsible for NO and IL-1 beta production in mixed glial cell cultures upon endotoxin stimulation . Moreover, in the presence of astrocytes the induction of iNOS, but not that of IL-1 beta in microglial cells is gradually inhibited.

Aust Dent J, 1996 Jun, 41(3), 188 - 92
A preliminary study of the effects of laser radiation on collagen metabolism in cell culture; Skinner SM et al.; A low power Ga-As pulse laser was used to stimulate cultured human embryonic fibroblast cells . Energy fluencies varied from 0-1 J/cm2 over a period of 1-4 days . Fibroblast procollagen production was monitored by the synthesis of {3H} hydroxyproline, and DNA replication was assessed by {3H} thymidine incorporation . Following laser treatment, controlled pepsin digestion measured the increase in cell biostimulation . Maximum increase in collagen production and cell biostimulation occurred after 4 episodes of laser treatment at 24-hour intervals . Laser doses between 0.099 and 0.522 J/cm2 had the most significant stimulatory effects on fibroblast function . Clinical efficacy of the low power Ga-As pulse laser may be related to enhanced connective tissue repair.

J Clin Microbiol, 1996 Jun, 34(6), 1572 - 5
Comparison of cell culture, mouse inoculation, and PCR for detection of Toxoplasma gondii: effects of storage conditions on sensitivity; James GS et al.; The sensitivity of detection of a wild-type strain of Toxoplasma gondii by cell culture, mouse inoculation, and PCR was determined following sample storage under conditions to which clinical specimens may be subjected during transport to the testing laboratory . Sample storage at -20 degrees C significantly decreased the sensitivity of mouse inoculation . The sensitivity of cell culture decreased with sample storage at 4 and -20 degrees C . The sensitivity of PCR was reduced by storage at 4 degrees C for 48 h, freezing, and heating . These findings have implications for the selection of appropriate methods for the direct detection of T . gondii organisms in suboptimally transported clinical samples.

Cytometry, 1996 Jun 1, 24(2), 123 - 30
Quantification of apoptotic cells with fluorescein isothiocyanate-labeled annexin V in chinese hamster ovary cell cultures treated with cisplatin; Boersma AW et al.; Plasma membrane binding of annexin V was used to detect and quantitate apoptotic cells induced by cytotoxic drug treatment in epithelial cell cultures . Chinese hamster ovary (CHO) cells were incubated for 2 h with the ID90 concentration of Cisplatin (20 microM), and 24, 48, 72, and 96 h later the unfixed cells were stained with fluorescein isothiocyanate (FITC)-conjugated annexin V . The fluorescence signal was quantitated by flow cytometry (FCM) . During the early phase of the apoptotic response, the annexin V-binding frequency histograms showed two separate cell populations, a dimly and a brightly fluorescent one . At t = 96 h after drug incubation, when the process of apoptosis was completed, only the brightly fluorescent population was present . A dose-effect relationship could be established between the Cisplatin concentration used in the 2 h incubation and the binding of annexin V on the cell membrane, as estimated by FITC fluorescence . The dimly and brightly fluorescent populations were sorted on the basis of annexin V binding, and assayed for 1) DNA breaks by in situ nick translation assay and DNA content by DNA-propidium iodine fluorescence in a bivariate analysis, 2) membrane integrity by dye exclusion, and 3) morphological characteristics of apoptosis . The dimly fluorescent cell population appeared to represent apoptotic cells in the early phase of the death process, as demonstrated by intact cell membranes, normal DNA content, few DNA breaks, and chromatin condensation . The brightly fluorescent cells predominantly had sub-G1 DNA content, nuclear fragmentation, leaky cell membranes, and probably represent late apoptotic cells . These results demonstrate that cytotoxic drug-induced apoptosis can be quantitated by annexin V binding and that by using this assay early and late apoptotic cells can be identified.

Tissue Cell, 1996 Jun, 28(3), 301 - 12
Immunohistochemical and histochemical characterisation of epithelial cells of rabbit lacrimal glands in tissue sections and cell cultures; Millar TJ et al.; The purpose of this study was to establish conditions for isolation and long term culture of acinar cells from the Harderian gland, and superior and inferior lacrimal glands of the rabbit and to compare the in vitro growth patterns of cultured cells from these glands . In order to determine the predominant cell type in the cultures, cells and tissue sections were stained using a variety of antibodies to cytokeratins, smooth muscle actin, and neuron specific enolase . Similarly, PAS and alcian blue histochemistry were used to test for the presence of mucins . The glands were excised and cells isolated using enzymatic digestion and then established in long term culture . Different media and substrata were trialed for suitability . When cultured on uncoated Costar plastic in DMEM/10%FBS, the pattern of cell growth was similar for all glands with distinct phases involving aggregation and migration out from the aggregates before cells died between 20 to 30 days . Immunohistochemical staining indicated that the cultures were of acinar cells with a small percentage of ductal cells . The acinar cells of the lacrimal glands in situ and in vitro stained with antibody MNF116 directed against cytokeratins 5, 6, 8 and 17 but did not stain for antibodies to cytokeratin 18 . The reverse staining pattern was true for the Harderian gland . Sections from the white lobe of the Harderian gland showed islets of serous secreting cells which showed positive staining when MNF116 was used . In situ, PAS positive cells were found in a small number of demilunes in the superior and inferior lacrimal glands and also in cells of the intercalated ducts . Surprisingly, in culture nearly all cells, including those isolated form the Harderian gland became PAS positive . In this study we have demonstrated that acinar cells from the Harderian and lacrimal glands of rabbit can be isolated and maintained in culture for 20 to 30 days, and that despite dramatic morphological changes, these cells retain their distinctive phenotype as indicated by antibody staining to specific cellular structural proteins such as cytokeratins and actin . However, the cultured cells also begin to produce mucins as indicated by PAS staining.

Prostate, 1996 Jun, 28(6), 364 - 71
Selection toward diploid cells in prostatic carcinoma derived cell cultures; Ketter R et al.; For elucidation of the growth-regulatory mechanisms in prostatic carcinoma, in vitro investigations on prostatic cell cultures are required . However, one major problem of cell culturing is the selection of particular cell types such that the cell lines representing only some of the features as compared with the tumor of origin . We studied the chromosomal composition of 20 prostatic tissue-derived cell cultures and 12 original (fresh) tissue specimens that were obtained from 13 patients with prostatic adenocarcinoma . Using fluorescence in situ DNA hybridization (FISH), evident clonal abnormalities were detected in 78% of the fresh cancer samples and in 47% of the cultured cancer samples . Of the seven cases revealing clonal abnormalities in the fresh cancer specimen, aneuploidy was detected in only two samples after cell culturing at the earliest passage studied . The aneuploid cell populations in the cultured samples were all lost during progressive subcultivation (after passage 4) . Interestingly, by performing FISH on cytogenetic preparations aneuploidy was confined to the interphases, with the metaphases being found to be diploid . This finding indicates that the aneuploid cells have a proliferation disadvantage in cell culture resulting in an overgrowth of diploid cells.

J Virol, 1996 Jun, 70(6), 3930 - 7
Dengue type 4 virus mutants containing deletions in the 3' noncoding region of the RNA genome: analysis of growth restriction in cell culture and altered viremia pattern and immunogenicity in rhesus monkeys; Men R et al.; The dengue type 4 virus (DEN4) genome contains a 384-nucleotide (nt) 3' noncoding sequence in which the last 81 nt, predicted to form a secondary structure, are thought to be essential for virus replication . Immediately upstream of the secondary structure, short RNA sequences that are conserved among mosquito-borne flaviviruses have been identified . A series of deletions that range from 30 to 262 nt were introduced into this upstream region of full-length DEN4 cDNA to create viable deletion mutants, some of which might prove to be useful for inclusion in a live attenuated virus vaccine . When studied by an infectious-center assay, most full-length RNA transcripts of the deletion constructs exhibited reduced infectivity when transfected into simian LLC-MK2 cells compared with the full-length RNA transcripts of wild-type parental virus . Deletion mutations that extended as far as the 5' boundary of the 3' noncoding region and whose 3' boundary did not extend beyond the last 113 nt of the 3' end were viable . With the exception of mutant 3'd 303-183, which contained a deletion of nt 303 to 183 from the 3' terminus, deletion mutants produced plaques that appeared late on simian LLC-MK2 cells or exhibited a small-plaque morphology on mosquito C6/36 cells compared with the wild-type virus . These mutants also replicated less efficiently and attained a lower titer in LLC-MK2 cells than parental wild-type virus . Significantly, mutant 3'd 303-183 grew to a high titer and was least restricted in growth . Mutant 3'd 303-183 and four other moderately to severely restricted mutants were selected for evaluation of infectivity and immunogenicity in rhesus monkeys . There was a suggestion that occurrence and duration of viremia were reduced for some of the deletion mutants compared with the wild-type virus . However, more convincing evidence for attenuation of some of the mutants was provided by an analysis of antibody response to infection . Mutant 3'd 303-183 induced an antibody response equivalent to that stimulated by wild-type virus, whereas other mutants induced low to moderate levels of antibodies, as measured by radioimmunoprecipitation and virus neutralization . The immunogenicity of these 3' DEN4 deletion mutants in monkeys appeared to correlate with their efficiency of growth in simian LLC-MK2 cells . One or more mutants described in this paper may prove to be useful for immunization of humans against disease caused by dengue virus.

J Neurochem, 1996 Jun, 66(6), 2300 - 10
Amyloid precursor protein metabolism in primary cell cultures of neurons, astrocytes, and microglia; LeBlanc AC et al.; Amyloid precursor protein (APP) gives rise by proteolytic processing to the amyloid beta peptide (A beta) found abundantly in cerebral senile plaques of individuals with Alzheimer's disease . APP is highly expressed in the brain . To assess the source of cerebral A beta, the metabolism of APP was investigated in the major cell types of the newborn rat cerebral cortex by pulse/chase labeling and immunoprecipitation of the APP and APP metabolic fragments . We describe a novel C-terminally truncated APP isoform that appears to be made only in neurons . The synthesis, degradation, and metabolism of APP were quantified by phosphorimaging in neurons, astrocytes, and microglia . The results show that although little APP is metabolized through the amyloidogenic pathways in each of the three cultures, neurons appear to generate more A beta than astrocytes or microglia.

J Biol Chem, 1996 May 24, 271(21), 12350 - 5
Nitrogen metabolism in Lignifying Pinus taeda cell cultures; van Heerden PS et al.; The primary metabolic fate of phyenylalanine, following its deamination in plants, is conscription of its carbon skeleton for lignin, suberin, flavonoid, and related metabolite formation . Since this accounts for approximately 30-40% of all organic carbon, an effective means of recycling the liberated ammonium ion must be operative . In order to establish how this occurs, the uptake and metabolism of various 15N-labeled precursors (15N-Phe, 15NH4Cl, 15N-Gln, and 15N-Glu) in lignifying Pinus taeda cell cultures was investigated, using a combination of high performance liquid chromatography, 15N NMR, and gas chromatograph-mass spectrometry analyses . It was found that the ammonium ion released during active phenylpropanoid metabolism was not made available for general amino acid/protein synthesis . Rather it was rapidly recycled back to regenerate phenylalanine, thereby providing an effective means of maintaining active phenylpropanoid metabolism with no additional nitrogen requirement . These results strongly suggest that, in lignifying cells, ammonium ion reassimilation is tightly compartmentalized.

Int J Cancer, 1996 May 16, 66(4), 578 - 86
Three-dimensional cell culture induces novel proliferative and metabolic alterations associated with oncogenic transformation; Kunz-Schughart LA et al.; To date, cell biological characteristics of oncogene-transfected cells have been investigated either in relatively homogeneous monolayer cultures or in heterogeneous tumors in vivo . To evaluate the emergence of cellular heterogeneity during tumor formation, we have established a multicellular spheroid system from an oncogene-dependent, genetically determined 2-stage carcinogenesis model for 3-dimensional growth under well-defined conditions . The effect of T24Ha-ras transfection on cellular growth, proliferation, cell viability and oxygenation was investigated using spontaneously immortalized (Rat1) and c-myc-transfected (M1) Fisher 344 rat embryo fibroblasts and a tumorigenic T24Ha-ras-transfected clone of each (Rat1-T1 and MR1) . Spheroid volume growth curves and {3H}thymidine autoradiographs clearly demonstrated that spheroids better reflect the degree of tumorigenicity in vivo as opposed to monolayer cultures . Studies on Rat1 and M1 aggregates showed that the potential for tumor formation of Rat1 cells might be manifested in vitro as an increased capability of the cells to survive in 3D culture . pO2 measurements confirmed that neither cell quiescence nor cell death in the pseudo-normal cell aggregate types is due to an oxygen deficiency . In contrast, depletion of oxygen coincided with necrotic cell death in Rat1-T1 spheroids and proliferation arrest in MR1 cultures . Cell-line-specific attributes in 3D culture that were not specifically related to ras transfection of the cells included histological structure, development of necrosis and thickness of viable cell rim . However, growth behavior, proliferation characteristics and their association with the oxygen supply might be correlated with the extent of transformation.

Int J Cancer, 1996 May 16, 66(4), 551 - 6
A comparative study of cytokine gene transcripts in normal and malignant breast tissue and primary cell cultures derived from the same tissue samples; Speirs V et al.; Using a differential centrifugation method followed by culture in selective medium, we have successfully isolated and maintained individual epithelial and stromal cells from normal (n = 10) and malignant (n = 6) human breast tissue and characterised their phenotype by immunocytochemistry . Further, we have studied expression of the cytokine genes IL-1beta, IL-6, IL-8 and TNF-beta in each cell fraction by RT-PCR and have compared these results with cytokine gene expression in tissue extracts from which primary cultures were derived . In breast tumours, there was near complete absence of IL-1beta in both whole tissue and cell fractions, and in normals it was present in only 3/10 tissue preparations, with increased expression in stromal (6/10) and epithelial (5/10) cell samples . IL-6 was constitutively expressed in all tumour-derived breast tissue samples but down-regulated in tumour cell cultures, with the opposite result in normal breast . Near identical levels of IL-8 expression were found throughout each preparation, irrespective of tissue origin . TNF-beta was expressed in all normal tissue samples, in 9/10 epithelial preparations but in only 6/10 stromal preparations . In tumours, TNF-beta was associated predominantly with whole tissue or stromal samples, with reduced expression in epithelial preparations . Our data confirm that primary cultures of normal and malignant human breast tissue can be successfully separated into epithelial and mesenchymal cell populations and their phenotype can be maintained in culture for up to 30 days . However, this cellular separation does alter the cytokine profiles; therefore, experimental findings with isolated cells should be treated with a caveat.

J Biotechnol, 1996 May 15, 46(3), 161 - 85
The importance of ammonia in mammalian cell culture; Schneider M et al.; Ammonia has been reported to be toxic and inhibitory for mammalian cell cultures for many years . Reduction of growth rates and maximal cell densities in batch cultures, changes in metabolic rates, perturbation of protein processing and virus replication have been reported . However, cellular mechanisms of ammonia toxicity are still the subject of controversy and are presented here . The physical and chemical characteristics of ammonia and ammonium are important, with the former capable of readily diffusing across cellular membranes and the latter competing with other cations for active transport by means of carrier proteins . The main source of the ammonia which accumulates in cell cultures is glutamine, which plays an important role in the metabolism of rapidly growing cells . Strategies to overcome toxic ammonia accumulation include substitution of glutamine by glutamate or other amino acids, nutrient control, i.e., controlled addition of glutamine at low concentrations, or removal of ammonia or ammonium from the culture medium by means of ion-exchange resins, ion-exchange membranes, gas-permeable membranes or electrodialysis.

Proc Natl Acad Sci U S A, 1996 May 14, 93(10), 4706 - 11
Reconstitution of the hippocampal mossy fiber and associational-commissural pathways in a novel dissociated cell culture system; Baranes D et al.; Synapses of the hippocampal mossy fiber pathway exhibit several characteristic features, including a unique form of long-term potentiation that does not require activation of the N-methyl-D-aspartate receptor by glutamate, a complex postsynaptic architecture, and sprouting in response to seizures . However, these connections have proven difficult to study in hippocampal slices because of their relative paucity (<0.4%) compared to commissural-collateral synapses . To overcome this problem, we have developed a novel dissociated cell culture system in which we have enriched mossy fiber synapses by increasing the ratio of granule-to-pyramidal cells . As in vivo, mossy fiber connections are composed of large dynorphin A-positive varicosities contacting complex spines (but without a restricted localization) . The elementary synaptic connections are glutamatergic, inhibited by dynorphin A, and exhibit N-methyl-D-aspartate-independent long-term potentiation . Thus, the simplicity and experimental accessibility of this enriched in vitro mossy fiber pathway provides a new perspective for studying nonassociative plasticity in the mammalian central nervous system.

Neurosci Lett, 1996 May 3, 209(1), 33 - 6
Induction of apoptosis and secondary necrosis in rat dorsal root ganglion cell cultures by oxidized low density lipoprotein; Papassotiropoulos A et al.; Neural cell degeneration underlies central and peripheral nervous system disorders . In this study we examined the influence of oxidized low density lipoprotein (Ox-LDL) on rat dorsal root ganglion (DRG) cells in culture . Methods used were cell morphology, lactate dehydrogenase (LDH) release, the TUNEL-reaction and DNA fragmentation . Exposure of DRG cells to Ox-LDL for 24 h led to elevation of LDH in the culture medium; short term exposure (4 h) induced apoptosis, evidenced by DNA fragmentation and a positive TUNEL-reaction . DRG cells modified LDL in the presence of Cu2+ to mildly oxidized and to a small extent to fully oxidized forms; these in situ-generated LDL oxidation products were strongly toxic . These results suggest that Ox-LDL is a neurotoxin; it initiates apoptotic cell injury which progresses to necrosis and cell death.

Am J Physiol, 1996 May, 270(5 Pt 1), E895 - 9
Paracrine stimulation of preadipocyte-enriched cell cultures by mature adipocytes; Considine RV et al.; We developed a double-chamber system in which to examine the effects of mature adipocytes on the growth and differentiation of preadipocytes and other cells in the adipose tissue . In the present study we found that mature adipocytes from both lean and obese subjects release a factor that stimulates the growth of preadipocyte-enriched and dedifferentiated adipocyte-enriched cell cultures . This growth stimulation was dependent on both time of exposure to mature cells and the number of mature cells in the coculture . Proliferation of the preadipocyte-enriched (n = 4) and dedifferentiated adipocyte-enriched cultures (n = 5) in the presence of mature adipocytes from obese subjects {body mass index (BMI) > 35} was 4.1- and 2.9-fold more (P < 0.05) than that in the presence of adipocytes from lean subjects (BMI < or = 25) . There was no difference in the growth of cultures enriched in preadipocytes or dedifferentiated adipocytes from lean or obese subjects in the absence of mature adipocytes . These observations demonstrate that mature adipocytes from obese patients stimulate the growth of preadipocyte-enriched cultures to a greater extent than those from lean individuals.

Am J Physiol, 1996 May, 270(5 Pt 1), C1503 - 10
Effects of hypercapnia on prostanoid and cAMP production by cerebral microvascular cell cultures; Parfenova H et al.; In the newborn pig cerebral circulation, arteriolar dilation in response to hypercapnia requires the presence of intact endothelium and is accompanied by an indomethacin-sensitive increase in cortical adenosine 3',5'-cyclic monophosphate (cAMP) . The effects of short-term hypercapnia on production of dilator prostanoids and cAMP were investigated using newborn pig cerebral microvascular smooth muscle cells and endothelial cells cultured both separately and in noncontact coculture . Microvascular smooth muscle cells respond to hypercapnia (pH 7.00 +/- 0.05 PCO2 75 +/- 3 mmHg) by a 1.3- to 1.7-fold increase in basal cAMP production that is not affected by indomethacin, whereas hypercapnia and 80 mM sodium propionate do not affect iloprost-stimulated cAMP production . Microvascular endothelial cells cultured on Millicel inserts respond to hypercapnia by a two- to fourfold increase in prostacyclin (as 6-keto-prostaglandin F1 alpha) and prostaglandin E2 production in both luminal and abluminal compartments . For noncontact coculture, Millicel inserts with endothelial cells (as hypercapnia-sensitive producers of prostanoids) were installed into cell culture dishes with aspirin-pretreated smooth muscle cells (as targets for endothelium-derived dilator prostanoids) . Exposure of noncontact microvascular cell cocultures to hypercapnia results in a three- to fourfold stimulation of prostanoid and cAMP production . Therefore, short-term hypercapnia increases cAMP production by microvascular smooth muscle cells via 1) a direct (prostanoid independent) mechanism and 2) an endothelial-dependent pathway that involves prostanoids . Endothelium-produced prostanoid signals are necessary for a full increase in cAMP production by cerebral microvascular smooth muscle cells in response to hypercapnia.

Int J Immunopharmacol, 1996 May, 18(5), 305 - 9
Anti-retroviral activity of methionine enkephalin and AZT in a murine cell culture; Sin JI et al.; Previously, this laboratory has demonstrated that azidothymidine used in combination with methionine enkephalin, an opioid pentapeptide, was more effective than AZT alone in inhibiting disease progression due to murine retrovirus infections . In order to study the mechanism(s) by which Met-ENK mediates-antiviral effects, when used in combination with AZT in Friend leukemia virus infected mice, an in vitro focus forming assay was used . AZT at 1 ng/ml inhibited FLV replication by 30-50% in the susceptible Mus dunni cell line . By contrast, the immunostimulatory neuropeptide, Met-ENK, displayed no direct inhibition of viral replication . This suggests that Met-ENK does not have any direct anti-retroviral activity . Subsequent testing of Met-ENK in the presence of AZT showed no ability of this peptide to promote inhibition of viral replication due to AZT . By contrast, in the presence of mouse spleen cells, as a source of lymphocytes, in vitro combination treatments using AZT and Met-ENK reduced FLV replication by 67%, compared to 47% using AZT alone . The inhibition due to Met-ENK was abrogated when spleen cells were pretreated with naloxone, an opioid antagonist . Therefore, we conclude that Met-ENK effects are mediated via opioid receptors on spleen cells and that the observed anti-FLV activity is dependent on the use of Met-ENK stimulated spleen cells in combination with AZT.

Pharmacology, 1996 May, 52(5), 321 - 8
Inhibition of proliferation of psoriatic and healthy fibroblasts in cell culture by selected Dead-sea salts; Levi-Schaffer F et al.; The effect of five selected minerals abundant in the Dead-sea brine was studied on proliferation of fibroblasts grown from psoriatic and healthy skin biopsy specimens in cell culture . The reason for carrying out this study was looking for the mechanism of the antiproliferative effect of selective Dead-sea minerals in improving the psoriatic condition . Psoriatic skin shave biopsy specimens (both from involved and uninvolved areas of the body) as well as healthy skin (obtained from amputated limbs) were incubated in tissue culture, and their outgrowing fibroblasts were used for this study . The number of cells and their cyclic AMP content were used as parameters for cell division and for proving the selective involvement of magnesium salts in the antiproliferative effect . It is shown that the inhibitory effects of magnesium bromide and magnesium chloride on cell growth were significantly stronger than those of their corresponding potassium salts or of sodium chloride . These results were obtained with both psoriatic and healthy skin fibroblasts, indicating that the inhibitory effect of the selected Dead-sea minerals is present in healthy and psoriatic skin cells.

J Virol Methods, 1996 May, 59(1-2), 155 - 60
Video imaging of firefly luciferase activity to identify and monitor herpesvirus infection in cell culture; Mettenleiter TC et al.; Expression of reporter genes incorporated into complex viral genomes is being used increasingly to monitor virus infection in cell culture and in the organism . One of the most sensitive markers is luciferase from Photinus pyralis which catalyzes a luminescent reaction that can be traced in cell and organ extracts in luminometers . A novel method is described for monitoring Iuciferase activity after infection of cells with recombinant herpesvirus (pseudorabies virus) which carries stably and expresses luciferase using ultra high sensitive photon-counting enhanced video image acquisition . The data show that firefly luciferase activity in virus-infected cells can be monitored without destruction of the cells using video equipment for either macroscopic image acquisition or microscopy . Resolution down to a single-cell level can thereby be achieved . This method increases greatly the potential of monitoring virus infection in real time with a non-destructive highly sensitive method in cell culture and should also help to assay viral spread in the animal.

Phytochemistry, 1996 May, 42(1), 199 - 203
Triacylated anthocyanins from Ajuga reptans flowers and cell cultures; Terahara N et al.; Four anthocyanins were isolated from Ajuga reptans flowers and one from the cell cultures . By FAB mass spectrometry measurements, the structures of these pigments were determined as delphinidin and cyanidin glucosides acylated with two cinnamic acids, while three of them were also malonylated . A delphinidin-based pigment in the crude extract from cell cultures was identical to the major flower pigment as shown by HPLC co-chromatography . Moreover, by application of 1H and 13C NMR consisting of DQF-COSY, NOESY, ROESY, 2D-HOHAHA, HSQC and HMBC methods, the structures of two new anthocyanins were identified as delphinidin and cyanidin 3-O-(2-O-(6-O-(E)-p-coumaryl-beta-D-glucopyranosyl)-(6-O-(E)-p- coumaryl)-beta-D-glucopyranosyl)-5-O-(6-O-malonyl-beta-D-glucopyranoside ) . The deacylated anthocyanins were confirmed as delphinidin and cyanidin 3-sophoroside-5-glucosides.

J Lipid Res, 1996 May, 37(5), 1076 - 85
Structural relationships between nascent apoA-I-containing particles that are extracellularly assembled in cell culture; Forte TM et al.; Apolipoprotein A-I (apoA-I) incubated with CHO cells assembles three major nascent lipid complexes with diameters of 7.3, 9, and 11 nm . Previous studies suggested that the smaller nascent particles were precursors for the larger nascent ones . To test this hypothesis, the 7.3, 9, and 11 nm apoA-I-lipid complexes formed by incubating CHO cells with lipid-free apoA-I were isolated and subsequently each subpopulation was re-incubated with cells in the absence of other subpopulations . The physical-chemical characteristics of each subpopulation were examined before and after re-incubation in an effort to understand relationships . if any, between the different nascent complexes . The 7.3, 9, and 11 nm complexes were unique in that each of the particles had pre-alpha mobility on agarose gels: this rapid migration was not altered by re-incubation with cells . Protein crosslinking studies indicated that the 7.3, 9, and 11 nm complexes possessed 2, 3, and 4 apoA-I molecules per complex, respectively; it is unlikely that the size of the particle and number of apoA-I molecules per particle played a role in the increased negative charge of the particles . The present study shows that smaller particles did not give rise to larger ones upon re-incubation with cells . Rather, the 11 and 9 nm particles both generated smaller discs (the 11 nm giving rise primarily to 9 nm discs and the 9 nm complex giving rise to 7.3 nm discs) suggesting that, during incubation with cells, the complexes are destabilized and remodeled into smaller, not larger, complexes . Surprisingly, the 7.3 nm complexes during re-incubation with cells were extremely stable and did not undergo size alteration . When the 7.3 nm particles were incubated with additional small quantities of lipid-free apoA-I (1-2 microgram/ml), larger discoidal complexes were generated suggesting that the formation of larger particles may be driven by the availability of lipid-free apoA-I.

J Endod, 1996 May, 22(5), 249 - 52
A commercially available cell culture device modified for dentin barrier tests; Schmalz G et al.; The suitability of a dentin barrier test based on a commercially available cell culture chamber was evaluated by testing the cytotoxicity of dental cements . The two chambers of the culture device as produced are separated by a membrane . This was replaced by a bovine dentin disk (500 micrometers thick) . Mouse fibroblasts were grown on the "pulpal" side of the dentin for 24 h; test materials were then placed into the "cavity" side of the upper chamber . The number of viable cells was determined after 24 h . After exposure to zinc phosphate cement at a powder/liquid ratio of 2:1, approximately 100% of cells survived . A ratio of 1:1 yielded 81% survival . Only 24% and 28% of the cells survived after exposure to Ketac Fil and Ketac Silver, respectively . The light-curing glass ionomer cement (vitrebond) and zinc oxide-eugenol killed all cells . These results agree with those obtained from a previous study, wherein the dentin barrier test device was constructed in our laboratory.

J Infect Dis, 1996 May, 173(5), 1252 - 5
Effect of stavudine on human immunodeficiency virus type 1 virus load as measured by quantitative mononuclear cell culture, plasma RNA, and immune complex-dissociated antigenemia; Griffith BP et al.; The antiviral effect of stavudine (2', 3'-didehydro-3'-deoxythymidine) against human immunodeficiency virus (HIV) type 1 was measured in 15 HIV-infected patients at baseline and at weeks 4, 10, 22, 34, and 52 of therapy . Patients received 0.1, 0.5, 1.0, or 2.0 mg/kg/day of stavudine . At all time points examined during the 52 weeks of therapy, the median virus titers in peripheral blood mononuclear cells were decreased 1-2 logs, and median immune complex-dissociated antigen levels were reduced 37%-67% compared with baseline values . Plasma RNA content measured by polymerase chain reaction was reduced approximately 0.5 log from baseline median values at both time points examined (weeks 10 and 52) . These data demonstrate that stavudine has a substantial and durable antiviral effect.

J Acquir Immune Defic Syndr Hum Retrovirol, 1996 May 1, 12(1), 1 - 5
Examining the molecular genetics of HTLV-I with an infectious molecular clone of the virus and permissive cell culture systems; Derse D et al.; Infectious molecular clones of HTLV-I proviruses have only recently been reported . The long wait for such provirus clones reflects the difficulties inherent in propagating HTLV-I in vitro, and thus a rigorous demonstration of infectivity has awaited improved cell culture systems and sensitive detection techniques for HTLV-I . An intact HTLV-I provirus, originating from an American ATL patient, was subcloned into a plasmid vector and was designated pCS-HTLV . Transient transfections of mammalian cells with pCS-HTLV resulted in the synthesis of viral proteins and mRNAs which were assembled into virions that had physical and morphological characteristics typical of HTLV-I particles . The ability of these virus particles to infect cells, replicate, and produce infectious progeny was demonstrated initially in short term, cell-free infection assays by monitoring the expression of specific viral mRNAs . These studies have been extended in cell culture systems that support continuous virus production . Primary T-lymphocytes have been infected either with cell-free supernatant fluids from, or by coculture with, cells transiently transfected with pCS-HTLV, giving rise to continuous, IL-2-dependent cell lines that have been in culture for >1 year . Furthermore, fetal rhesus lung cells (FRhL) were shown to be permissive for HTLV-I replication and sustained virus expression after infection with pCS-HTLV . Continuous FRhL cell lines now have been established that express various HTLV-I proviruses and mutants . These provirus clones and cell lines provide us with the means to address long-standing questions dealing with the biology of HTLV-I.

Hepatology, 1996 May, 23(5), 1098 - 104
Heat serum inactivation as a mandatory procedure for antiactin antibody detection in cell culture; Cancado EL et al.; In autoimmune hepatitis (AIH), the smooth-muscle antibody is specific for polymerized actin . Detection of antiactin antibody (AAA) has been hampered by technical problems . We have investigated AAA in 30 sera from patients with liver diseases and smooth-muscle antibody . AAA was detected by indirect immunofluorescence in 1:40, 1:80, and 1:160 dilutions . Five techniques were performed using fibroblasts: with vinblastine (A); without drugs (B); with sodium citrate (C); without drugs but with heat serum inactivation (D); and with sodium citrate and heat serum inactivation (E) . For comparative analysis, we considered: the total number of AAA-positive sera regardless of the dilution in which reactivity was observed, as well as in each dilution separately; and the comparison of AAA intensity between 1:40 x 1:80, 1:40 x 1:160, and 1:80 x 1:160 dilutions . AAA was more positive in techniques B, C, D, and E than in A (P < .001) in general, and in each dilution separately . AAA was more positive in technique D than in B in 1:40 (P = .0005) and 1:80 dilutions (P = .03), as well as in E than in C (P = .0001) in 1:40 dilution . Techniques B and D yielded results similar to C and E, respectively . AAA staining was significantly more intense in 1:80 and 1:160 than in 1:40 dilution in A, B, and C; it was both significantly less intense in 1:80 and 1:160 than in 1:40 dilution and in 1:80 than in 1:160 in techniques D and E . We concluded that heat inactivation increased AAA seropositivity/intensity in 1:40 and 1:80 dilutions, preventing false-negative results; actin polymerization with sodium citrate did not enhanced AAA seropositivity/intensity . The technique with vinblastine was the least effective.

J Cell Physiol, 1996 May, 167(2), 185 - 95
Isolation of vascular smooth muscle cell cultures with altered responsiveness to the antiproliferative effect of heparin; Caleb BL et al.; Smooth muscle cell (SMC) hyperplasia in the arterial wall is an important component of both atherogenesis and post-vascular surgical restenosis . One naturally-occurring group of molecules which can suppress SMC proliferation in animal models and in cell culture systems are the complex carbohydrates of the heparan sulfate class, including heparin . In this communication, we have used retrovirus vectors to introduce several oncogenes into SMC: SV40 Large T antigen (SVLT), polyoma virus Large T antigen (PyLT), v-myc, and adenovirus E1a . We analyzed a total of 11 cultures . A combination of Western blot analysis, immunoprecipitation, and indirect immunofluorescence confirmed the expression of the infected oncogenic protein in each culture we isolated . All four oncogenes permitted the maintenance of a normal SMC phenotype, as assessed by the general morphology of cells in the light microscope and the presence of SMC-specific alpha-actin in an immunofluorescence assay . Doubling times in infected cells ranged from 20 to 33 hr, and final cell densities in infected cultures ranged from 4 x 10(4) to 5 x 10(5) cells per cm2 . By comparison, the parent line had a doubling time of 30 hr and reached a final cell density of 1 x 10(5) cells per cm2 . Despite the differences sometimes observed in these proliferation parameters, neither one was strongly correlated with heparin responsiveness . PyLT, v-myc, and E1a all produced SMC cultures or lines which retained sensitivity to the antiproliferative activity of heparin (ED50 = 50 micrograms/ml) . In contrast, SVLT expression yielded SMC lines which were highly resistant to heparin (ED50 > 300 micrograms/ml) . These results suggest that altered responsiveness to heparin is dependent upon which oncogenic protein is being expressed in the cells . The availability of cloned, immortal SMC lines with a wide range of heparin responsiveness should aid in the understanding of the cellular and molecular mechanism of action of this potentially important growth regulator and therapeutic agent.

Exp Cell Res, 1996 May 1, 224(2), 354 - 64
Use of the dissociating enzyme thermolysin to generate viable human normal intestinal epithelial cell cultures; Perreault N et al.; The regulation of intestinal cell proliferation, migration, and differentiation has been the subject of numerous studies . However, in human, progress in this field has been traditionally hampered by the lack of normal epithelial cell models . The aim of the present study was to define conditions in order to isolate, and more importantly to grow in a continuous manner, human small intestinal epithelial cells . A number of mechanical and/or enzymatic dissociation methods have been tested to isolate viable epithelial cells from the fetal small intestine . Cultured cells were characterized by indirect immunofluorescence and Western blot analysis . It was found that the use of thermolysin (50 microgram/ml, 2-3 h at 37 degrees C) can be advantageously applied to the isolation of viable epithelial cells free from contaminating fibroblasts when obtained from the 17- to 19-week fetal ileum . Furthermore, this procedure allowed the generation of continuously growing human intestinal epithelial cell cultures, which retain the ability to express specific cytokeratins as well as intestinal cell markers over a number of passages . This study shows that normal epithelial cell cultures can be relatively easily and reproducibly generated from the human fetal small intestine.

Brain Res, 1996 Apr 22, 717(1-2), 135 - 46
Polyamine amides are neuroprotective in cerebellar granule cell cultures challenged with excitatory amino acids; Green AC et al.; Primary cultures of rat cerebellar granule cells have been used to assess the potential neuroprotective effects of philanthotoxins and argiotoxin-636 (ArgTX-636) . These polyamine amides are potent antagonists of ionotropic L-glutamate (L-Glu) receptors . In granule cells loaded with fluo-3, ArgTX-636 and philanthotoxin-343 (PhTX-343) antagonised increases of intracellular free calcium concentration ({Ca2+}i) that were stimulated by N-methyl-D-aspartate (NMDA) . The antagonism was use-dependent . Antagonism by PhTX-343 was fully reversible, but recovery following antagonism by ArgTX-636 was slow and only partial during the time-course of an experiment . Neither compound inhibited K(+)-induced increases in {Ca2+}i . In excitotoxicity studies with cerebellar granule cells, the release of lactate dehydrogenase (LDH) and morphological observations were used to assess cell death . A 20-30 min exposure to 500 microM NMDA, 100 microM L-Glu or 500 microM kainate was sufficient to kill > 90% of the cells after 18-20 h . When added 5 min prior to, and during agonist exposure, PhTX-343 and ArgTX-636 provided total neuroprotection . ArgTX-636 was about 20-30 fold more potent than PhTX-343 against NMDA, but was approximately equipotent with PhTX-343 against a kainate challenge . Neither of the toxins showed any inherent toxicity even at 400 microM and 100 microM respectively . Some analogues of PhTX-343 are more potent, both in terms of antagonism of NMDA-stimulated increases of {Ca2+}i and neuroprotection, than PhTX-343 and ArgTX-636.

Mol Cell Endocrinol, 1996 Apr 19, 118(1-2), 37 - 46
Gelatinase A secretion and its control in peritubular and Sertoli cell cultures: effects of hormones, second messengers and inducers of cytokine production; Hoeben E et al.; Extracellular matrix components as well as enzymes and enzyme-inhibitors controlling the turn-over of these components play an important role in the local control of testicular function . Zymographic analysis was used to study the secretion and the control of the secretion of gelatinase A (MMP-2) and B (MMP-9) by primary cultures of rat Sertoli cells and by subcultures of peritubular cells . Data on gelatinase A were complemented by measurement of the corresponding mRNA by Northern blot analysis . The agonists investigated included hormones (FSH, testosterone), second messengers (dbcAMP, phorbolester and a Ca(2+)- ionophore), interleukin-1 beta (IL-1 beta) and inducers of cytokine production (Concanavalin A: ConA; lipopolysaccharide: LPS; double stranded RNA: PIC) . It is demonstrated that Sertoli cells originally secrete both gelatinase A and B . When maintained in serum-free medium, however, they rapidly lose the ability to secrete gelatinase B . After 3 days of culture gelatinase A remains the only measurable gelatinase in both Sertoli and peritubular cell cultures . The production in peritubular cells, however, exceeds that in Sertoli cells some 25-fold . This was confirmed by a 30-fold difference in the level of steady-state gelatinase A mRNA levels . Gelatinase A secretion and gelatinase A mRNA were stimulated by ovine FSH in Sertoli cells and by dbcAMP and ConA in both Sertoli and peritubular cells . IL-1 beta displayed measurable but limited stimulatory effects in both cell types . Interestingly, in peritubular cells but not in Sertoli cells, ConA stimulated the production of a lower MW species probably representing an activated form of gelatinase A . It is concluded that both the amounts of gelatinase A produced, the levels of the corresponding mRNA and the regulation differ in cultured peritubular cells and Sertoli cells . The lectin concanavalin A is a novel and potent inducer of gelatinase A . It resembles cytochalasin D in selectively inducing an activated form of gelatinase A in peritubular cells . The mechanism responsible for this selective effect warrants further investigation.

Proc Natl Acad Sci U S A, 1996 Apr 16, 93(8), 3598 - 601
Protein-free cell culture on an artificial substrate with covalently immobilized insulin; Ito Y et al.; Insulin was immobilized on a surface-hydrolyzed poly(methyl methacrylate) film . Chinese hamster ovary cells overexpressing human insulin receptors were cultured on the film in the absence of serum or soluble proteins . Small amounts of immobilized insulin (1-10% of the required amount of free insulin) were sufficient to stimulate cell proliferation . In addition, the maximal mitogenic effect of immobilized insulin was greater than that of free insulin . Immobilized insulin activated the insulin receptor and downstream signaling proteins, and this activation persisted for longer periods than that obtained with free insulin, probably explaining the greater mitogenic effect of the immobilized insulin . Finally the immobilized-insulin film was usable repeatedly without marked loss of activity.

Virology, 1996 Apr 15, 218(2), 389 - 95
Inhibition of HIV-1 in cell culture by synthetic humate analogues derived from hydroquinone: mechanism of inhibition; Schneider J et al.; Humic acids are natural constituents of soil and ground water and mainly consist of mixtures of polycyclic phenolic compounds . A similar complex of compounds with a mean size of about 1000 Da, designated HS-1500, was synthesized by oxidation of hydroquinone . HS-1500 inhibited HIV-1 infection of MT-2 cells with an IC50 of 50-300 ng/ml and showed a mean cell toxicity of about 600 micrograms/ml . Inhibition of HIV-induced syncytium formation was observed at 10-50 micrograms/ml . Treatment of free and cell-attached HIV with HS-1500 irreversibly reduced its infectivity, whereas the susceptibility of target cells for the virus was not impaired by treatment prior to infection . The HIV envelope protein gp120SU bound to sepharose-coupled HS-1500 and could be eluted by high salt and detergent . HS-1500 interfered with the CD4-induced proteolytic cleavage of the V3 loop of virion gp120SU . Furthermore, binding of V3 loop-specific antibodies was irreversibly inhibited, whereas binding of soluble CD4 to gp120SU on virus and infected cells was not affected . In conclusion, our data suggest, that the synthetic humic acid analogue inhibits the infectivity of HIV particles by interference with a V3 loop-mediated step of virus entry.

J Biol Chem, 1996 Apr 12, 271(15), 9033 - 8
Cortisol inhibits the synthesis of insulin-like growth factor-binding protein-5 in bone cell cultures by transcriptional mechanisms; Gabbitas B et al.; Glucocorticoids inhibit the synthesis of insulin-like growth factor-binding protein-5 (IGFBP-5) in osteoblasts, but the mechanisms involved are unknown . IGFBP-5 stimulates bone cell growth, and its inhibition by glucocorticoids may be relevant to the action of this binding protein on bone formation . We tested the effects of cortisol on IGFBP-5 expression in cultures of osteoblast-enriched cells from fetal rat calvariae (Ob cells) . Cortisol decreased IGFBP-5 polypeptide levels in the extracellular matrix and caused a time- and dose-dependent decrease in IGFBP-5 mRNA . IGFBP-5 transcripts were markedly decreased by cycloheximide, and further suppressive effects of cortisol could not be determined . Cortisol did not modify the decay of IGFBP-5 mRNA in transcriptionally arrested Ob cells . Cortisol decreased IGFBP-5 hnRNA, the rate of IGFBP-5 transcription, and the activity of the murine IGFBP-5 promoter by 35% in transient transfection experiments . Deletion analysis showed that the region responsive to cortisol is from base pairs -70 to +22, and E-box-binding proteins or c-Myb-related nuclear factors may be involved in its regulation . In conclusion, cortisol inhibits IGFBP-5 transcription in Ob cells through the Myb-binding domain . This effect may be partly responsible for the effect of glucocorticoids on bone formation.

J Biol Chem, 1996 Apr 5, 271(14), 8416 - 23
Expression of the three alternative forms of the sphingolipid activator protein precursor in baby hamster kidney cells and functional assays in a cell culture system; Henseler M et al.; Sphingolipid activator proteins (SAPs) are non-enzymatic glycoproteins required for lysosomal degradation of various sphingolipids with short oligosaccharide chains by their respective exohydrolases . Four of these (SAP-A to SAP-D or saposins A to D) are derived from a common precursor by proteolytic processing . Alternative splicing of the SAP-precursor gene results in insertion of additional 6 or 9 bases of exon 8' or 8, respectively, into the SAP-B coding region of the transcribed mRNAs . To examine the features of the three different SAP-precursor proteins (prosaposins), the respective cDNAs were stably expressed in baby hamster kidney cells . Pulse-chase experiments with transfected cells and endocytosis studies on human fibroblasts showed that synthesis, transport, and maturation of all SAP-precursor led to formation of the four mature SAPs (SAP-A to SAP-D) . In order to determine the biological function of the three different SAP-B isoforms, SAP-precursor-deficient human fibroblasts were loaded with recombinant SAP-precursor proteins with or without 2- and 3-amino acid insertions, respectively, purified from the medium of the baby hamster kidney cells . They were found to stimulate at nanomolar concentrations the turnover of biosynthetically labeled ceramide, glucosylceramide, and lactosylceramide . Since the physiological function of SAP-B is to stimulate the degradation of sulfatide by arylsulfatase A (EC 3.1.6.1) and globotriaosylceramide by beta-galactosidase (EC 3.2.1.23) loading studies with the respective exogenously labeled lipids on SAP-precursor-deficient fibroblasts were performed . Addition of different purified SAP-precursors to the medium of the lipid-loaded fibroblasts showed positive stimulation of the lipid degradation by all three SAP-B isoforms derived from the SAP-precursors . These findings establish that all three forms of the SAP-B can function as sulfatide/globotriaosylceramide activator.

Eur J Neurosci, 1996 Apr, 8(4), 801 - 8
Evidence for modulation of dopamine-neuronal function by tachykinin NK3 receptor stimulation in gerbil mesencephalic cell cultures; Alonso R et al.; Primary cultures of gerbil mesencephalon were used for studying the modulation exerted by tachykinin NK(3) receptor activation on the activity of dopamine (DA) neurons . Dopamine neurons were identified by their ability to take up {(3)H}DA in a nomifensine-dependent manner . Moreover, tyrosine hydroxylase immunohistochemistry revealed that these neurons accounted for 5-7% of the total cell population . The NK(3) receptor agonists, senktide (EC(50) = 0.58 nM) and {MePhe(7)}neurokinin B (EC(50) = 3 nM), increased spontaneous {(3)H}DA release in a concentration-dependent manner . In contrast, tested at a supramaximal concentration (IC(50) = 0.89 nM), neither septide nor substance P were found to affect {(3)H}DA release . The senktide-evoked {(3)H}DA release was not observed when extracellular Ca(2+) was chelated, but was unaffected by nomifensine . This indicates that this increase in {(3)H}DA outflow resulted more from an exocytotic process than from reversal of carrier-mediated DA uptake . Moreover, the senktide effect was unaffected by the Na+ channel blocker tetrodotoxin, a result suggesting a direct action of senktide on DA neurons . The non-peptide NK(3) receptor antagonist, SR 142801, shifted or blocked (IC(50) = 0.89 nM) the senktide-evoked {(3)H}DA release, while its (-)-antipode, SR 142806, was 80-fold less potent, in agreement with binding data . Selective antagonists for Nk1 (SR 140333) or Nk2 (SR 48968) receptors failed to reduce the senktide effect . Light scanning microscopic analysis of mesencephalic cells loaded with the Ca(2+) sensitive dye, fluo-3, showed that senktide induced a rise in cytosolic Ca(2+) in 8-10% of the cell population . The senktide-induced elevation in intracellular Ca(2+) was rapid in onset and transient (at 10-8 M) or more sustained with no further increase in fluorescence intensity (at 10(-7) M) . The proportion of senktide-responsive cells was not significantly modified when extracellular Ca(2+) was chelated, but was reduced by 87% in the presence of SR 142801 and by 75% in cultures that were pre-treated with the DA neurotoxin 1-methyl-4-phenylpyridinium . The present study shows that enhancement of spontaneous {(3)H}DA release and intracellular Ca(2+) mobilization may be observed after NK(3) receptor stimulation and that both biochemical events are likely to occur in DA neurons.

Int J Biochem Cell Biol, 1996 Apr, 28(4), 421 - 30
Differential solubilization of osteoblastic alkaline phosphatase from human primary bone cell cultures; Radisson J et al.; Mineralization of cartilage and bone requires alkaline phosphatase activity . In order to study the enzymatic properties of bone alkaline phosphatase in bone disease and more particularly in patients with osteoporosis and osteoarthritis, we investigated the solubilization of alkaline phosphatase from primary bone cell cultures derived from human bone explants . To study the release of alkaline phosphatase from membranes, several detergents at a concentration above the critical micellar concentration and cholesterol were used . Solubilized alkaline phosphatase was characterized by enzymatic activity and electrophoresis analysis . Almost all the alkaline phosphatase was solubilized using non-ionic detergent as n-octylglucoside and hecameg . In comparison with initial membranous activity, the solubilized activity was increased by a factor, i.e . 2 +/- 0.05 (SEM, n = 3) (with n-octylglucoside), i.e . 2.1 +/- 0.05 (SEM, n = 3) (with Hecameg) . With an ionic detergent (sodium dodecylsulfate), zwitterionic detergent ((cholamido propyl) dimethylammonio 1 propane sulfonate) and cholesterol, a fraction of alkaline phosphatase was resistant to solubilization . Electrophoresis studies showed that released alkaline phosphatase was a glycosylphosphatidylinositol protein (amphipatic form) with 140 kDa as apparent molecular weight . A hydrophilic form was obtained by treatment with a specific lipase . This study showed differential solubilization of osteoblastic alkaline phosphatase from human primary bone cell cultures . Better extractibility and higher activation of this membrane anchored enzyme were obtained with non-ionic detergents.

Pflugers Arch, 1996 Apr, 431(6), 814 - 27
Action potentials, ion channel currents and transverse tubule density in adult rabbit ventricular myocytes maintained for 6 days in cell culture; Mitcheson JS et al.; Adult rabbit ventricular myocytes were cultured in a basic medium (Medium 199) for up to 6 days to assess preservation of morphology and ion channel currents . In culture, cells remained rod shaped and striated but their ends became progressively rounded . Cell cross-sectional area declined slightly (by 14%) over the first 24 h, in contrast, whole-cell capacitance (which reflects external surface membrane plus membrane infoldings) decreased by 42% over the same time . Using whole-cell patch-clamp, we observed that the typical "N" shape steady-state current-voltage (I-V) relation became flattened after 24 h in culture . L-type Ca channel density was assessed as barium flux (IBa,L) via the channel . IBa,L (normalised to cell capacitance) declined by 50% after 24 h and recovered partially by days 4 and 6 . The density of inward rectifier K current declined by 54% after 24 h and showed no recovery subsequently . In contrast, there was no significant decline in the density of transient outward K current after 24 h, but it declined subsequently by 65% after 6 days . We speculate that the time course of change in each ion channel density may reflect a change in pattern of ion channel expression, or differential membrane loss since the density of transverse tubules decreased by 57% after 6 days in culture . These results suggest that even by 24 h in culture, ion channel density in myocytes has changed substantially from the acutely isolated state.

Appl Environ Microbiol, 1996 Apr, 62(4), 1424 - 7
Detection of infectious enteroviruses by an integrated cell culture-PCR procedure; Reynolds KA et al.; Rapid detection of infectious enteroviruses in environmental samples was made possible by utilizing an integrated cell culture-reverse transcriptase PCR approach . By this method, the presence of infectious enterovirus was confirmed within 24 h, compared with > or = 3 days by cell culture alone . The combined methodology eliminated typical problems normally associated with direct reverse transcriptase PCR by increasing the equivalent volume of environmental sample examined and reducing the effects of inhibitory compounds.

J Neurotrauma, 1996 Apr, 13(4), 223 - 31
Hemoglobin potentiates excitotoxic injury in cortical cell culture; Regan RF et al.; Excessive activation of glutamate receptors may contribute to neuronal loss after a traumatic or ischemic central nervous system insult . Such injuries are often associated with hemorrhage and extravasation of hemoglobin, a prooxidant and putative neurotoxin . In this study, we investigated the effect of nontoxic concentrations of hemoglobin on the neurotoxicity of the synthetic glutamate receptor agonists NMDA, AMPA, and kainate in primary murine cortical cultures . Continuous exposure to each excitotoxin alone for 24-28 h produced concentration-dependent neuronal death (EC(50) about 12 mu M for AMP(+)A, 50 mu M for kainate, and 12 mu M for NMDA) . Hemoglobin 0.25-1.0 mu M consistently potentiated the neurotoxicity of low concentrations of AMPA and kainate, increasing neuronal loss by about 150% at 6 mu M AMPA and by about 90% at 30 mu M kainate . This effect was attenuated by the iron chelator deferoxamine and the alpha-tocopherol analogue trolox . Hemoglobin coexposure had less impact on slowly triggered NMDA neurotoxicity, significantly increasing neuronal death only at agonist concentrations that alone produced little or no injury . Hemoglobin pretreatment had no effect on the rapidly triggered excitotoxicity induced by brief exposure to high concentrations of NMDA . These results suggest that hemoglobin may contribute to neuronal loss after CNS hemorrhage by exacerbating excitotoxicity . At moderate levels of agonist exposure, this effect may be somewhat selective for the AMPA/kainate component of injury.

J Steroid Biochem Mol Biol, 1996 Apr, 58(1), 117 - 21
Activity of progesterone and anti-progestins in a rat mammary primary cell culture system; Taylor JA et al.; A primary culture system of virgin rat mammary epithelial cells, grown in a serum-free medium, was developed as a means of assaying the efficacy of compounds with known anti-progestational properties . Cells were grown in 24-well plates on hydrated collagen gels and could be cultured for at least seven days . Experiments were routinely stopped three days after overnight attachment of cells using fibronectin (4 micrograms/ml) . DNA synthesis, measured by thymidine incorporation, was significantly increased by the addition of ovine prolactin (43 nM; P < 0.01) or progesterone (0.15 microM; P < 0.05) or both (P < 0.01) to the basal medium . When added to medium containing progesterone plus prolactin (complete medium), RU486 (mifepristone) and ZK98734 (lilopristone) significantly depressed DNA synthesis in a dose-dependent manner using doses ranging from 0.015 microM to 15 microM . Maximum inhibition was achieved at 15 microM for both compounds . DNA synthesis was 24.5 +/- 2.6% (mean +/- SEM, n = 4) and 32.0 +/- 2.2% (n = 3) of that in complete medium for RU486 and ZK98734, respectively (both P < 0.001) . There was no inhibitory effect of either compound in basal medium or basal medium plus prolactin, indicating the absence of toxicity and that the inhibitory effect is specific for a progesterone-mediated process.

Matrix Biol, 1996 Apr, 15(1), 21 - 9
Stimulation of collagenase (matrix metalloproteinase-1) synthesis in histiotypic epithelial cell culture by heparin is enhanced by keratinocyte growth factor; Putnins EE et al.; The role of heparin and heparan sulfate in the control of epithelial collagenase production was investigated utilizing a histiotypic cell culture model . The effect of keratinocyte growth factor (KGF), a heparin-binding growth factor, on collagenase secretion was also examined . Heparin, and, to a lesser extent, heparan sulfate induced release of a 58-kDa, gelatin-degrading enzyme which was subsequently identified as the collagenase, matrix metalloproteinase-1 . The increase in collagenase secretion by heparin was further enhanced by the addition of KGF . KGF alone did not have any effect . Analysis of secreted radiolabelled proteins showed that the increase in collagenase activity was not due to a general increase in protein synthesis . Synthesis of collagenase protein was specifically increased by heparin and further increased by KGF plus heparin . Heparin and heparan sulfate in combination with KGF may thus have important roles in the regulation of epithelial cell collagenase under conditions such as inflammation and wound healing.

Eur J Clin Invest, 1996 Apr, 26(4), 322 - 4
Quantitative and qualitative alterations of dystrophin are expressed in muscle cell cultures of Xp21 muscular dystrophy patients (Duchenne and Becker type); Mongini T et al.; The authors studied skeletal muscle cell cultures from four control subjects, two patients with Duchenne muscular dystrophy, two Duchenne carriers and three patients with Becker muscular dystrophy with different phenotypes . Western blotting was performed on well-differentiated myotubes and compared with the results obtained in muscle tissue . In all cultures the band of dystrophin closely corresponded to the one observed in muscle tissue: both quantitative and qualitative defects were observed . This confirms the early expression of the Xp21 gene defect in uninnervated muscle cultures and supports the usefulness of muscle cultures both in diagnostic procedure and as a model to study the disease.

Eur Respir J, 1996 Apr, 9(4), 808 - 20
Airway smooth muscle cell culture: application to studies of airway wall remodelling and phenotype plasticity in asthma; Hirst SJ; Chronic persistent asthma is characterized by poorly reversible airway obstruction . Histopathological studies of airways removed postmortem from patients with severe asthma reveal marked inflammatory and architectural changes associated with airway wall thickening . Increased airway smooth muscle content, occurring as a result of hyperplastic and/or hypertrophic growth, is believed to be one of the principal contributors to airway wall thickening . Intense interest is building to discover the mechanisms responsible for these long-term structural changes . In vitro cell culture offers a powerful and exacting approach to cellular and molecular studies of the long-term regulation of airway smooth muscle function . This review discusses the methodologies for establishing and maintaining cell cultures of airway smooth muscle . It also describes the characteristics of these cells in culture and addresses the potential importance of phenotype plasticity and its possible relationship to altered smooth muscle function in vivo . Drawing on parallels from vascular studies, this review focuses, in particular, on the synthetic nature of the airway smooth muscle cell, emphasizing its potential to alter the composition of the extracellular matrix environment and orchestrate key events in the process of chronic airway remodelling.

Exp Anim, 1996 Apr, 45(2), 161 - 70
Effects of phenobarbital on aniline metabolism in primary liver cell culture of rats with ethionine-induced liver disorder; Noguchi M et al.; In experiment 1, the amount of aniline (AN) metabolites in the primary cell culture medium of the liver cells obtained from ethionine (ET)-treated rats was compared with that of the control (normal) rats . Although the metabolites detected in both groups were p-aminophenol (p-AP), N-acetyl-p-AP (AAP), acetoanilide (AAN), AAP-glucuronide (AAPG), phenylhydroxylamine sulfate (PHAS) and p-AP-glucuronide (p-APG), the amount of AAP was lower and that of p-APG was markedly higher in the ET-treated rats than in the control rats . In experiment 2, phenobarbital (PB) was orally administered to the ET-treated and control rats at a dose of 100 mg/kg . The time course changes in AN metabolites in the primary cell culture medium of liver cells obtained at 2 or 48 hr after PB treatment were compared with those without PB treatment . In the ET-treated rats, the amount of PHAS was slightly higher at 2 hr after PB treatment, and that of AAP was lower and that of p-APG was higher at 48 hr after PB treatment as compared with those without PB treatment . In the control rats, the amounts of AAP, AAN, p-AP and p-APG at 2 hr after PB treatment remained lower than those without PB treatment, and that of AAP was markedly lower and that of p-APG was higher at 48 hr after PB treatment as compared with those without PB treatment . These findings indicated greater detoxication in the primary liver cell culture in the ET-treated rats than in the control rats . Furthermore, detoxication was greater in the primary cell culture of liver cell obtained from the ET-treated rats after PB treatment than from those without PB treatment, because the production of acetylates (AAP) decreased and p-APG increased (induction of conjugated enzyme) in the PB treatment group.

Eur J Clin Chem Clin Biochem, 1996 Apr, 34(4), 305 - 10
Glutamate-induced efflux of protein, neuron-specific enolase and lactate dehydrogenase from a mesencephalic cell culture; Gross J et al.; A mixed mesencephalic cell culture damaged by glutamate was used as a model to study the efflux of lactate dehydrogenase and neuron-specific enolase from neuronal cells into the culture medium . Glutamate toxicity was induced in sister cultures by 15 min exposure to 100 mumol/l glutamate in a Ca2+ containing salt solution . Cell injury was monitored 24 h later by measuring the lactate dehydrogenase activity and the neuron-specific enolase content in the cells and in the culture medium . The neuronal cell damage is reflected by an efflux of neuron-specific enolase and lactate dehydrogenase from the cells and an increase of lactate dehydrogenase catalytic activity concentration and neuron-specific enolase mass concentration in the culture medium . It was found that the efflux fraction calculated from estimations of the cells was clearly higher than the efflux fraction calculated from estimations of the amount of enzymes found in the culture medium . Calculations of the recovery of lactate dehydrogenase and neuron-specific enolase and experiments designed to study the efflux of lactate dehydrogenase and neuron-specific enolase during incubation and washing showed that higher amounts of neuron-specific enolase are released than lactate dehydrogenase . A close correlation was found between the glutamate-induced changes of the neuron-specific enolase efflux fraction, based on enzyme determinations of the cells, and the change of the microscopically counted neuron-specific enolase immunoreactive cell numbers . This indicates that the determination of the neuron-specific enolase efflux fraction (cells) is an accurate and sensitive marker of damaged neurons . The lactate dehydrogenase efflux fraction seems to be less sensitive for the quantitation of neuronal cell damage; in addition, it depends not only on the neuronal damage but also on the proportion of neurons in the cell culture.

J Cell Physiol, 1996 Apr, 167(1), 89 - 94
Nitric oxide-induced perturbations in a cell culture model of the blood-brain barrier; Hurst RD et al.; The actions of an intracellular nitric oxide generator compound on the properties of a co-culture model of the blood-brain barrier are described . Addition of the iron-sulphur cluster nitrosyl Roussin's black salt (RBS, heptanitrosyl-tri-mu3-thioxotetraferrate (1-)) resulted in a rapid and dose-dependent (50-250 microM) decline in the electrical resistance displayed by co-cultures of vascular endothelial cells and C6 glioma cells . The breach in barrier integrity elicited by RBS (250 microM) could be prevented by either haemoglobin (100 microM), methylene blue (200 microM), or by photon-induced inactivation of RBS . In contrast, the nitric oxide synthase inhibitor nitro-L-arginine methyl ester (250 microM) caused no inhibition in the decline in resistance of RBS-exposed cultures . Addition of 8-bromo-guanosine-cyclic monophosphate (500 microM) did not mimic the actions of RBS . Exposure to intense light of co-cultures manifesting a high transcellular electrical resistance resulted in a reduction in tissue resistance which could be prevented by the presence of haemoglobin (100 microM) . We conclude that nitric oxide liberated from RBS results in a reversible diminution in the integrity of the endothelial cell barrier in the co-culture system, and we suggest that light-sensitive endogenous nitric oxide generator compounds may be present in intact cells . Possible roles of nitric oxide in blood-brain-barrier function are considered.

Eur J Endocrinol, 1996 Apr, 134(4), 497 - 500
Bovine granulosa cell culture for assessment of potency and specificity of antibodies to pregnant mares' serum gonadotrophin; Kuran M et al.; Antibodies to pregnant mares' serum gonadotrophin (PMSG) neutralize the effect of PMSG in vivo and increase the number of transferable embryos when administered at the optimum time relative to the preovulatory luteinizing hormone (LH) surge in PMSG-stimulated cows . The objective of the present study was to investigate the possible use of bovine granulosa cells in a serum-free culture system as a bioassay for antibodies to PMSG . Granulosa cells (2-3 x 10(5) viable cells) were cultured with varying doses of PMSG and/or an anti-PMSG for 4 days . Whilst progesterone production (ng/micrograms DNA) of granulosa cells was stimulated by PMSG (p < 0.01) in a dose-dependent manner, increasing amounts of anti-PMSG neutralized (p < 0.01) this stimulatory effect of either follicle-stimulating hormone or LH on progesterone production of bovine granulosa cells in vitro . The bovine granulosa cell culture system is a potential in vitro bioassay method for testing the specificity and the neutralizing capacity of different anti-PMSG preparations.

J Cell Biol, 1996 Apr, 133(1), 185 - 97
Partial laminin alpha2 chain restoration in alpha2 chain-deficient dy/dy mouse by primary muscle cell culture transplantation; Vilquin JT et al.; Laminin-2 is a component of skeletal and cardiac basal lamina expressed in normal mouse and human . Laminin alpha2 chain (LAMA2), however, is absent from muscles of some congenital muscular dystrophy patients and the dystrophia muscularis (dy/dy) mouse model . LAMA2 restoration was investigated following cell transplantation in vivo in dy/dy mouse . Allogeneic primary muscle cell cultures expressing the beta-galactosidase transgene under control of a muscular promoter, or histocompatible primary muscle cell cultures, were transplanted into dy/dy mouse muscles . FK506 immunosuppression was used in noncompatible models . All transplanted animals expressed LAMA2 in these immunologically-controlled models, and the degrees of LAMA2 restoration were shown to depend on the age of the animal at transplantation, on muscle pretreatment, and on duration time after transplantation in some cases . LAMA2 did not always colocalize with new or hybrid muscle fibers formed by the fusion of donor myoblasts . LAMA2 deposition around muscle fibers was often segmental and seemed to radiate from the center to the periphery of the injection site . Allogeneic conditionally immortalized pure myogenic cells expressing the beta-galactosidase transgene were characterized in vitro and in vivo . When injected into FK506-immunosuppressed dy/dy mice, these cells formed new or hybrid muscle fibers but essentially did not express LAMA2 in vivo . These data show that partial LAMA2 restoration is achieved in LAMA2-deficient dy/dy mouse by primary muscle cell culture transplantation . However, not all myoblasts, or myoblasts alone, or the muscle fibers they form are capable of LAMA2 secretion and deposition in vivo.

Neuroreport, 1996 Mar 22, 7(4), 913 - 7
Effects of neurotrophins on early auditory neurones in cell culture; Malgrange B et al.; During the first week of postnatal development, the innervation of the organ of Corti changes from an immature to an adult pattern . Dissociated cell cultures of early postnatal spiral ganglia were used to investigate the effects of nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) on maturing auditory neurones . BDNF was the most potent stimulator of neuritogenesis, NT-3 provided the strongest support for neuronal survival, while NGF supported limited neuritogenesis, and only at pharmacological levels . These findings suggest that both BDNF and NT-3 participate in the postnatal maturation of cochlear innervation and that NGF is most probably not involved in this process.

J Chromatogr A, 1996 Mar 15, 727(2), 223 - 30
Determination of cytidine 5'-monophospho-N-acetylneuraminic acid pool size in cell culture scale using high-performance anion-exchange chromatography with pulsed amperometric detection; Fritsch M et al.; A simultaneous determination of cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) and its metabolic products, cytidine, CMP and Neu5Ac by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using a Carbo-Pac PA1 column is described . Preparation of the samples involved a single purification step of the crude cell extract on DEAE-Sepharose . The method is adequate to quantify the amount of CMP-Neu5Ac produced by one culture dish; equivalent to 6.10(6) cells . In addition, a method for desalting and recovery of the separated material was developed to determine the cellular concentration of CMP-Neu5Ac in Madin Darby canine kidney (MDCK) cells . The addition of 5 mM N-acetylmannosamine to the culture medium gave rise to a 6.4-fold elevation of this value.

Neurosci Lett, 1996 Mar 15, 206(2-3), 207 - 11
Regulation of glucocorticosteroid receptor expression in rat hippocampal cell cultures by nerve growth factor; Sarrieau A et al.; Dispersed hippocampal cells cultured in serum-free conditions were used to study the effects of nerve growth factor (NGF) on the expression of type I (mineralocorticosteroid or MR) and type II (glucocorticosteroid or GR) corticosteroid receptors . Cells, plated at a density of 1.2 x 10(6) cells/ml in 60 mm Petri dishes, were mainly identified as neurons (90-95%) and maintained for at least 2 weeks . A 7-day treatment with 10-50 ng NGF/ml induced a concentration-dependent decrease of GR binding (40% decrease) compared to untreated cells . In contrast, MR density was unaffected by a 7-day treatment with 50 ng NGF/ml . Data are discussed as possible direct and/or indirect effects of NGF at the level of both neuronal and glial cells.

Virology, 1996 Mar 15, 217(2), 442 - 51
The equine herpesvirus 1 IR6 protein is nonessential for virus growth in vitro and modified by serial virus passage in cell culture; Osterrieder N et al.; The IR6 protein of different plaque isolates from three passages of the equine herpesvirus 1 strain Rac was investigated . Southern blot and DNA sequence analyses revealed that plaque isolates from the 12th passage (RacL11 and RacL22) retained both copies of the IR6 gene, whereas two different genotypes were observed by the 185th passage: RacM24 still harbored both copies of the IR6 gene, whereas RacM36 deleted one of the two copies . In the 256th passage (RacH), both copies of the IR6 gene were absent . As compared to the wild-type IR6 protein, both the RacM24 and RacM36 IR6 protein displayed amino acid exchanges at positions 34, 42, 110, and 134 of the 272 amino acid polypeptide . It is shown that (i) the IR6 protein is nonessential for virus growth in vitro . (ii) In RacL11-infected equine and rodent cells, the typical rod-like appearance of the IR6 protein could be detected from 6 hr p.i., whereas in RacM24- and RacM36-infected cells formation of these structures was not observed . (iii) The RacL11 IR6 gene product was present in both the nuclei and cytoplasmic fraction of infected cells . In contrast, the IR6 protein of both RacM24 and RacM36 colocalized with cytoplasmic membrane vesicles . (iv) The RacL11 and RacL22 IR6 protein is present in viral nucleocapsids, whereas that of RacM24 and RacM36 is not incorporated into virions . (v) The RacL11 IR6 gene product aggregated to disulfide-linked oligomers, whereas the RacM24 and RacM36 IR6 protein showed only marginal oligomerization . (vi) In COS7 cells transfected with constructs expressing either the full-length RacL11-IR6 protein or a truncated form lacking the 81 carboxyterminal amino acids, the formation of rod-like structures was observed, indicating that another viral protein is not necessary for aggregation of the IR6 protein . In contrast, the IR6 protein expressed from constructs derived from either RacM24 or RacM36 failed to form these structures . (vii) Analyses of chimeric RacL11-RacM24 IR6 proteins suggest a crucial role for amino acid Leu-134 in the ability of the IR6 protein to form the rod-like structures.

Biochem Biophys Res Commun, 1996 Mar 7, 220(1), 181 - 5
The induction of cyclo-oxygenase-2 in human pulmonary epithelial cell culture (A549) activated by IL-1beta is inhibited by tyrosine kinase inhibitors; Akarasereenont P et al.; Cyclo-oxygenase (COX) exists as two isoforms . In endothelial cells, the induction of COX (COX-2) elicited by endotoxin or inflammatory cytokines is mediated by tyrosine kinase . Here we have investigated whether the induction of COX-2 elicited by IL-1beta in human pulmonary epithelial cells (A549) is mediated by tyrosine kinase . The activity of COX-2 was assessed by measuring the accumulation of PGE2 by radioimmunoassay . The expression of COX-2 protein was detected by immunoblot using specific antibodies to COX-2 . Untreated A549 cells contained no COX-2 protein and released low levels of PGE2 (<0.3 ng/ml for 24h) . A549 cells treated with IL-1beta (0.01 to 10 ng/ml) contained COX-2 protein and released greater amounts of PGE2 . The increased COX-2 protein and activity in response to IL-1beta (10 ng/ml) was inhibited by the tyrosine kinase inhibitors tyrphostin (AG126; 0.015 to 15 microM) or erbstatin (0.004 to 4 microM) . Thus, the induction of COX-2 by IL-1beta in epithelial cells is mediated by tyrosine kinase.

Shi Yan Sheng Wu Xue Bao, 1996 Mar, 29(1), 39 - 47
{CD4-CD8- to CD4-CD8+ transition induced by anti-CD3 mAb in vitro cell culture system}; Tian L et al.; In our experiments, we have observed the effect of anti-CD3 mAb in inducing the differentiation of CD4-CD8- (double negative, DN) thymocytes into CD4+ CD8+ (double positive, DP) cells in an in vitro cell culture system including IL-7 for maintaining the growth of TN thymocytes . When TN thymocytes were stimulated by immobilized anti-CD3 mAb for 3 days, CD4-CD8+ cells generated, which were mostly immediate precursors of CD4+CD8+ thymocytes according to their surface TCR beta expression . The transition from DN to DP thymocytes accompanied with downregulation of interleukin 2 receptor (IL-2 R) alpha chain, and the TCR alpha beta-CD3 expression induced by IL-7 was also inhibited . Anti-CD3 mAb was effective only before the appearance of functional TCR-alpha beta-CD3 complex . Taking together, the results strongly suggest that anti-CD3 mAb induced DN thymocyte defferentiation is through the pre-TCR complex.

AIDS Res Hum Retroviruses, 1996 Mar 1, 12(4), 315 - 23
Detection of unintegrated HIV type 1 DNA in cell culture and clinical peripheral blood mononuclear cell samples: correlation to disease stage; Nicholson WJ et al.; This article reports on the development of PCR as a sensitive method of detecting both linear and circular forms of HIV-1 unintegrated viral DNA (UVD) . The method was developed in a cell line study designed to follow the sequential synthesis of these forms over time . In all T lymphoid lineage cell lines, the full-length linear UVD (LUVD) was synthesized prior to both 1 and 2 LTR forms of circular UVD (CUVD), although all forms were detected by 12 hr postinoculation . Analysis of unstimulated PBMC samples from HIV-positive patients showed a significant difference in the presence of detectable CUVD forms and CDC groups II and IV (p < 0.001) and CDC groups III and IV (p < 0.001) . No significance was demonstrated between CDC groups II and III (p > 0.5), linking the presence of CUVD forms to clinical disease and immunodeficiency . We propose that circular unintegrated forms of HIV-1 DNA may play a role in the development of acquired immunodeficiency syndrome.

J Clin Microbiol, 1996 Mar, 34(3), 759 - 61
Isolation of group B porcine rotavirus in cell culture; Sanekata T et al.; While group A and C rotaviruses have been grown in cell culture, group B rotavirus has never been cultured . In this study we successfully isolated porcine group B rotavirus in swine kidney cells . Pancreatin treatment is essential for the propagation of group B rotavirus.

J Clin Microbiol, 1996 Mar, 34(3), 664 - 70
Isolation of the equine granulocytic ehrlichiosis agent, Ehrlichia equi, in tick cell culture; Munderloh UG et al.; The equine granulocytic ehrlichiosis agent, Ehrlichia equi, is closely related or identical to the human granulocytic ehrlichiosis (HGE) agent . Both are suspected of being transmitted by ticks . We have successfully isolated E . equi in a cell line, IDE8, derived from a putative vector, the tick Ixodes scapularis . Peripheral blood leukocytes from an experimentally infected horse were inoculated onto IDE8 monolayers . Cultures were incubated in a candle jar at 34 degrees C in tick cell culture medium with NaHCO3 and an organic buffer {3-(N-morpholino)-propanesulfonic acid} (MOPS) . Within 2 weeks, infected cells were detected in Giemsa-stained culture samples, and the organisms subsequently spread to uninfected cells in the cultures . E . equi was passaged serially by transferring a portion of an infected culture to new cell layers every 2 to 3 weeks . The identity of the organisms was confirmed by PCR using oligonucleotide primers specific for E . equi and the HGE agent and by immunocytology . Homologous equine antibodies and human anti-HGE convalescent serum recognized E . equi grown in tick cell culture . Electron microscopy revealed electron-lucent and -dense ehrlichia-like forms developing within host cell endosomes . E . equi passaged twice in tick cell culture retained infectivity and pathogenicity for the equine host, as demonstrated by intravenous inoculation of a suspension of infected tick cells and subsequent reisolation from peripheral blood, in fulfillment of Koch's postulates . The horse developed severe clinical signs, i.e., fever, inappetence, thrombocytopenia, icterus, and limb edema, typical of granulocytic equine ehrlichiosis, within 1 week.

Protein Expr Purif, 1996 Mar, 7(2), 137 - 42
Purification of recombinant insect transferrin from large volumes of cell culture medium using high capacity Ni(2+)-dipicolylamine gel; Winzerling JJ et al.; We report the purification of secreted recombinant Manduca sexta transferrin from Spodoptera frugiperda (Sf9) cell culture medium in a single step using high capacity Ni(2+)-dipicolylamine (DPA)-Novarose gel . Although the original sample was highly diluted (approximately 10 micrograms transferrin/ml medium) and the cell culture medium contained 10% surfactant (Pluronic F68) and a lipid emulsion, we were able to recover the recombinant transferrin (1 mg protein/100 ml) under gentle elution conditions with 70% yield at > 90% homogeneity . This work demonstrates the versatility of immobilized metal ion affinity chromatography using a high metal ion capacity gel to purify a recombinant protein and illustrates the potential of this affinity technique for protein separations from large volumes of cell culture media that contain surfactants.

Z Orthop Ihre Grenzgeb, 1996 Mar-Apr, 134(2), 117 - 24
{Biocompatibility of a new ionomer bone cement in endoprosthetics . In vitro testing using a mixed bone cell culture}; Meyer U et al.; Periosteal derived bovine osteoblasts and osteoclasts migrated in culture onto an ionomeric cement, currently evaluated as a cement for orthopaedic implants . Cell cultures were maintained for 4 weeks and used to study the in-vitro behaviour of cells on the material surface . Osteoblasts and osteoclasts colonized the substrate in monolayers and exhibited their phenotypic morphology . Differentiation into both cell lines were demonstrated by immunostaining . Staining for aluminium in cells growing on the bone cement showed an uptake and storage of aluminium in the cells . EDAX-microanalysis revealed high concentrations of Al and Si in the periosteal tissue . Despite uptake of Al in the cells, signs of toxicity were not apparent in the cell culture system.

J Pharm Pharmacol, 1996 Mar, 48(3), 277 - 80
Uptake and transport characteristics of chloroquine in an in-vitro cell culture system of the intestinal mucosa, Caco-2; Augustijns PF; The transepithelial transport and uptake of chloroquine were studied in cultured human intestinal Caco-2 cell layers, to investigate whether a specific mechanism facilitates the flux of chloroquine . Due to ionization of chloroquine at the pH of the intestinal lumen, the fraction of the neutral form, which is required for partitioning into biological membranes, is very low, while oral bioavailability has been reported to be nearly complete . Several observations, such as concentration-dependent uptake and temperature-dependent transepithelial flux, suggest the presence of carrier mediated transport . However, alternative mechanisms may be invoked to explain these observations . It is suggested that concentration dependence can originate from ion-trapping in acidic compartments of the cell or non-specific binding to cell components, while temperature-dependent transport can, at least partly, be explained by the temperature dependence of the acid dissociation constants of chloroquine . No differences were observed in the transepithelial flux of the enantiomers of chloroquine . pH-dependent uptake as well as pH-dependent transepithelial transport suggest that the translocation of chloroquine occurs according to the fraction of neutral molecules . From the data obtained in this study, it is concluded that chloroquine crosses the gastrointestinal barrier by passive diffusion . The extensive area of the gastrointestinal tract probably compensates for the low fraction of the neutral molecule . An interesting finding of this study was the concentration-dependent increase in transepithelial electrical resistance across monolayers incubated with chloroquine at the apical side.

Arch Oral Biol, 1996 Mar, 41(3), 243 - 52
Selected salivary-gland cell culture and the effects of isoproterenol, vasoactive intestinal polypeptide and substance P; Horie K et al.; To establish a selected salivary-gland cell culture and determine the effect of neuropeptides, monolayers were cultured using 3T3 cells as a feeder layer . To confirm the origin of these cultured cells, amylase production was examined by electron microscopy and periodic acid-Schiff staining, together with immunocytochemical analysis of myosin, anti-cytokeratin (CK-1, CK 10/13, CK MNF116, CK LMW, CK HMW and CK-19) and amylase antibody . The cultured cells demonstrated secretion granules containing amylase and presented features characteristic of acinar cells, which they retained until passage two . By using a feeder layer in conjunction with a newly formulated culture medium, the selectability of these cells was improved . Changes in proliferation of cultured salivary-gland cells in the presence of selected neurotransmitters were also examined . Isoproterenol enhanced cellular proliferation . On the other hand, vasoactive intestinal polypeptide and substance P, which increase the weight of salivary glands in vivo, showed no significant enhancement of proliferation.

Phytochemistry, 1996 Mar, 41(5), 1259 - 63
Expression of poplar phenylalanine ammonia-lyase in insect cell cultures; McKegney GR et al.; A cDNA encoding one of the phenylalanine ammonia-lyase genes from Populus trichocarpa x deltoides was inserted into a baculovirus expression vector and the PAL protein was successfully expressed in insect cell cultures . High levels of active holoenzyme were obtained that could be purified in a single chromatographic step . Site-directed mutagenesis and expression of the mutant enzyme confirmed that conversion of the putative active site serine202 residue to alanine is sufficient to destroy the catalytic activity of PAL.

J Invest Dermatol, 1996 Mar, 106(3), 510 - 6
Stimulation versus inhibition of keratinocyte growth by 1,25-Dihydroxyvitamin D3: dependence on cell culture conditions; Gniadecki R; 1,25-Dihydroxyvitamin D3 (1,25{OH}2D3) inhibits proliferation of keratinocytes in vitro and psoriatic epidermal cells in vivo and is considered to be a negative regulator of keratinocyte growth . It has been recently observed, however, that 1,25(OH)2D3 and its active analogs stimulate epidermal proliferation after topical application in mice . In this study we show that 1,25(OH)2D3, depending on the culture conditions, can either stimulate or inhibit DNA synthesis in human keratinocytes . In cells cultured with 0.15 mM calcium in the absence or with low levels (0.1 ng/ml) of epidermal growth factor, exposure to 10(-11) - 10(-6) M 1,25(OH)2D3 imposed cell cycle block in the late G1 phase . When keratinocytes were cultured in the presence of high extracellular calcium concentration (1.8 mM), 1,25(OH)2D3 in concentrations of 10(-11) - 10(-9) M stimulated cell growth by increasing the proportion of cells entering S phase . 1,25(OH)2D3 also stimulated growth of keratinocytes cultured in low calcium concentrations when the cells were previously suspended for a short time in a semisolid medium . Growth stimulation was absent in the presence of the anti-E-cadherin antibody, which is known to inhibit calcium-dependent differentiation . These results suggest that keratinocytes committed to terminal differentiation by an elevation of calcium concentration or suspension in a semisolid medium respond to 1,25(OH)2D3 with an increase in DNA synthesis . In contrast, proliferating undifferentiated keratinocytes may be the main target for the anti-proliferative activity of 1,25(OH)2D3.

J Virol, 1996 Mar, 70(3), 1981 - 9
Deduced consensus sequence of Sindbis virus strain AR339: mutations contained in laboratory strains which affect cell culture and in vivo phenotypes; McKnight KL et al.; The consensus sequence of the Sindbis virus AR339 isolate, the prototype alphavirus, has been deduced . THe results presented here suggest (i) that a substantial proportion of the sequence divergence evident between the consensus sequence and sequences of laboratory strains of AR339 has resulted from selection for efficient growth in cell culture, (ii) that many of these changes affect the virulence of the virus in animal models, and (iii) that such modified genetic backgrounds present in laboratory strains can exert a significant influence on genetic studies of virus pathogenesis and host range . A laboratory strain of Sindbis virus AR339 was sequenced and cloned as a cDNA (pTRSB) from which infectious virus (TRSB) could be derived . The consensus sequence was deduced from the complete sequences of pTRSB and HRsp (E . G . Strauss, C . M . Rice, and J . H . Strauss, Virology 133:92-110, 1984), from partial sequences of the glycoprotein genes of three other AR339 laboratory strains, and by comparison with the sequences of the glycoprotein genes of three other AR339 sequence . HRsp differed form the consensus sequence by eight coding changes, and TRSB differed by three coding changes . In the 5' untranslated region, HRsp differed from the consensus sequence at nucleotide (nt) 5 . These differences were likely the result of cell culture passage of the original AR339 isolate . At three of the difference loci (one in TRSB and two in HRsp), selection of cell-culture-adaptive mutations was documented with Sindbis virus or other alphaviruses . Selection in cell culture often results in attenuation of virulence in animals . Considering the TRSB and HRsp sequences together, one noncoding difference from the consensus (an A-for-G substitution in the 5' untranslated region at nt 5) and six coding differences in the glycoprotein genes (at E2 amino acids 1, 3, 70, and 172 and at E1 amino acids 72 and 237) were at loci which, either individually or in combination, significantly affected alphavirus virulence in mice . Although the levels of virulence of isogenic strains containing either nt 5 A or nt 5 G did not differ significantly in neonatal mice, the presence of nt 5 A greatly enhanced the effect of a second attenuating mutation in the E2 gene . These results suggest that minimal differences in the "wild type" genetic background into which an additional mutation is introduced can have a dramatic effect on apparent virulence and pathogenesis phenotypes . A cDNA clone of the consensus AR339 sequence, a sequence devoid of occult attenuating mutations introduced by cell culture passage, will allow the molecular genetic examination of cell culture and in vivo phenotypes of a virus which may best reflect the sequence of Sindbis virus AR339 at the time of its isolation.

Invest Ophthalmol Vis Sci, 1996 Mar, 37(4), 501 - 10
Gangliosides of migrating and nonmigrating corneal epithelium in organ and cell culture; Yang Z et al.; PURPOSE: To identify major gangliosides - the sialated glycolipids - of corneal epithelium; to determine which specific gangliosides, if any, are synthesized in a higher amount or are downregulated during corneal epithelial cell migration; and to determine what role, if any, they play in the modulation of corneal epithelial cell proliferation . METHODS: {3H}-galactose-labeled and unlabeled glycolipids of migrating and nonmigrating rabbit corneal epithelium in cell and/or in organ culture were chromatographed on DEAE Sephadex to isolate gangliosides . The gangliosides eluted from the ion-exchange column were further characterized by thin-layer chromatography (TLC), glycosidase digestions, and TLC-immunostain analysis . A {3H}-thymidine incorporation assay was used to determine the effect of exogenous gangliosides on corneal epithelium cell proliferation . RESULTS: Upon TLC of the acidic fraction eluted from the DEAE column, only two radiolabeled glycolipids (GL1 and GL2), migrating as a doublet, were detected . Regardless of whether the epithelia were prepared by cell culture or organ culture, both GL1 and GL2 were present in a significantly higher amount in migrating compared to nonmigrating epithelia . Further characterization of GL1 and GL2 identified them as gangliosides known as GM3 . TLC-immunostain analysis, as well as orcinol staining of thin-layer chromatograms of gangliosides of unlabeled cells, revealed that GM3 also accumulates in a higher amount in migrating compared to nonmigrating epithelial cell cultures . Exogenous addition of GM3, but not various other gangliosides, inhibited corneal epithelial cell proliferation in a dose-dependent manner . CONCLUSIONS: GM3 is the major ganglioside present in corneal epithelium, and its levels are elevated during corneal epithelial cell migration . It is suggested that the ganglioside plays a role in events that modulate corneal epithelial cell proliferation.

Exp Neurol, 1996 Mar, 138(1), 121 - 43
Basic fibroblast growth factor affects neuronal migration and differentiation in normotypic cell cultures from the cochleovestibular ganglion of the chick embryo; Hossain WA et al.; To study the role of basic fibroblast growth factor (FGF-2) in the development of sensory neurons, the cochleovestibular ganglion of the chicken embryo provides a well-characterized structure . This permits use of morphological markers in a cell culture preparation comparable to the normal embryo (normocytic) . Otocysts were explanted from white leghorn embryos at Hamburger-Hamilton Stages 14-16, when ganglion cell precursors normally start migrating from the otic epithelium . The cultures were supplemented with either fetal bovine serum or human recombinant FGF-2 (in defined medium or serum) for 2 or 5 days . FGF-2 increased explant growth, neuroblast migration, and neurite outgrowth 2- to 10-fold in the first 2 days . Neuronal morphology appeared within 2-3 days with FGF-2 but required at least 4-5 days with serum . FGF-2 in defined medium stimulated early migration and differentiation, but without serum led to degeneration after 5 days . In serum, growth was later and slower but continued for at least 3 weeks . When explants were cultured in serum with a neutralizing antibody to FGF-2, but no FGF added, neuroblast migration and elongation were decreased by 2- to 4-fold, compared to serum alone . Immunocytochemistry demonstrated FGF receptor sites on the migrating ganglionic neuroblasts, on their processes and growth cones, and in the incipient ganglion and otic epithelium at Stages 15-17, both in the embryo and in vitro . The findings suggest that FGF-2 stimulates early migration and differentiation of ganglion cells by activating the receptors of neuroblasts or their precursors in the embryonic otocyst . However, other factors must sustain their later development.

Neuroreport, 1996 Feb 29, 7(3), 710 - 2
Liposome-mediated NGF gene transfection increases ChAT activity in CNS cell cultures; Le W et al.; Liposome-mediated NGF transfection has been shown to increase the expression and secretion of NGF in primary rat septo-hippocampal cell cultures . Here we report that along with increased NGF expression, the activity of choline acetyltransferase, the synthetic enzyme for acetylcholine, is increased by 18% within 2 days, by 41% within 4 days and by 32% within 8 days after NGF gene transfection in septo-hippocampal cell cultures . This result further confirms that biologically active NGF is functionally expressed in septo-hippocampal cells when transfected with cDNA for NGF completed with liposomes.

Epilepsy Res, 1996 Feb, 23(1), 1 - 14
Remacemide HCl and its metabolite, FPL 12495AA, limit action potential firing frequency and block NMDA responses of mouse spinal cord neurons in cell culture; Wamil AW et al.; The novel anticonvulsant, remacemide HCl {(+/-)-2-amino-N-(1-methyl-1,2-diphenylethyl)acetamide monohydrochloride; FPL12924AA}, and a desglycinated metabolite {(+/-)-1-methyl-1,2-diphenylethylamine monohydrochloride; FPL 12495AA} reversibly limited sustained high-frequency repetitive firing (SRF) of sodium-dependent action potentials by mouse spinal cord neurons in monolayer dissociated cell culture . Limitation occurred with an IC50 of 7.9 X 10(-6) M for remacemide and 1.2 X 10(-6) M for FPL 12495AA (P < 0.05 vs . remacemide) . Stereoisomers of the desglycinate limited SRF with IC50 values of 3.3 X 10(-6) M and 3.5 X 10(-6) M for the S(+) and R(-) compounds, respectively . The concentration of racemic desglycinate and of either stereoisomer that produced limitation in all neurons tested was 10(-4) M . Maximal rate of rise (Vmax) of action potentials decreased progressively until firing ceased during 400-ms depolarizing pulses . Efficacy of remacemide, but not of the desglycinate, increased with time (maximum at 16-36 h) . The limitation was voltage dependent . In addition, reduction of Vmax and action potential failure occurred during stimulation with 400-ms pulses and trains of brief (1 ms) depolarizations at different frequencies . These findings suggest an effect on voltage-sensitive sodium current that generates the action potential upstroke . Remacemide and the desglycinate also significantly reduced the amplitude of neuronal responses to pressure application of NMDA in use-dependent manner at concentrations equal to the IC50 values for limitation of action potential firing . Resting potential and input resistance were not changed significantly by either drug . Limitation of high-frequency firing of action potentials by both remacemide HCl and FPL 12495AA may contribute to the anticonvulsant efficacy of these compounds at concentrations overlapping the range required to block glutamatergic hyperexcitability.

Photodermatol Photoimmunol Photomed, 1996 Feb, 12(1), 1 - 6
Phototoxicity due to sulphonamide derived oral antidiabetics and diuretics: investigations in a cell culture model; Selvaag E et al.; A number of sulphonamide-derived oral antidiabetics (chlorpropamide, glibenclamide, glipizide, gliquidone, glymidine, tolazamide and tolbutamide) and diuretics (bemetizide, bendroflumethiazide, benzylhydrochlorothiazide, bumetanide, butizide, chloratalidone, furosemide, hydrochlorothiazide, hydroflumethiazide, indapamide, piretanide, polythiazide, trichlormethiazide and xipamide) were investigated for phototoxicity in a cell culture model . Cell death dependent on ultraviolet A fluence and test substance concentration was observed in the presence of the oral antidiabetics glibenclamide and gliquidone, as well as the diuretics bemetizide, bendroflumethiazide, benzyl-hydrochlorothiazide, bumetanide, butizide, hydrochlorothiazide, hydroflumethiazide, piretanide, polythiazide and trichlormethiazide . Bendroflumethiazide was phototoxic at 5x10(-5) M and higher concentrations, bemetizide, benzylhydrochlorothiazide, bumetanide and hydroflumethiazide were phototoxic at 2.5x10(-4) M and higher concentrations, and the oral antidiabetics glibenclamide and gliquidone as well as the diuretics butizide, hydrochlorothiazide, piretanide, polythiazide and trichlormethiazide were phototoxic at 5(-4) M and higher concentrations . Electron microscopic investigations showed swelling of mitochondria and endoplasmic reticulum as well as aggregation of euchromatin when the cells were irradiated in the presence of photosensitizers.

Microsc Res Tech, 1996 Feb 1, 33(2), 232 - 9
Use of bone cell cultures to study skeletal pathology; Jackson ME et al.; We describe procedures for the isolation, culture, and analysis of neonatal osteoblasts from osteopetrotic (toothless (tl) and osteopetrosis {op}) rats and normal littermates . Normal osteoblasts produce and mineralize an extracellular matrix indistinguishable from that of well-characterized fetal rat osteoblasts in vitro . Mutant (tl and op) cultures show an early abnormal pattern of cell proliferation and a later premature, extensive mineralization which mimic the mutant phenotype in vivo . In cocultures with normal osteoclasts, mutant (tl) osteoblasts also show a greatly reduced ability to orchestrate bone resorption, as revealed by pit formation in bone slices, in response to physiologic mediators . These phenomena in vitro are consistent with the behavior of mutant osteoblasts and osteoclasts in vivo and suggest that more definitive microscopic analyses of osteoblasts from each mutation in vitro will provide insights on the roles of osteoblasts in the compromised bone resorption which characterizes the osteopetroses as well as their role in osteoclast ontogeny . This study shows that when their behavior is confirmed in vivo, bone cell cultures offer rigorous systems for understanding skeletal cell dysfunction in normal and pathological development.

Dev Dyn, 1996 Feb, 205(2), 135 - 49
Clonal cell cultures from adult spinal cord of the amphibian urodele Pleurodeles waltl to study the identity and potentialities of cells during tail regeneration; Benraiss A et al.; The urodele amphibians are nearly the only adult vertebrates able to regenerate their missing or amputated tail . The most striking feature of this model lies in the ability of the spinal cord (SC) to differentiate, within the regenerating tail, a new ependymal tube from which the SC and the peripheral nervous system originate . A fundamental question is whether, in response to tail excision, the ependymoglia of the old SC stump behaves as an embryonic neuroepithelium . To evaluate this possibility, cell lines from primary cell cultures of adult SC were established for the first time in newts, and two cell clones, immunochemically characterized as ependymoglial cell populations, could be obtained . To analyze the potentialities of these clonal cells, after transplantation in tail regenerates, cell-marking experiments, using either in vitro transfection with lacZ gene or the lineage tracer lysinated rhodamine dextran (LRD), were performed . One to 2 weeks postimplantation, most of labeled derivatives were identified as melanocytes . Interestingly, labeled cells were also seen integrated in the ependymoglia of the regenerating SC . Two to 6 weeks after implantation in young regenerates, we also observed LRD-labeled elongated cells close to nerves or myofibers which were unambiguously identified as Schwann cells by galactocerebroside staining . Taken together, these findings showed that clonal cells derived from adult newt SC cultures could largely find, in regenerate mesenchyme, suitable environmental conditions to differentiate into melanocytes or Schwann cells . Because these two cells types arise from neural crest cells during embryo-genesis, this supports the interesting view that multipotent cells are still present in the SC of adult urodeles.

Biol Reprod, 1996 Feb, 54(2), 446 - 52
Insulin-like growth factor I inhibits aromatization induced by follice-stimulating hormone in rat sertoli cell culture; Rappaport MS et al.; Sertoli cells in the testis and granulosa cells in the ovary convert androgen to estrogen under the primary control of FSH . Insulin-like growth factor I (IGF-I) markedly augments FSH-stimulated estrogen production in the rat granulosa cell . In this study we examined the regulation of aromatase by FSH and characterized the effects of IGF-I on FSH-induced estrogen production by Sertoli cells cultured from the testes of 16-day-old rats . FSH stimulated aromatization of androstenedione in Sertoli cell culture and achieved maximal effectiveness by 12 h of treatment . Analysis of aromatase mRNA by reverse transcription-polymerase chain reaction indicated a marked induction by FSH within 3 h of treatment that was dependent on FSH concentration . IGF-I inhibited FSH-stimulated aromatization dose-dependently; inhibition was approximately 50% by 6 h of cotreatment (p < 0.01) . IGF-I was ineffective if added more than 3 h after addition of FSH . Aromatase mRNA was reduced by IGF-I (37 +/- 12%, p < 0.01), coincident with the decrease in estrogen production . To further address the mechanism of IGF-I inhibition, potential interactions with the cAMP and protein kinase C (PKC) signaling pathways were examined . IGF-I inhibited aromatase activity induced by dibutyryl cAMP and inhibited FSH-stimulated estrogen production in the presence of 3-isobutyl-1-methylxanthine, suggesting that IGF-I action was independent of cAMP production . Phorbol-12-myristate-13-acetate (PMA) and IGF-I were additive in their inhibition of FSH . However, down-regulation of PKC prevented PMA inhibition of FSH but not inhibition by IGF-I . We conclude that IGF-I specifically inhibits FSH-induced aromatization in the Sertoli cell in marked contrast to the effects of IGF-I on rat granulosa cells . Although IGF-I and PMA both inhibit aromatase induction, the independence of the IGF-I effect from PKC down-regulation suggests that the initial action of IGF-I is independent of PKC . As IGF-I treatment similarly alters FSH stimulation of both estrogen production and aromatase mRNA, it is likely that the effect of IGF-I on estrogen production in the Sertoli cell is a result, at least in part, of a decrease in aromatase mRNA.

Lab Invest, 1996 Feb, 74(2), 557 - 70
Automated monitoring of apoptosis in suspension cell cultures; Kravtsov VD et al.; Cell death by apoptosis is often accompanied by extensive DNA cleavage at internucleosomal linker sites . Thus, the foremost techniques for estimating apoptosis are based on biochemical, electrophoretic, or flow cytometry analysis of nuclear DNA . However, apoptosis is also associated with a chain of morphologic changes in the nuclear and cytoplasmic structures that are easily recognizable using light microscopy . We suggest that changes in morphology of cells undergoing apoptosis might cause characteristic changes in their optical properties . It follows that continuous measuring of the OD of cells undergoing apoptosis would enable the study of the kinetics of cell death . We recently described an automated microculture kinetic (MiCK) assay that provides multiple OD measurements in nondisrupted cell cultures . In the present study the MiCK assay was employed to follow OD changes in HL-60 and OCI/AML-3 myelogenous leukemia cells and murine thymocytes exposed to ethanol, hydrogen peroxide, etoposide, cisplatin, doxorubicin, or hyperthermia; i.e., divers stimuli known to induce cell death via apoptosis or necrosis . The MiCK assay revealed prominent differences between the optical properties of the cells undergoing the two different modes of death . Plotting the OD data accumulated during the assay period against time betrayed characteristic patterns of either "apoptotic" or "necrotic" OD curves . The best fit slope of the indicative of apoptosis steep rising component of the apoptotic curve, correlated directly with the percentage of morphologically apoptotic cells in the culture . Criteria for graphical estimate of apoptosis were suggested and used to study apoptosis induced in HL-60 cells by the chemotherapy compounds etoposide and cisplatin . The MiCK assay demonstrated markedly varying time courses of the cell apoptotic response to these two drugs . In both cases, however, the time of graphically predicted peaks of apoptosis correlated with the time of morphologically and electrophoretically recognized peaks of apoptosis . Adaptability of the MiCK assay to a 96-well microplate format opens the way for large-scale studying of cell apoptotic response to various stimuli . An important technical advantage of the automated MiCK assay of apoptosis is that it does not require additional laboratory procedures after microcultures are initiated.

Biomaterials, 1996 Feb, 17(3), 237 - 42
Tissue engineering and autologous transplant formation: practical approaches with resorbable biomaterials and new cell culture techniques; Sittinger M et al.; The engineering of living tissues in vivo requires new concepts in cell culture technology . In contrast to conventional cell cultures, the development of tissues depends on a three-dimensional arrangement of cells and the formation or synthesis of an appropriate extracellular matrix . Special emphasis is given to the major role of the extracellular matrix and cell differentiation in an artificial tissue . New technical approaches of in vitro tissue engineering are compared to the natural development of tissues in vivo . Current methods using resorbable biomaterials, tissue encapsulation and perfusion culture are discussed . Major consideration is given to scaffold structures of biomaterials that define a three-dimensional shape of a tissue or guide matrix formation . The different goals of tissue engineering such as in vitro models and transplant production are taken into account in the described techniques . Practical concepts comprising cell multiplication and differentiation in subsequent steps for future clinical applications are outlined.

Planta Med, 1996 Feb, 62(1), 51 - 3
Production of hypericin, pseudohypericin and flavonoids in cell cultures of various Hypericum species and their chemotypes; Kartnig T et al.; Suspension cultures were established from the shoots of sterile germinated seeds of various provenances of seven Hypericum (H.) species in a half strength modified Murashige and Skoog liquid medium . In most strains of H . perforatum (18 provenances) and all strains of H . maculatum (6 provenances) as well as in the cultures of H . tomentosum, H . bithynicum, H . glandulosum and H . balearicum, hypericin and pseudohypericin could be proven, however, in extremely varying amounts . In general, the pseudohypericin content was significantly higher than that of hypericin . The flavonoid patterns, comprising monomeric quercetin derivatives and dimeric apigenin derivatives, varied among strains over a wide range.

Burns, 1996 Feb, 22(1), 35 - 9
Human keratinocyte isolation and cell culture: a survey of current practices in the UK; Daniels JT et al.; A survey was conducted to establish current techniques for isolation and culture of human keratinocytes . A questionnaire was sent to all units thought to be involved in keratinocyte culture, a total of 34 individuals; 62 per cent of those surveyed responded to the questionnaires . The proportion of individuals using high-calcium medium to culture keratinocytes was 53 per cent, while 47 per cent used low-calcium serum-free medium . The majority of replies followed trends dependent on the culture method employed . Details of anatomical donor skin site, keratinocyte isolation and culture were compared . In particular, the problems associated with the use of commercially prepared low-calcium, serum-free medium were reported . The basic principles of keratinocyte culture reported in the literature were seen amongst all the replies received . It is interesting to note the variations in methods adopted as techniques are passed on and continuously modified to suit the requirements of the individual worker . This survey also highlights the difficulties that can occur when using mass-produced complex media.

Exp Neurol, 1996 Feb, 137(2), 255 - 62
Elevated potassium enhances glutamate vulnerability of dopaminergic neurons developing in mesencephalic cell cultures; Andreeva N et al.; This study examines the effects of high K+ concentration on the growth and development of mesencephalic cells and their glutamate vulnerability . Mesencephalic cell cultures obtained from Wistar rat embryos on the 14th gestational day were maintained for 14 days in medium with either normal (4.2 mM) or elevated (24.2 mM) potassium concentration . There was no significant difference due to various K+ concentration in cell growth and survival up to day in vitro (DIV) 13-14 . In order to test the glutamate (Glu) vulnerability, cultures were treated with 100 mu M Glu for 15 min in salt solution on the DIV 3,6,8 and 13 . Glu-induced neuronal damage was estimated 24 h later by measuring the neuron-specific enolase (NSE) content in the culture medium and by counting the number of tyrosine hydroxylase-immunoreactive (TH-IR) neurons . Glu had no damaging effect on the cells on DIV 3, but became pronounced beyond DIV 6 . Elevated potassium concentration 24.2 mM) in the culture medium during development significantly increased neuronal vulnerability to Glu treatment, indicated by a higher increase of NSE content in the medium and by a more pronounced Glu-induced decrease of the number of TH-IR cells . The Glu-induced decrease of the number of TH-IR cells and of NSE-IR cells let us conclude that dopaminergic neurons are more vulnerable to glutamate than other neurons from mesencephalic culture.

Endocrinology, 1996 Feb, 137(2), 431 - 7
Transcriptional and posttranscriptional regulation of interstitial collagenase by platelet-derived growth factor BB in bone cell cultures; Varghese S et al.; Platelet-derived growth factor (PDGF), a bone cell mitogen, stimulates bone collagen degradation and does not enhance bone matrix apposition rates . The mechanism of the effect on collagen degradation is unknown, and it could involve changes in interstitial collagenase synthesis . We tested the effects of PDGF on interstitial collagenase expression in cultures of osteoblast-enriched cells from fetal rat calvariae (Ob cells) . After 4-8 h of treatment, PDGF BB at 0.3 nM increased steady state collagenase messenger RNA (mRNA), whereas PDGF AA had no effect . The effect of PDGF BB on collagenase transcripts was dose dependent . PDGF BB increased the levels of immunoreactive collagenase after 6 h, whereas the levels were decreased after 16 h . Stimulation of collagenase mRNA by PDGF BB was dependent on de novo protein synthesis and activation of protein kinase C . PDGF BB prolonged the half-life of collagenase mRNA in transcriptionally arrested cells . PDGF BB initially increased and subsequently decreased the rate of collagenase gene transcription and the levels of collagenase heterogeneous nuclear RNA . In conclusion, PDGF BB regulates interstitial collagenase in Ob cells by transcriptional and posttranscriptional mechanisms, and this effect may contribute to its stimulatory actions on bone collagen degradation.

J Virol, 1996 Feb, 70(2), 1091 - 9
Identification and characterization of the bovine herpesvirus 1 UL7 gene and gene product which are not essential for virus replication in cell culture; Schmitt J et al.; The UL7 gene of bovine herpesvirus 1 (BHV-1) strain Schonboken was found at a position and in a context predicted from the gene order in the prototype alphaherpesvirus herpes simplex virus type 1 . The gene and flanking regions were sequenced, the UL7 RNA and protein were characterized, and 98.3% of the UL7 open reading frame was deleted from the viral genome without destroying productive virus replication . Concomitant deletion of nine 3' codons from the BHV-1 UL6 ORF and 77 amino acids from the carboxy terminus of the predicted BHV-1 UL8 protein demonstrated that these domains are also not essential for function of the respective proteins . The UL7 open reading frame encodes a protein of 300 amino acids with a calculated molecular mass of 32 kDa . Comparison with UL7 homologs of other alphaherpesviruses revealed a high degree of homology, the most prominent being to the predicted UL7 polypeptide of varicella-zoster virus, with 43.3% identical amino acids . A monospecific anti-UL7 serum identified the 33-kDa (apparent-molecular-mass) UL7 polypeptide which is translated from an early-expressed 1.7-kb RNA . The UL7 protein was localized in the cytoplasm of infected cells and could not be detected in purified virions . In summary, we describe the first identification of an alphaherpesviral UL7-encoded polypeptide and demonstrate that the UL7 protein is not essential for replication of BHV-1 in cell culture.

Brain Res, 1996 Jan 29, 707(2), 282 - 7
Expression pattern of messenger RNAs for prostanoid receptors in glial cell cultures; Kitanaka J et al.; Expression level of messenger RNAs (mRNAs) for prostanoid EP3, FP, and TP receptors was investigated in cultured rat astrocytes, oligodendrocytes, and microglia, as well as in meningeal fibroblasts, rat glioma C6 cells, rat pheochromocytoma PC12 cells, whole brain, and several peripheral tissues by reverse transcriptase-polymerase chain reaction . Cultured astrocytes and oligodendrocytes expressed mRNAs for 3 prostanoid receptors examined . In contrast, cultured microglia and pheochromocytoma PC12 cells expressed EP3 and TP receptor mRNAs, but not FP receptor mRNA . Glioma C6 cells expressed only TP receptor mRNA among 3 prostanoid receptors with the same expression level as that in astrocytes . Cultured meningeal fibroblasts expressed 3 receptor transcripts, and their expression levels were lower than those in astrocytes . Expression level of mRNA for each prostanoid receptor in cultured glial cells was higher than that in whole brain . These observations suggest that each prostanoid has its specific roles in each glial cell type of the brain.

Brain Res, 1996 Jan 22, 707(1), 88 - 99
Transforming growth factor alpha differentially affects GABAergic and cholinergic neurons in rat medial septal cell cultures; Mazzoni IE et al.; The effects of transforming growth factor alpha (TGF alpha) on low and high density cultures of fetal (embryonic day 17) rat medial septal cells were investigated and in some instances, compared to those of epidermal growth factor (EGF) . In high density cultures, TGF alpha induces a significant increase in the number of astroglia and microglia . While the effects of TGF alpha on the astroglia are more pronounced when compared to EGF, those on the microglia are less notable . In addition, TGF alpha produces a time- and dose-dependent decrease in the activity of choline acetyltransferase (EC 2.3.1.6) and a proportional decrease in the number of acetylcholinesterase-positive neurons in these high density cultures . However, although both EGF and TGF alpha decreased choline acetyltransferase activity maximally at the same concentration (10 ng/ml), the latter was consistently more potent . TGF alpha does not affect cholinergic cell survival but the expression of their chemical phenotype and does so indirectly via the glial cells . On the other hand, TGF alpha directly induces a dose- and time-dependent increase in glutamic acid decarboxylase activity in these high density cultures without affecting the number of glutamic acid decarboxylase immunoreactive neurons . In low density cultures, TGF alpha acts as a general neuronal survival factor, affecting both cholinergic and GABAergic neurons . Here TGF alpha's neurotrophic activity is more evident than its effects on their chemical phenotype . These results suggest that TGF alpha exerts distinct and differential effects on the biochemical expression of two neuronal populations in the developing medial septum maintained in high density culture . Finally, as TGF alpha acts as a general neuronal survival factor in low density cultures, cell to cell interactions appear to be important in the ultimate response of these cells to this growth factor.

Int J Cancer, 1996 Jan 17, 65(2), 238 - 45
Modulation of MR-visible mobile lipid levels by cell culture conditions and correlations with chemotactic response; Delikatny EJ et al.; A transformed murine fibroblast cell line has been used to assess which criteria govern the appearance of a lipid pool that is mobile on the MR time scale . A high-resolution proton MR signal arising from neutral lipids, including triglyceride and cholesteryl esters, has previously been associated with membrane events in stimulated, transformed and malignant cells . We report that the attenuation of cellular proliferation by confluence or low pH caused significant increases in MR-visible lipid and that the lipid signal could be amplified at high density by the removal of serum . A significant decrease in chemotactic response accompanied the culture of cells at high density, but chemotactic response was not generally linked to alteration of the lipid signal . The appearance of the signal was also not correlated with the proportion of cells in any phase of the cell cycle . Significant changes in the MR-visible pools of the lipid metabolites choline, phosphocholine and glycerophosphocholine were measured under the culture conditions employed with 2D MRS and suggest that MR-visible lipid may arise from the catabolism of phospholipids.

Biochem Biophys Res Commun, 1996 Jan 17, 218(2), 490 - 4
Glucocorticoid regulation of androgen binding protein expression in primary Sertoli cell cultures from rats; Lim K et al.; Glucocorticoids are known to inhibit testicular function, and its receptor is also localized in the Sertoli cells . To evaluate possible role of glucocorticoid in Sertoli cells, the effects of dexamethasone on the expression of androgen binding protein (ABP) have been investigated in primary Sertoli cell cultures . Dexamethasone increased ABP mRNA levels, with maximal stimulation reached at 36 hr . The induction of ABP mRNA was dependent on the low concentration (10(-8) and 10(-7) M) of dexamethasone but gradually reduced in the cells treated with high concentration (10(-6) and 10(-5) M) . Dexamethasone-induced ABP mRNA level was no change in the cells after addition of cycloheximide but almost reduced by actinomycin-D pretreatment . Steady-state levels of ABP mRNA gradually increased in the Sertoli cells prepared from 14- and 21-days of age corresponding to rat puberty, and ABP mRNA was induced by dexamethasone . These results suggest that ABP gene is transcriptionally regulated by dexamethasone in primary Sertoli cell cultures.

Virology, 1996 Jan 15, 215(2), 207 - 10
Evidence for phenotypic mixing with reovirus in cell culture; Rozinov MN et al.; From pairwise mixed infections of different reovirus wild-type isolates (T3 Dearing plus T1 Lang or plus T2 Jones) the progeny virus is phenotypically mixed, i.e., progeny viral particles contain proteins derived from both parents but the corresponding genes derived from only one parent . Experiments with differential inactivation of virus progeny of mixed infections by monoclonal antibodies or by 33% ethanol reveal phenotypic mixing of two outer shell proteins, sigma 1 and probably mu 1 . Phenotypic mixing of the mu 1 protein, the product of the M2 gene, is a possible explanation for the recent observation of M2 gene-linked dominant interference between reovirus isolates.

Blood, 1996 Jan 15, 87(2), 545 - 56
Retention of quiescent hematopoietic cells with high proliferative potential during ex vivo stem cell culture; Young JC et al.; Human CD34+/Thy-1+/Lin- hematopoietic cells purified from bone marrow (BM) or mobilized peripheral blood (MPB) are highly enriched for pluripotent stem cells . Ex vivo expansion of this population is proposed as a means of providing accelerated short-term, as well as long-term, engraftment after myeloablative therapy . Here we demonstrate that primitive quiescent cells are retained in bulk expansion cultures of CD34+/Thy-1+/Lin- cells and that the cell production capacity of the expanded cell product can largely be attributed to cells exhibiting quiescent behavior during culture . CD34+/Thy-1+/Lin- cells from adult BM or MPB were labeled with the fluorescent membrane dye PKH26, followed by in vitro culture of 10(4) cells on a murine stromal layer in the presence of interleukin (IL)-3, IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF) . With each subsequent cell division, PKH26 fluorescence is reduced by roughly half, which allows tracking of the number of cell divisions . Progenitor cells present after a 2-week expansion period were sorted into CD34+/Lin-/dyebright and CD34+/Lin-/dyedim fractions and then cultured in a 4-week single-cell proliferation assay to characterize the proliferative capacity of each group . Fifty-nine percent of progenitors remaining dyebright after bulk culture (four or fewer cell divisions) were observed to proliferate in single cell culture, and produced an average of 1,780 cells per plated cell . In contrast, only 26% of dyedim (more than four divisions) progenitors were observed to proliferate and displayed a lower average proliferative capacity of 225 cells per plated cell . Similar behaviors were observed after a second consecutive cycle of bulk culture, indicating that quiescent cells with high proliferative capacity existed in culture for at least 4 weeks . Single CD34+/Lin-/dyebright progenitors purified from bulk cultures were observed to produce as many as 1,000 CD34 positive progeny during single cell culture, and these progeny included multilineage colony forming cells . These data demonstrate that among CD34 positive cells recovered after in vitro bulk culture, a higher proliferative capacity correlated with quiescent behavior . The described culture method provides quantitation of the cell producing capacity of individual cells in hematopoietic cell mixtures and may prove useful for predicting engrafting potential in products intended for cellular therapy.

Gynakol Geburtshilfliche Rundsch, 1996, 36(3), 143 - 8
{Diagnosis of Chlamydia trachomatis infections in routine ambulatory care of a gynecologic-obstetrical clinic: comparison of genome, antigen and cell culture detection methods for various indications}; Schultze D et al.; OBJECTIVE: Comparison of four different assays for routine diagnosis of urogenital Chlamydia trachomatis infections . METHODS: Samples from 285 female patients were tested using each of the following tests: PCR (Amplicor Chlamydia trachomatis), ELFA (VIDAS Chlamydia), cell culture and direct immunofluorescence assay (DFA) . RESULTS: C . trachomatis was detected by PCR in 13 endocervical swab specimens obtained from 189 female patients (6.9%) . Among 13 PCR-positive samples, 10 tested positive by cell culture and DFA, and 8 were positive by ELFA . For 3 patients with pregnancy-related complications, a positive result was obtained by PCR only . Each of the 96 urethral swabs proved to be negative . CONCLUSIONS: PCR is more sensitive than cell culture, DFA and ELFA, especially in the context of C . trachomatis infection during pregnancy . In addition and in order to avoid false-negative PCR results, a careful collection of epithelial cells infected with C . trachomatis is imperative.

Dev Biol Stand, 1996, 88, 49 - 56
Experience with viral contamination in cell culture; Garnick RL; Genentech has had direct experience with two contaminations of large-scale cell cultures by Minute Virus of Mice (MVM) . No definitive source of either contamination has been identified, although the conclusions of the investigation were consistent with a raw material used in cell culture as the source . Effective analytical barriers were developed following the first contamination using polymerase chain reaction (PCR) and cell culture-based methodology for MVM, and these were employed on a routine basis . This approach was effectively used to detect and contain the second contamination, thus dramatically minimizing its impact . Close interactions with regulatory authorities were critical in the positive management of these situations.

Oncol Res, 1996, 8(10-11), 435 - 47
The human breast cell DNA synthesome: its purification from tumor tissue and cell culture; Coll JM et al.; In this report, we describe for the first time the isolation and purification of a multiprotein complex for DNA replication from MDA MB-468 human breast cancer cells . This complex, which we designate the DNA synthesome, fully supports the in vitro replication of simian virus 40 (SV40) origin-containing DNA in the presence of the viral large T-antigen . Since the SV40 virus utilizes the host's cellular proteins for its own DNA replication, our results indicate that the DNA synthesome may play a role not only in viral DNA synthesis but in human breast cell DNA replication as well . Our studies demonstrate that the following DNA replication proteins constitute the DNA synthesome: DNA polymerase alpha, DNA primase, DNA polymerase delta, proliferating cell nuclear antigen, replication protein A, replication factor C, DNA topoisomerases I, II, and DNA polymerase epsilon . In addition, we successfully isolated the DNA synthesome from human breast tumor tissue as well as from xenografts from nude mice injected with the human breast cancer cell line MCF-7 . The DNA synthesome purified from the breast cancer tissues fully supports SV40 DNA replication in vitro . Furthermore, our results obtained from a novel forward mutagenesis assay suggest that the DNA synthesome isolated from a nonmalignant breast cell line mediates SV40 DNA replication by an error-resistant mechanism . In contrast, the DNA synthesome derived from malignant breast cells and tissue exhibited a lower fidelity for DNA synthesis in vitro . Overall, our data support the role of the DNA synthesome as mediating breast cell DNA replication in vitro and in vivo.

Rev Chir Orthop Reparatrice Appar Mot, 1996, 82(8), 724 - 36
{Cell culture and orthopedic surgery . II . Medical applications . Diagnosis, biomaterials evaluation, therapy}; Anselme K et al.; Currently, cell cultures are used for 3 kinds of applications in orthopaedics: diagnosis: they can help to diagnose hereditary diseases like Marfan syndrome, osteogenesis imperfecta, or some osteochondrodysplasia . biomaterial evaluation: cell cultures bring information to ensure safety and efficacy of medical devices following the European Community's directive concerning medical devices . Results of in vitro biomaterial evaluation must be related to cell types (osteoblasts or fibroblasts), cell species (human or rat) and methods used (primary cell line or immortalised cell line) . therapy: first clinical applications of cell cultures have been published recently . They concern cultured autologous chondrocytes reimplantation . Bone cells cultured on biomaterials have been tested in animal reimplantation experiments . Animal cells mediated gene therapy experiments are now developed on muscle cells . Cell cultures allow also to determine the best therapeutic way to cure bone tumors . For the moment, these applications are still limited but in the next years, they could develop considerably . Therefore, orthopaedic surgeons must keep interest in this new field.

Rev Chir Orthop Reparatrice Appar Mot, 1996, 82(8), 709 - 23
{Cell culture and orthopedic surgery . 1 . Principles, methods, fundamental applications}; Anselme K et al.; Many reasons, in 1996, may incite orthopaedic surgeons to take an interest in cell culture . Firstly, cell culture bring informations on bone cell physiology, and the physiological properties of the skeletal cells are involved in the success of the orthopaedic surgeon's act . Secondly, in orthopaedics field's literature, more and more articles using cell culture techniques are published and surgeons need some basic knowledges to understand these results . Then, in the first part of this work, we detail the essential rudiments; vocabulary, cell culture methods, and cell physiology notions . In the second part of this work, we resume the progress realized these last years with cells cultured from the skeleton . Using these informations, surgeons will be able to appreciate the potential uses of cell cultures for diagnosis, biomaterial evaluation and for the development of new therapies.

Chin J Biotechnol, 1996, 12(3), 201 - 6
Simple spinner bottle with rotating basket packed with carriers for hybridoma cell culture; Chen Y et al.; r-69B is a mouse-mouse hybridoma cell line, producing monoclonal antibody IgG against human r-IFN . It was cultured for 21 days in the 1.0-L spinner bottle which was assembled with a rotating basket packed with the 8.0-g Fibra-Cel carriers . The agitation was 100 r/min . The results showed that 53.5% of the cells can be trapped within the carriers in the basket and the cell concentration and MAb was about double those in the suspension culture . The spinner bottle could be assembled simply and used in general laboratories . It also could be used for different kinds of cells, including anchorage-dependent and independent cells.

Connect Tissue Res, 1996, 35(1-4), 343 - 9
Characterization of the apatite crystals of bone and their maturation in osteoblast cell culture: comparison with native bone crystals; Rey C et al.; Calcium phosphate crystals deposited in the organic matrix synthesized by chick bone osteoblasts in culture were studied by x-ray and electron diffraction, Fourier transform infrared spectroscopy and chemical composition . The amounts of mineral phase deposited with time and the extent of calcification (% of mineral phase in the tissue) were also determined as a function of time, as were the nature of the changes in the short range order of the crystals . The amount of mineral deposited and the extent of calcification increased with time; the tissue not only contained more crystals of apatite, but the extent of calcification also increased with time as it does in vivo . After 30 days of culture the extent of calcification in the cell culture matrix was similar to that in late chick embryonic and early postnatal chick tibiae . The nature of the CO3 and HPD4 environments were similar to those found in vivo although the concentrations of these ions and the changes in their concentrations with time appeared to develop more slowly in cell culture than they do in vivo . However, the general overall pathway of maturation was similar in cell culture to that observed in vivo.

Tsitologiia, 1996, 38(6), 646 - 9
{The cytogenetic study of established Syrian hamster cell lines . III . An analysis of the NOR-marker chromosomes in Syrian hamster cell cultures by using differential chromosome Ag-->G restaining}; Urmanova MA et al.; Using silver staining --> G-banding restaining procedure it was found that the Syrian hamster chromosomes had nucleolar organizers on subtelocentric chromosomes 2, 6, 9, 10 and 13, and on acrocentric chromosomes 16, 17 and 19 . In this case, NORs may be recognized only on one of homologous chromosomes, and no more than 9 NO-chromosomes may be found in one metaphase plate . In HaK, BHK-21 (C-13) BKK and BHK-21 (C-13) C cell lines, chromosomes 6, 9, 16, 17 and 19 have NORs, and in each line there are specific marker chromosomes with NOR . The markers consist of the material of chromosomes 6, 9, 16 and 17, with only chromosomes 16 and 17 being incorporated into rearrangements by their NORs . The silver staining --> G-banding restaining procedure makes possible not only identification of NO-chromosomes, but also refinement of the marker origins.

J Vet Diagn Invest, 1996 Jan, 8(1), 3 - 10
Detection of porcine reproductive and respiratory syndrome virus in cell cultures and formalin-fixed tissues by in situ hybridization using a digoxigenin-labeled probe; Larochelle R et al.; A nonradioactive in situ hybridization method is described for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) in cell cultures and in formalin-fixed paraffin-embedded tissue sections originating from experimentally infected pigs and from 1 field case . A 174 bp cDNA probe targeting the viral RNA encoding the nucleocapsid protein of a Canadian PRRSV isolate was generated by polymerase chain reaction . The cDNA probe was labeled by random priming with digoxigenin-dUTP using a commercially available kit . The ability of the digoxigenin-labeled probe to specifically detect PRRSV RNA was tested on cultured cells infected with 6 Canadian PRRSV isolates, a US PRRSV isolate and the European Lelystad isolate . The probe detected all Canadian PRRSV isolates tested as well as the US PRRSV isolate but did not detect the Lelystad isolate . In addition, when tested on formalin-fixed paraffin-embedded tissue sections from pigs experimentally infected with several Canadian isolates and from a field case, a strong signal without background staining was obtained . Our results indicate that nonradioactive in situ hybridization could represent a useful tool for the detection of PRRSV in routinely fixed and processed tissues . In situ hybridization could also be used to differentiate infection by North American and European Lelystad-like PRRSV isolates.

Biomaterials, 1996 Jan, 17(1), 23 - 9
Evaluation of implant materials (hydroxyapatite, glass-ceramics, titanium) in rat bone marrow stromal cell culture; Ozawa S et al.; Bone marrow stromal cells of femora from young adult rats produce bone-like mineralized tissue in culture . We evaluated three implant materials (hydroxyapatite (HA), glass-ceramics (GC) and commercial pure titanium (Ti)), as to their ability to provide an environment for marrow cells to differentiate into osteoblasts and function as suitable for mineralized tissue formation . We did this by measuring the DNA content, alkaline phosphatase (ALP) activity and calcium (Ca) content in culture, and the expression of osteopontin and bone sialoprotein by means of analysis of gene expression using Northern hybridization . DNA measurement showed no difference between each material, but ALP activity and Ca content in the culture on HA and GC were higher than on Ti and the control . An analysis of the gene expression revealed the highest expression of osteopontin and bone sialoprotein in the culture on HA . Mineralized nodule formation (both in area and number) was the most predominant on HA, followed by that on GC . These results showed that HA and GC provided a favourable situation for marrow cells to differentiate osteoblasts, resulting in a large amount of mineralized tissue formation on these surfaces . Present in vitro results could explain the rapid bone bonding of HA and GC in vivo.

Biomed Pharmacother, 1996, 50(8), 373 - 5
Expression of tissue-type plasminogen activator, plasminogen activator inhibitor and von Willebrand factor in the supernatant of endothelial cell cultures in response to the seeding of adenocarcinoma cell line HRT-18; Morganti M et al.; Plasminogen activator inhibitor (PAI-1), tissue type plasminogen activator (tPA) and von Willebrand factor (vWF) concentrations were measured by ELISA in the supernatant of the following cultures: endothelial cells from human umbilical vein (HUVEC); human colon cancer cells (HRT-18); and co-culture cells of HUVEC + HRT-18 . No measurable amount of the three substances was found in the supernatant of HRT-18 cell culture . Compared to the value in the HUVEC supernatant, in the UVEC/HRT-18 co-cultures, tPA concentration was significantly lower (P = 0.0047), PAI-1 significantly higher (P = 0.026) and vWF also significantly higher (P = 0.0048) . These data indicate that HRT-18 tumor cells do not produce tPA, PAI-1 and vWF; however, these tumor cells induce endothelial cells to change the production of these substances . As a consequence, the interaction between tumor and endothelial cells in vivo may lead to depression of fibrinolysis and enhancement of platelet adhesion.

Biomed Pharmacother, 1996, 50(8), 369 - 72
Detection of minimal but significant amount of von Willebrand factor in human omentum mesothelial cell cultures; Morganti M et al.; The presence of von Willebrand factor (vWF) in human mesothelial cells is a controversial issue . The aim of this paper was to investigate the presence of vWF in human mesothelial cell cultures by means of multiple specific techniques and to compare the amount of vWF to that in endothelial cell cultures . Morphological evidence that vWF is present in the cell cytoplasm in human omentum mesothelial cells (HOMC) has been obtained by vWF staining by means of anti-vWF antibodies and immunofluorescence . The vWF concentration value (measured by ELISA) was 0.02 ng/mL in the supernatant of HOMC (160,000 ng/mL cells/mL after 12-48 hours) and between 0.08-0.12 ng/mL in the supernatant of endothelial cell cultures (160,000 cells/mL) . Ethidium-bromide staining of PCR products recorded the typical vWF signal both in the endothelial and in the mesothelial cell cultures, although it was noticeably more intense in the endothelial cell culture . These data show that minimal but significant amounts of vWF are present in human mesothelial cell cultures.

Planta, 1996, 200(3), 289 - 95
Characterization of seven basic endochitinases isolated from cell cultures of Citrus sinensis (L.); Mayer RT et al.; Seven endochitinases (EC 3.2.1.14) (relative molecular masses 23,000-28,000 and isoelectric points 10.3-10.4) were purified from nonembryogenic Citrus sinensis L . Osbeck cv . Valencia callus tissue . The basic chitinase/lysozyme from this tissue (BCLVC) exhibited lysozyme, chitinase and chitosanase activities and was determined to be a class III chitinase . While BCLVC acted as a lysozyme at pH 4.5 and low ionic strength (0.03) it acted as a chitinase/chitosanase at high ionic strengths (0.2) with a pH optimum of ca . 5 . The lysozyme activity of BCLVC was inhibited by histamine, imidazole, histidine and the N-acetyl-D-glucosamine oligosaccharide (GlcNAc)3 . The basic chitinase from cv . Valencia callus, BCVC-2, had an N-terminal amino acid sequence similar to tomato and tobacco AP24 proteins . The sequences of the other five chitinases were N-terminal blocked . Whereas BCLVC was capable of hydrolyzing 13.8-100% acetylated chitosans and (GlcNAc)4-6 oligosaccharides, BCVC-2 hydrolyzed only 100% acetylated chitosan, and the remaining enzymes expressed varying degrees of hydrolytic capabilities . Experiments with (GlcNAc)2-6 suggest that BCLVC hydrolysis occurs in largely tetrasaccharide units whereas hydrolysis by the other chitinases occurs in disaccharide units . Cross-reactivities of the purified proteins with antibodies for a potato leaf chitinase (AbPLC), BCLVC, BCVC-3, and tomato AP24 indicate that these are separate and distinct proteins.

J Gynecol Obstet Biol Reprod (Paris), 1996, 25(5), 479 - 84
{Prevalence of Chlamydia trachomatis among men consulting in urology . Comparative study between cell culture and sperm PCR}; Aynaud O et al.; OBJECTIVE: Genital Chlamydia trachomatis infection can be difficult to diagnose, depending on the precise site of infection . Given the lack of major clinical signs and symptoms in many cases and the risk of male and female infertility . Chlamydia trachomatis is a public health problem . It can be difficult to detect this pathogen in sperm by means of cell culture, because of seminal fluid toxicity for cell lines . New techniques such as the polymerase chain reaction (PCR) can be used to detect genomic DNA . STUDY DESIGN: We studies 81 patients by applying the Amplicor CT PCR test to sperm, in comparison with cell culture on sperm and urethral samples . RESULTS: The prevalence of Chlamydia trachomatis infection was not significantly different (3.7% vs 5%) in the urethral cell culture and PCR methods, respectively (p > 0.05) . In contrast, PCR was significantly more sensitive than sperm cell culture (5% vs 1.2%; p < 0.03) . Moreover, we have not detected of genital chlamydiose among the infertile men . CONCLUSION: These findings suggest that PCR detection of Chlamydia trachomatis can dispense with the need for urethral sampling and cell culture in selected male patients.

Acta Neurochir (Wien), 1996, 138(8), 1002 - 7
A case of pituitary somatotroph adenoma with concomitant secretion of growth hormone, prolactin, and adrenocorticotropic hormone--an adenoma derived from primordial stem cell, studied by immunohistochemistry, in situ hybridization, and cell culture; Matsuno A et al.; Somatotroph adenomas often secrete prolactin (PRL) besides growth hormone (GH) and are sometimes immunostained for other anterior pituitary hormones or their subunits, such as thyroid-stimulating hormone (TSH) beta-subunit and glycoprotein hormone alpha-subunit (alpha SU) . However, somatotroph adenomas showing hypersecretion of adrenocorticotropic hormone (ACTH) are extremely rare . There have been, to our knowledge, only five published reports on somatotroph adenomas accompanied by excessive ACTH secretion . Here we report a case of intracavernously invading somatotroph macro-adenoma with high serum GH, PRL, and ACTH levels . We examined the case using immunohistochemistry (IHC), in situ hybridization (ISH), and cell culture, and confirmed GH, PRL, and ACTH, as well as alpha SU, production, and the expression of Pit-1 protein by the adenoma, which is known as a transcriptional factor for GH, PRL, and TSH, not for ACTH . Therefore, the presence of unknown transcriptional factor other than Pit-1, common to GH, PRL, and ACTH, may be speculated to be expressed in this adenoma . In our previous study, we had found plurihormonal mRNA expression, especially for ACTH, the beta-subunit of follicle-stimulating hormone and luteinizing hormone in some somatotroph adenomas, using non-radio-isotopic ISH, and suggested that these adenomas might be derived from plurihormonal primordial stem cells . Our present case is significant from the viewpoint of histogenesis of pituitary adenomas, because it further supports the cell origin of somatotroph adenomas from plurihormonal primordial stem cells, and moreover it suggests the presence of unknown transcriptional factor other than Pit-1, common to GH, PRL, and ACTH.

Free Radic Biol Med, 1996, 21(3), 297 - 306
Conventional cell culture media do not adequately supply cells with antioxidants and thus facilitate peroxide-induced genotoxicity; Leist M et al.; Commercially available calf serum did not supply the cultured murine fibroblast cell line L929 with amounts of selenium and alpha-tocopherol sufficient to protect against peroxide damage . Supplementation of the culture medium with 30 microM alpha-tocopherol or 50 nM sodium selenite led to a substantial increase of cellular alpha-tocopherol concentrations from 18 +/- 3.0 to 3179 +/- 93.0 pmol/10(6) cells or cellular selenium concentrations from 0.17 +/- 0.02 to 1.75 +/- 0.16 ng/10(6) cells, respectively . L929 fibroblasts grown in selenite-containing medium also had markedly raised activities of both cytosolic glutathione peroxidase (from 11 +/- 0.9 to 67.2 +/- 4.2 mU/10(7) cells) and phospholipid hydroperoxide glutathione peroxidase (from 0.2 to 9.5 +/- 0.9 mU/10(7)cells) . Supplementation with alpha-tocopherol inhibited single-strand breaks induced by low concentrations of H2O2 only, whereas an adequate selenium supply almost completely inhibited single-strand breaks induced by up to 30 microM H2O2 and also significantly reduced H2O2-induced cell death . An inadequate selenium supply and corresponding increase of GPx activity upon selenite supplementation was also observed with other cell lines, for instance, D10N, ECV-304, HepG2, and THP-1 . Our data strengthen the relevance of standardized and adequate supplementation of tissue culture media with antioxidants to improve viability and genetic stability of cultured cells in general and in particular, if they are oxidatively challenged.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 1996 Jan, 81(1), 63 - 9
Attenuation of the natural course of herpes simplex virus infection in human oral epithelial cell cultures by smokeless tobacco extracts suggests the possibility of a synergistic mechanism for carcinogenesis; Murrah VA et al.; High prevalence of both tobacco use and latent herpes simplex virus type 1 suggests the opportunity for synergism between these agents as cocarcinogens . In this study, postprimary human oral epithelial cell cultures were infected with herpes simplex virus type 1 pretreated with 2% extracts of either loose leaf, moist, or dry snuffs . Cultures were subsequently periodically exposed to the tobacco . Parameters measured included percentage of cultures undergoing active virus production, onset and time course of cytopathic effects, and concentration of virus released into the media over time . Results showed inhibition of both herpes simplex virus-mediated cell lysis and viral replication by tobacco extracts . This is the first time that these phenomena have been demonstrated in normal human oral epithelial cells . The work described here provides evidence to support a hypothesis that herpes simplex virus type 1 and smokeless tobacco may act synergistically in oral carcinogenesis.

Dev Neurosci, 1996, 18(1-2), 73 - 86
Cell culture approaches to understanding the actions of steroid hormones on the insect nervous system; Levine RB et al.; During metamorphosis of the hawkmoth, Manduca sexta, ecdysteroids regulate the dendritic remodeling and programmed death of identified motoneurons . These changes contribute to the dramatic reorganization of behavior that accompanies metamorphosis . As a step toward elucidating cellular and molecular mechanisms by which ecdysteroids affect neuronal phenotype, we have investigated the responses of Manduca motoneurons to ecdysteroids in vitro . Following dendritic regression at the end of larval life, thoracic leg motoneurons placed in culture respond to ecdysteroids by an increase in branching complexity, similar to events in vivo . Growth cone structure is affected markedly by ecdysteroids . At pupation, a rise in ecdysteroids triggers the segment-specific death of proleg motoneurons: the same segmental pattern of death is observed when motoneurons from different segments are removed from the nervous system and exposed to ecdysteroids in vitro . These studies provide strong evidence that Manduca motoneurons are direct targets of steroid action and set the stage for further studies of the specific mechanisms involved.

J Appl Toxicol, 1996 Jan-Feb, 16(1), 49 - 54
Potential chemoprotectant activity of mechanism-based glycosidase inhibitors against ricin toxicity in Chinese hamster ovary and macrophage J774A.1 cell cultures; Hassoun EA et al.; The abilities of the triacetylated galacto- and gluco-derivatives of 2-deoxy-2-fluoro-D-pyranosyl fluoride as well as alpha- and beta-N-bromoacetyl-D-galactopyranosylamine to inhibit the cytotoxicity of ricin in vitro in macrophage J774A.1 and Chinese hamster ovary (CHO) cell lines were determined . Leakage of lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) from the cells into the culture media were used as indicators of ricin cytotoxicity . The potential chemoprotectants were used in concentrations ranging from 10(-8) to 10(-4) g ml-1 . Of the four potential mechanism-based, site-specific glycosidase inhibitors that were tested, 3,4,6-tri-O-acetyl-2-deoxy-2-fluoro- beta-D-glucopyranosyl fluoride exhibited the greatest chemoprotectant activity . The ricin-induced LDH release was inhibited in a concentration-dependent manner by this compound, with the LDH leakage returning to control values in the presence of the highest concentration of this chemoprotectant in both cell cultures when given 4 h prior to ricin . This compound exhibited a small but significant inhibition of AST release from both cell cultures when given simultaneously with ricin . 3,4,6-Tri-O-acetyl-2-deoxy-2-fluoro-beta-D-galactopyranosyl fluoride exhibited a small but significant chemoprotective effect only at the highest concentration in both cell cultures when given simultaneously with ricin . Both the alpha- and beta-isomers of N-bromoacetyl-D-galactopyranosylamine exhibited activity against ricin toxicity in the CHO cell line, with the beta-isomer exhibiting greatest activity . The beta-isomer exhibited greater cytotoxicity in the absence of ricin, as demonstrated by the release of both enzymes from the cultured CHO cells . Further studies will be required to assess the utility of these compounds as chemoprotectants against ricin toxicity in vivo.

J Appl Toxicol, 1996 Jan-Feb, 16(1), 43 - 8
Potential chemoprotectant activity of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (DDC) against ricin toxicity in Chinese hamster ovary and macrophage J774A.1 cell cultures; Hassoun EA et al.; The protein toxin ricin is one of the most toxic substances known . No specific chemoprotective agents are available against ricin or similar protein toxins . Previous investigations have suggested that deoxynucleoside analogs may be effective in decreasing the toxicity of ricin . We have therefore examined the effects of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (DDC) in decreasing the cytotoxicity of ricin in J774A.1 macrophage and Chinese hamster ovary (CHO) cells in culture by assessing the release of lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) from the cells, as well as decreased cell viability . The results clearly indicate that DDC provided partial protection against ricin toxicity in both cell culture systems based on the leakage of LDH and AST . Concentration-dependent effects between 10(-10) and 10(-4) g ml-1 were produced . Under similar conditions, AZT had no effect on the toxicity of ricin in these two cell culture systems . When assessing cell viability, DDC almost doubled the viability of both CHO and J774A.1 cells at a concentration of 10(-4) g ml-1 in the presence of ricin . The results demonstrate that DDC but not AZT exhibits a chemoprotective effect against ricin toxicity in the two cell culture systems employed.

J Cell Biochem Suppl, 1996, 24, 24 - 31
Cell culture methods for the establishment of the NCI series of lung cancer cell lines; Oie HK et al.; More than 200 human small cell lung cancer and non-small cell lung cancer cell lines were established over 15 years mainly by utilizing the serum-free, hormone and growth factor supplemented, defined media HITES and ACL4 . Use of modified, established cell culture techniques such as the mechanical spillout method for the releasing of cell aggregates from tumor tissue, ficoll gradient centrifugation for the separation of tumor cells from erythrocytes and tissue debris, and an apparatue consisting of a platinum tubing attached to a suction flask for removal of spent medium have greatly contributed to the success in culturing tumor cells . Characterization of these lung cancer cell lines have extended our knowledge of lung cell biology . Studies elucidating the nutritional requirements of lung cancer cell growth may be helpful for the manipulation of these tumors in patients.

Scand J Rheumatol, 1996, 25(4), 233 - 8
Cytokine production (IL-6 and TNF alpha) in whole blood cell cultures of patients with systemic lupus erythematosus; Swaak AJ et al.; Whole blood cell culture has great advantage over isolated peripheral blood mononuclear cell culture, because it needs only small amounts of blood and is fast to perform . The current report focuses on the measurement of IL-6 and TNF alpha produced by peripheral blood monocytes of patients with systemic lupus erythematosus (SLE) in the whole blood cell culture system . After an initial triggering with lipopolysaccharide (LPS), a specific stimulus for monocytes, a decreased production of IL-6 relative to the controls was observed . Dividing our SLE patients according to treatment with corticosteroids, overall the IL-6 production was decreased in the patients treated with corticosteroids . TNF alpha production was comparable with normals, with the exception of an increased spontaneous production and using LPS stimulus of 4 pg/ml . In the patients treated with corticosteroids a decreased TNF production was observed, in contrast to the non-treated patients in which an increased TNF production was found compared with the controls using LPS doses higher than 62 pg/ml . The impaired acute phase reaction (APR) that has been described in the literature, might be explained by our observation of a decreased production of mainly IL-6 . However, also this study showed that treatment has a strong impact on ex vivo IL-6 and TNF production.

Antimicrob Agents Chemother, 1996 Jan, 40(1), 179 - 85
Effects of nifedipine, metronidazole, and nitric oxide donors on spore germination and cell culture infection of the microsporidia Encephalitozoon hellem and Encephalitozoon intestinalis; He Q et al.; Two species of microsporidia, Encephalitozoon hellem and Encephalitozoon intestinalis, were isolated from AIDS patients and cultured in green monkey kidney cells . A spore germination assay and a cultured-cell infection assay were used to test the efficacy of candidate antiparasitic agents . The calcium channel blocker nifedipine, metronidazole, and two nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside, were tested in the two assays . Nifedipine (10(-8) M) significantly inhibited E . hellem spore germination in three of four germination media . Metronidazole (10(-5) M) inhibited germination weakly and significantly inhibited E . intestinalis germination in a single germination medium . The inhibitory effect of nifedipine and metronidazole used together was greater than the sum of the effects of the drugs used alone in all E . hellem germination assays . The NO donors also inhibited spore germination . The inhibitory effect of nifedipine and metronidazole could be reversed by washing the spores, while that of the NO donors was not reversible . In early cultured-cell infections, both nifedipine (10(-8) M) and metronidazole (10(-5) M) significantly reduced the number of cells being infected . As the infection spread, these agents were less effective . Some inhibition of the spread of the infection was also demonstrated with the NO donors at a concentration (10(-5) M) not obviously toxic to the cultured cells . These data suggest that combination drug therapy targeting spore germination and intracellular parasite development is promising.

Rev Latinoam Microbiol, 1996 Jan-Mar, 38(1), 17 - 23
Detection of Chlamydia psittaci in enteric subclinical infections in adult sheep, through cell culture isolation; Escalante-Ochoa C et al.; Ovine chlamydiasis is a well known disease in countries with a good practice of breeding sheep herds, not so in Mexico . Aiming to determine Chlamydia psittaci presence in sheep herds in Mexico, we sampled 10 different farmlands in 5 geographical zones on the highlands, gathering a total of 267 viable samples from adult ewes in SPG (92 of them were obtained in a second sampling carried out in five randomly chosen farms) . Samples were treated and inoculated on L-929 cells grown in Iscove's supplemented medium; identification of the characteristic intracytoplasmic inclusions was performed by Giemsa stain, and indirect immunofluorescence in suspected samples . Isolation was successful in 92.88% of the trials . No significant differences were observed among the studied herds nor between the two samplings . This high incidence leads to consider seriously the possible pathogenic role of C . psittaci in Mexico.

Tsitologiia, 1996, 38(3), 319 - 24
{Intercellular communication studied by using the gap junctions in a primary cell culture of human aortic intima}; Andreeva ER et al.; The rate of gap junction communication has been investigated in the primary culture of highly differentiated mesenchymal cells (subendothelial smooth muscle cells isolated from grossly normal and atherosclerotic areas of human aorta), and in poorly differentiated cells of mesenchymal origin (adult human skin fibroblasts as well as skin fibroblasts and aortic smooth muscle cells derived from human fetus) . In cell cultures isolated from grossly normal and atherosclerotic aorta the number of cells coupled via gap junction increased with cell density and reached the plateau at a cell density of 50-70 cells/mm2 . In cultures of normal aortic cells the number of coupled cells was significantly higher than in cultures of atherosclerotic cells . Gap junctional communication between cells loaded with lipid inclusions was lower that that between cells free of excess of intracellular lipids . In cultures of human skin fibroblasts the rate of intercellular communication was comparable to that in cultures of atherosclerotic cells . There was practically no cell-to-cell communication in cultures of fetal cells . It is hypothesized that the reduced gap junctional communication in atherosclerotic human aorta is associated with alterations in the degree of smooth muscle cell differentiation.

Bioelectromagnetics, 1996, 17(1), 48 - 57
Cell culture dosimetry for low-frequency magnetic fields; Hart FX; Calculations of the current density and electric field distributions induced in cell cultures by an applied low-frequency magnetic field have assumed that the medium is uniform . This paper calculates these distributions for a more realistic, inhomogeneous, anisotropic model in which the cells are regarded as conducting squares surrounded by insulating membranes . Separate parameters are used to specify the resistivities of the cell interior, the cell membrane parallel to its surface, the cell membrane perpendicular to its surface, and the intercellular junction parallel to the membrane . The presence of gap junctions connecting the interiors of adjacent cells is also considered . For vertical applied magnetic fields, the induced currents and field distributions may deviate considerably from the homogeneous medium model if there is sufficiently tight binding of the cells to each other . The presence of gap junctions can produce relatively large transmembrane electric fields or intracellular current densities . These considerations are generally less important for horizontal applied fields . A simple microscopic model of the cell surface is also discussed.

Breast Cancer Res Treat, 1996, 38(3), 313 - 24
Expression of L-PHA-binding proteins in breast cancer: reconstitution and molecular characterization of beta 1-6 branched oligosaccharides in three-dimensional cell culture; Taeda Y et al.; Expression of beta 1-6 branched oligosaccharides in human breast cancer cells was investigated in vivo and in vitro . Lectin histochemical and lectin blotting analyses of surgically resected specimens were performed using L-PHA (phaseolus vulgaris leukoagglutinin) lectin, which binds to beta 1-6 oligosaccharides . The glycoproteins bearing beta 1-6 oligosaccharides of breast cancer tissues were found to be 170 kD and 120 kD in molecular weight, and the former appeared to be an epitope of carcinoembryonic antigen (CEA) . The beta 1-6 oligosaccharides were expressed in both cancer cell lines at the outer layer of the colonies when cultured in type I collagen, but not in agarose gel . No correlation was observed between beta 1-6 expression and cell cycle . The beta 1-6 oligosaccharides did not coincide with breast cancer-associated antigens, such as CEA, MUC1, and cathepsin D . The beta 1-6 oligosaccharides of these cell lines were markedly inhibited when swainsonine, a mannosidase II inhibitor, was added to the culture medium . The 120 kD molecule, which was obtained from MCF-7 cells cultured in type I collagen gel, was consistent with that of breast cancer tissues and was similar to lysosome-associated membrane glycoproteins (LAMPs) . The results suggest that the glycoproteins bearing beta 1-6 branched oligosaccharides in human breast cancer incorporate an epitope of CEA and human LAMPs and that the expression of LAMPs may depend on their surrounding matrices and may play an important role in cancer invasion or metastasis.

Avian Dis, 1996 Jan-Mar, 40(1), 109 - 13
Apoptosis in cell cultures induced by infectious bursal disease virus following in vitro infection; Tham KM et al.; Chicken embryo fibroblasts (CEFs) and Vero cells infected with infectious bursal disease virus (IBDV) exhibited the biochemical feature of apoptosis . Agarose gel electrophoresis of DNA extracted from IBDV-infected cells revealed the characteristic laddering pattern of DNA fragmentation, which was more intense in infected CEFs than in Vero cells . The appearance of apoptotic nucleosomal DNA fragments in IBDV-infected CEFs was independent of virus replication and occurred at an early stage following in vitro infection.

Avian Dis, 1996 Jan-Mar, 40(1), 103 - 8
Primary cell culture of turkey intestinal epithelial cells; Ali A et al.; Primary cell culture has been widely used in various types of studies and proven useful for the isolation and identification of avian pathogens . Difficulties in growing intestinal epithelial cells in vitro have limited their use for such studies . In the present study, a co-culture system was developed for the primary culture of intestinal epithelial cells . A monolayer obtained from 14-to- 16-day-old turkey embryo intestinal fibroblasts was used as a feeder layer . Feeder layers from turkey embryo fibroblasts and from a continuous cell line (mouse 3T3 fibroblasts) were also employed but were not as successful . The intestinal epithelial cells were isolated by dissociation from the intestinal tracts of 1-day-old turkey poults and grown on the feeder layers . Growth and maintenance media were supplemented with various components, including fetal calf serum, chicken serum, hormones, and other growth factors . The epithelial cells grown on feeder layers from the intestinal fibroblasts allowed the intestinal epithelial cells to be maintained in vitro for periods of 7 to 10 days . This technique may prove useful for various applications, including isolation of enteropathogens, and for basic studies of the intestinal tract concerning such subjects as physiology, immunology, and toxicology.

Arch Virol, 1996, 141(2), 331 - 44
Rapid and sensitive polymerase chain reaction based detection and typing of foot-and-mouth disease virus in clinical samples and cell culture isolates, combined with a simultaneous differentiation with other genomically and/or symptomatically related viruses; Vangrysperre W et al.; Reverse transcription followed by the polymerase chain reaction method (PCR) allowed the detection of foot-and-mouth disease virus (FMDV), regardless of the serotype . A primer set corresponding to highly conserved regions of the 2B sequence was selected . By combining in a single reaction tube specific primer pairs for FMDV, swine vesicular disease virus, (SVDV), encephalomyocarditis virus (EMCV) and bovine viral diarrhea virus (BVDV), all four viruses could be identified and differentiated in one amplification reaction, based on the different lengths of the respective amplified segments . In a similar way, FMDV types O, A, C, SAT 2 and Asia 1 could be identified and differentiated, using primers selected from the ID (VP1) genome region . All results were confirmed by direct sequencing of the PCR product . The very fast RNA extraction, reverse transcription and PCR permitted us to read the agarose gels within three hours after samples (cell culture isolates as well as clinical material) arrived, which is of great importance in case of an FMDV suspicion . Furthermore, a very high sensitivity was achieved (less than one PFU) . Therefore, our powerful detection assay by means of PCR for FMDV as well as for SVDV, EMCV and BVDV, has advantages compared to the presently used procedures.

Prostate Suppl, 1996, 6, 62 - 6
Responses of LNCaP prostatic adenocarcinoma cell cultures to LY300502, a benzoquinolinone human type I 5alpha-reductase inhibitor; Sutkowski DM et al.; We evaluate the metabolic inhibitory, antiproliferative, and antisecretory effects of LY300502, a benzoquinolinone human-specific type I-selective steroid 5alpha-reductase inhibitor in LNCaP human prostatic adenocarcinoma cell cultures . Reductive metabolism of {3H-T} in the LNCaP cells was inhibited in a concentration-dependent manner by LY300502 (IC50 approximately 5.77 nM) . The proliferative responses of LNCaP cells to LY300502 were examined in the presence of 0.1 NM testosterone (T), a concentration that stimulates maximal LNCaP cell numbers 40% above control levels . LY300502 significantly anatagonized T-induced stimulation of LNCaP cellular proliferation at concentrations greater that 10 nM (P<0.05), and at 1,000 nMcompletely blocked the mitogenic effects of T on LNCaP cells . In the absence of androgen, LY300502 had no effect on LNCaP cellular proliferation . In the presence of 100 nM T, an androgen concentration that maximally stimulates in vitro PSA production, LY300502 significantly antagonized T-induced PSA secretion at a concentration equal to or greater than 30 nM (P<0.05) . These studies provide the basis for additional investigations into the pathophysiologic significance of type I 5alpha-reductase to prostatic cancer and the potential utility of selective inhibitors as therapeutic agents.

Biosens Bioelectron, 1996, 11(3), 271 - 80
Determination of glutamine and glutamic acid in mammalian cell cultures using tetrathiafulvalene modified enzyme electrodes; Mulchandani A et al.; Tetrathiafulvalene (TTF) mediated amperometric enzyme electrodes have been developed for the monitoring of L-glutamine and L-glutamic acid in growing mammalian cell cultures . The detection of glutamine was accomplished by a coupled enzyme system comprised of glutaminase plus glutamate oxidase, while the detection of glutamic acid was carried out by a single enzyme, glutamate oxidase . The appropriate enzyme(s) were immoblized on the Triton-X treated surface of tetrathiafulvalene modified carbon paste electrodes by adsorption, in conjunction with entrapment by an electrochemically deposited copolymer film of 1,3-phenylenediamine and resorcinol . Operating conditions for the glutamine enzyme electrode were optimized with respect to the amount of enzymes immoblized, pH, temperature and mobile phase flow rate for operation in a flow injection (FIA) system . When applied to glutamine and glutamic acid measurements in mammalian cell culture in FIA, the results obtained with enzyme electrodes were in excellent agreement with those determined by enzymatic analysis.

J Nutr, 1996 Jan, 126(1), 251 - 8
The Caco-2 cell culture system can be used as a model to study food iron availability; Garcia MN et al.; To assess the usefulness of the Caco-2 cell culture system as a model to study the availability of dietary iron, preliminary experiments were performed to determine the optimal conditions for iron uptake and transport . Iron uptake of radioactive ferrous sulfate was optimal at pH 5.5 using a 2:1 molar ratio of ascorbic acid to iron and a 1-h incubation time . Under these experimental conditions, we studied the effect on iron uptake of adding supernatants from homogenates of different meat sources, soybean protein isolates, egg albumen and bovine serum albumin . Iron uptake was 6.3 +/- 1.7% from meat, which was significantly greater (P < 0.001) than the values of 1.2 +/- 0.3% from soybean protein, 1.3 +/- 0.3% from egg albumen and 0.8 +/- 0.1% from bovine serum albumin . Iron uptake was also significantly higher from digested meat samples than from undigested meat when the protein concentration was equalized . Measurements of iron uptake and protein concentration from fractions obtained after preparative isoelectric focusing of meat and soybean protein extracts showed two peaks of higher protein concentration and iron uptake in meat, apparently not found in soybean protein, that contained the factor(s) responsible for the higher iron uptake by the cells . In view of these observed similarities with iron absorption studies in humans, we conclude that the Caco-2 cell culture system is a useful in vitro model to study food iron availability.

J Gen Virol, 1996 Jan, 77 ( Pt 1), 119 - 22
Phenotypic mixing with recombinant haemagglutinin of high cleavability mediates multi-cycle replication of human influenza virus in cell culture; Kaverin NV et al.; When CV-1 cells expressing haemagglutinin (HA) of fowl plague virus A/FPV/34/Rostock(H7) (FPV) from an SV40-based recombinant vector were superinfected with the human influenza virus A/FM/1/47(H1N1)(FM1), phenotypically mixed progeny virus was observed . It contained cleaved FPV HA and uncleaved FM1 HA, was infectious without trypsin treatment and its infectivity was neutralizable by anti-FPV serum . When superinfection of H7 HA-expressing CV-1 cells was performed at a low multiplicity of infection, multi-cycle replication occurred . Control cells preinfected with an SV40-based recombinant not expressing FPV HA did not allow multi-cycle replication . Multi-cycle replication of FM1 virus was also observed when cells were preinfected with a vector expressing a highly cleavable mutant of influenza virus A/Port Chalmers/1/73(H3) HA carrying an insert of four arginine residues at the cleavage site . This was not the case when cells expressing uncleaved wild-type H3 HA were used . The results show that by phenotypic mixing with recombinant HA of high cleavability, a human influenza virus can be obtained in infectious form from cells lacking a suitable protease to activate this virus.

Endocrinology, 1996 Jan, 137(1), 65 - 71
Corticotropin-releasing factor (CRF) and glucocorticoids modulate the expression of type 1 CRF receptor messenger ribonucleic acid in rat anterior pituitary cell cultures; Pozzoli G et al.; Previous studies involving radioreceptor and functional assays have shown that CRF and glucocorticoids are able to modulate CRF receptors of the brain and anterior pituitary . In this study, we analyzed the effects of CRF, vasopressin (AVP), dexamethasone (DEX), and corticosterone on the regulation of CRF receptor (CRF-R1) messenger RNA (mRNA) levels in cultured rat anterior pituitary cells . CRF decreased CRF-R1 mRNA levels in a time- and concentration-dependent manner . In the presence of 10 nM CRF, CRF-R1 mRNA levels decreased within 1 h (to 65 +/- 3% of the control value; P < 0.01) with a maximal effect after 3 h (to 28 +/- 1% of the control value; P < 0.001) . The concentration dependence of the inhibitory effect of CRF at 3 h correlated with that required for ACTH secretion (half-maximal at approximately 0.03 nM) . Treatment with a maximal (100 nM) dose of AVP or a submaximal (0.1 nM) dose of CRF for 3 h reduced CRF-R1 mRNA levels to 66 +/- 3% and 53 +/- 6% of the control value, respectively . In the presence of both AVP and CRF, CRF-R1 mRNA levels were 32 +/- 3% of the control value . The incubation of cells for 3 h with 10 microM forskolin to activate adenylate cyclase or with 20 nM 12-0-tetradecanoylphorbol-13-acetate to activate protein kinase C resulted in a decrease in receptor mRNA levels to 40 +/- 9% (P < 0.01) and 28 +/- 8% (P < 0.001) of the control value, respectively, suggesting that the effects of CRF and AVP may be mediated by these pathways . DEX (20 nM) also caused a dose- and time-dependent decrease in mRNA levels . Maximal inhibition was observed after 3 h (to 31 +/- 6% of the control value; P < 0.001), with a partial recovery of mRNA levels at 24 or 48 h . Corticosterone similarly inhibited the accumulation of CRF-R1 mRNA in a dose- and time-dependent manner, but, in contrast to DEX, CRF-R1 mRNA levels returned almost to control levels after 24 h . These results indicate that the ability of CRF, AVP, and glucocorticoids to modulate the responses of corticotropes to CRF may be due in part to the actions of these agents on CRF-R1 mRNA accumulation.

J Neurochem, 1996 Jan, 66(1), 369 - 77
Characterization of agonist-induced down-regulation of NMDA receptors in cerebellar granule cell cultures; Resink A et al.; Exposure of cerebellar granule cells to NMDA in culture at 5 days in vitro, when cells are not yet vulnerable to NMDA, evoked a pronounced reduction in NMDA receptor activity, measured by NMDA-induced 45Ca2+ influx, and counteracted the normal developmental increase in NMDA receptors . The effect was concentration and time dependent, the half-maximal effect being reached at about 45 microM and by 4-5 h . The decrease in NMDA receptor function was accompanied by a significant reduction in the protein level of the obligatory NMDA receptor subunit (NR) NR1 . Both parameters remained at a low level as long as the agonist was present . However, receptor down-regulation was reversible, as receptor protein levels and NMDA responses were restored to control values upon NMDA removal, this process requiring protein synthesis . NMDA treatment also elicited a decrease in NR1, NR2A, and NR2B subunit messenger RNA (mRNA) levels . However, in comparison with NMDA receptor proteins, the decrease was faster, and NMDA receptor mRNA content recovered to control levels within 24 h in spite of the presence of NMDA . Concerning the mechanisms of agonist-induced regulation of NMDA receptor expression, it seems that protein kinase C-mediated protein phosphorylation is not involved, whereas inhibition of Ca2+/calmodulin-dependent kinase II/IV by KN-62 does depress NMDA receptor expression even in the absence of NMDA.

Neuroreport, 1995 Dec 29, 7(1), 93 - 6
BDNF or IGF-I potentiates free radical-mediated injury in cortical cell cultures; Gwag BJ et al.; Free radical-mediated damage to cultured cortical neurons was induced by a 24 h exposure to Fe2+ (30 microM) or an inhibitor of gamma-glutamylcysteine synthetase, L-buthionine-{S,R}-sulfoximine (BSO, 1 mM) . As expected, neuronal death was blocked by inclusion of the free radical scavenger trolox during the Fe2+ or BSO exposure . However, unexpectedly, pretreatment of the cultures with BDNF or IGF-I markedly potentiated neuronal death . This growth factor-potentiated death was still blocked by trolox, but was insensitive to glutamate antagonists . Concurrent addition of cycloheximide with the growth factors prevented injury potentiation . Present findings suggest that growth factors may increase free radical-induced neuronal death by mechanisms dependent upon protein synthesis.

J Biochem Toxicol, 1995 Dec, 10(6), 315 - 21
Chromium-induced production of reactive oxygen species, DNA single-strand breaks, nitric oxide production, and lactate dehydrogenase leakage in J774A.1 cell cultures; Hassoun EA et al.; The involvement of oxidative stress in the toxicity of chromium (VI) and chromium (III) has been proposed . We have therefore examined the effects of these cations on the production of superoxide anion, nitric oxide (NO), and DNA single strand breaks (SSB) in J774A.1 macrophage cells in culture as well as the effects on lactate dehydrogenase (LDH) leakage and cell viability . Following a 48 hour incubation, over twofold increases in superoxide anion and NO production were observed at concentrations of approximately 0.30 and 50 microM for Cr (VI) and Cr (III), respectively . The patterns of cell viability and LDH leakage paralleled superoxide anion and NO production for Cr (VI) and Cr (III) . A 50% decrease in viability was observed at approximately the concentrations that produced a twofold increase in superoxide and NO production . Concentration-dependent increases in DNA-SSB were observed after incubation with Cr (III) with maximum increases occurring at a concentration of approximately 60 microM . Cr (VI) had no effect on the incidence of DNA-SSB at any of the tested concentrations . The results indicate that Cr (VI) and Cr (III) are toxic to the J774A.1 cell line, and the toxicity may be due at least in part to an oxidative stress induced by the production of reactive oxygen species.

In Vitro Cell Dev Biol Anim, 1995 Dec, 31(11), 840 - 5
Isolation and characterization of a novel human prostatic stromal cell culture: DuK50; Roberson KM et al.; A novel human prostatic stromal cell culture, designated DuK50, has been passed in vitro > 12 mo . Tissue cultures were obtained from material harvested within a normal region of a radical prostatectomy specimen . These monolayers exhibited normal fibroblastic characteristics with each cell having a flattened, elongated appearance . Karyotypic analysis revealed a normal, male 46, XY chromosomal content with no numerical or structural abnormalities . DNA analysis using a Cell Analysis Systems Image Analyzer confirmed a euploid DNA content (7.9 pg DNA) . Cellular markers for verification of stromal cell type were performed by immunohistochemical techniques . DuK50 stained positive for vimentin and fibronectin . Immunostains for epithelial cytokeratins and prostate-specific antigen were negative, which ruled out contamination with prostatic epithelial cells . Negative immunostaining with desmin monoclonal antibody and light staining with smooth muscle actin alpha is consistent with the staining pattern of myofibroblasts . Response to various androgens, measured by a microculture tetrazolium assay technique, revealed a significant growth stimulation of DuK50 . Soft agar invasiveness assays and tumorigenicity studies in nude mice were negative . DuK50 exhibits a rapid doubling time with excellent plating efficiency, thrives in a readily available media supplemented with fetal bovine serum, and passes with routine trypsin protocols . The availability of this prostatic stromal cell culture may facilitate studies on this cell type's role in growth factor modulation, drug and steroid metabolism, and stromal-epithelial interactions in the prostate.

J Pharmacol Toxicol Methods, 1995 Dec, 34(4), 219 - 23
A rapid assay for measuring the metabolism of {3H}-retinoic acid in cell cultures; Garrabrant TA et al.; A simple method was developed to detect the metabolism of {3H}-retinoic acid to polar products using intact tumor cells in culture . Unaltered {3H}-retinoic acid was separated from more polar metabolites using C18-bonded solid phase extraction cartridges . Separation of unaltered retinoic acid and polar metabolites was confirmed by HPLC . The murine mammary carcinoma cell line TA3 Ha used in these studies converted 40% to 50% of added radioactive retinoic acid to polar metabolites released into the culture medium during a 4-hr incubation period . Metabolism of {3H}-retinoic acid by TA3 Ha cells was inhibited by the cytochrome P-450 inhibitors ketoconazole, clotrimazole, and liarozole . The simplicity and rapidity of this assay should make it useful for evaluating compounds as inhibitors of retinoic acid metabolism.

J Muscle Res Cell Motil, 1995 Dec, 16(6), 619 - 28
Expression of myosin heavy chain and of myogenic regulatory factor genes in fast or slow rabbit muscle satellite cell cultures; Barjot C et al.; We investigated the myogenic properties of rabbit fast or slow muscle satellite cells during their differentiation in culture, with a particular attention to the expression of myosin heavy chain and myogenic regulatory factor genes . Satellite cells were isolated from Semimembranosus proprius (slow-twitch muscle; 100% type I fibres) and Semimembranosus accessorius (fast-twitch muscle; almost 100% type II fibres) muscles of 3-month-old rabbits . Satellite cells in culture possess different behaviours according to their origin . Cells isolated from slow muscle proliferate faster, fuse earlier into more numerous myotubes and mature more rapidly into striated contractile fibres than do cells isolated from fast muscle . This pattern of proliferation and differentiation is also seen in the expression of myogenic regulatory factor genes . Myf5 is detected in both fast or slow 6-day-old cell cultures, when satellite cells are in the exponential stage of proliferation . MyoD and myogenin are subsequently detected in slow satellite cell cultures, but their expression in fast cell cultures is delayed by 2 and 4 days respectively . MRF4 is detected in both types of cultures when they contain striated and contractile myofibres . Muscle-specific myosin heavy chains are expressed earlier in slow satellite cell cultures . No adult myosin heavy chain isoforms are detected in fast cell cultures for 13 days, whereas cultures from slow cells express neonatal, adult slow and adult fast myosin heavy chain isoforms at that time . In both fast and slow satellite cell cultures containing striated contractile fibres, neonatal and adult myosin heavy chain isoforms are coexpressed . However, cultures made from satellite cells derived from slow muscles express the slow myosin heavy chain isoform, in addition to the neonatal and the fast isoforms . These results are further supported by the expression of the mRNA encoding the adult myosin heavy chain isoforms . These data provide further evidence for the existence of satellite cell diversity between two rabbit muscles of different fibre-type composition, and also suggest the existence of differently preprogrammed satellite cells.

Clin Infect Dis, 1995 Dec, 21(6), 1495 - 7
Diagnosis of ornithosis by cell culture and polymerase chain reaction in a patient with chronic pneumonia; Essig A et al.; We report the case of a woman who had pneumonia due to Chlamydia psittaci . A Chlamydia species was determined to be the causative agent of the pneumonia because it was isolated from bronchoalveolar lavage fluid, because it could be detected in lung biopsy specimens by the direct immunofluorescence technique, and because Chlamydia-specific antibodies could be detected by ELISA and microimmunofluorescence . The infectious agent could not be identified at the species level with use of serological techniques, but the isolate was determined to be C . psittaci by PCR with use of species- and genus-specific sequences within the chlamydial lipopolysaccharide biosynthesis gene gseA . The case reported herein exemplifies the problems encountered in diagnosing ornithosis and shows that isolation of the etiologic agent followed by identification of the species by PCR is helpful in diagnosing this rare disease . In addition, the findings in our case show that laboratory personnel who are conducting tests for Chlamydia pneumoniae should be aware of the risk of accidentally isolating highly infectious C . psittaci organisms.

Onderstepoort J Vet Res, 1995 Dec, 62(4), 227 - 33
The use of sucrose-acetone-extracted Rift Valley fever virus antigen derived from cell culture in an indirect enzyme-linked immunosorbent assay and haemagglutination-inhibition test; Paweska JT et al.; A sucrose-acetone-extracted, Madin-Darby-bovine-kidney (MDBK)-derived Rift Valley fever virus (RVFV) antigen was tested both in an indirect ELISA and a haemagglutination-inhibition test for its ability to detect serum antibodies to RVFV . Optimal conditions for antigen concentration, serum and conjugate dilutions for the ELISA were established by checkerboard titration . The specificity and sensitivity of ELISA were determined by the use of paired pre- and post-vaccination sheep-serum samples . Compared with the virus neutralization test, the overall ELISA specificity and sensitivity were 97.4 and 97.3%, respectively . There was a 100% correlation between the results obtained in haemagglutination-inhibition tests with a RVFV sucrose-acetone-extracted antigen derived from hamster liver, and from MDBK cells . A total of 10 582 field-serum samples (84 cattle, 3,659 sheep, 6,839 goats) collected in 1994-1995 from animals of unknown vaccination status in different regions of South Africa were tested with ELISA for antibodies against RVFV . There were no seropositive cattle, 0.16% seropositive sheep and 0.12% seropositive goats . This study demonstrates the potential diagnostic application of cell-culture-derived, sucrose-acetone-extracted RVFV antigen in an indirect ELISA and HI test.

Onderstepoort J Vet Res, 1995 Dec, 62(4), 217 - 22
Detection of bluetongue virus and African horsesickness virus in co-infected cell cultures with NS1 gene probes; Venter EH et al.; The serogroup specificity of the bluetongue virus (BTV) NS1 and VP3 gene probes was confirmed by means of northern blot hybridization . Under high-stringency conditions both probes hybridized to 22 BTV serotypes (18 South African serotypes, BTV3 from Cyprus and BTV16 from Pakistan) but not to serotypes that originate from Australia and India . Furthermore, NS1 gene probes of BTV and African horsesickness virus (AHSV) were used in a dot-spot in situ hybridization procedure to differentiate between BTV and AHSV in co-infected cell cultures . The method detects viral RNA directly i glutaraldehyde-fixed infected cell cultures without prior nucleic-acid extraction or purification . AHSV could be detected in cells infected with AHSV at a multiplicity of infection of 10(-4) PFU/cell in the presence of a hundred excess of co-infecting BTV . The method may have an application in epidemiological surveys to detect different orbiviruses in the same Culicoides population.

J Med Virol, 1995 Dec, 47(4), 370 - 7
Establishment and characterization of a carrier cell culture producing high titres of polyoma JC virus; Nukuzuma S et al.; This report concerns a carrier cell culture (designated JCI) infected persistently with JC virus (JCV) . Immunostaining with an anti-JCV antiserum revealed that JCI was a carrier culture in which only a small fraction of the cells (approximately 1.5%) produced the virus . The JCV titre was increased strikingly by incubating confluent JCI cells for 4-6 days in medium containing a low concentration of fetal bovine serum (2%) . Viral genomes cloned from the persistently infected JCI cells were heterogeneous with respect to size, but most clones had an alteration of the same regulatory region (designated CR-JCI) . Transfection experiments with a chimeric JCV DNA (Mad-1/CR-JCI), in which the regulatory region was CR-JCI and the other region was derived from an infectious JCV (Mad-1) DNA, showed that CR-JCI was less efficient in inducing viral growth than the regulatory regions of IMR-32-adapted JCVs . The transfected cells could be readily subcultured, and they continued to produce JCV . It is concluded that a decrease in the activity of the JCV regulatory region is of importance for the maintenance of the carrier state of JCI cells.

J Bone Miner Res, 1995 Dec, 10(12), 2011 - 6
Aluminum inhibits both initiation and progression of mineralization of osteoid nodules formed in differentiating rat calvaria cell cultures; Bellows CG et al.; Osteoid nodules form in cultures of fetal rat calvaria (RC) cells grown in medium containing 10% fetal bovine serum (FBS) and 50 microns/ml of ascorbic acid . When 10 mM beta-glycerophosphate (beta-GP) is added, the nodules mineralize in two phases: an initiation phase that is dependent upon alkaline phosphatase activity for cleavage of beta-GP to inorganic phosphate (P(i)) and a progression phase that proceeds independently of the activity of alkaline phosphatase and does not require exogenous phosphate . We have used this system to investigate the effects of aluminum (Al3+)on mineralization . When AlCl3 was added to culture medium at concentrations of 0, 3, 10, 30, 100, and 300 muM, the total concentrations of aluminum were 0.98, 6.07, 16.82, 40.19, 88.45, and 284.52 muM, respectively . The corresponding free Al3+ concentrations, assessed after ultrafiltration, were found to be 1.11, 1.75, 3.40, 6.22, 5.38, and 12.11 muM . In cultures in which osteoid was formed and mineralization initiated in the presence of added Al+ (3-300 muM), a dose-dependent inhibition of mineralization occurred . Osteoid formed in the presence of added Al3+ mineralized normally when Al3+ was removed from cultures at the time of initiation of mineralization with beta-GP . In osteoid nodules grown in the absence of Al3+, addition of Al3+ (3-300 muM) at the start of the initiation phase of mineralization resulted in a dose-dependent inhibition of mineralization . Addition of Al3+ to cultures after mineralization had been initiated in the absence of Al3+ inhibited progression of mineralization at added Al3+ concentrations of 10 muM and above . Al3+ did not decrease the conversion of beta-GP to P(i) and caused a small but significant increase in alkaline phosphatase activity at added concentrations of 100 muM or greater . The data show that Al3+ inhibits both the initiation and progression phases of mineralization starting at added concentrations of 3-10 muM (approximately 1.7-3.4 muM free Al3+) and that mineralization of osteoid formed in the presence of Al3+ is unaffected if Al3+ is removed prior to the initiation of mineralization.

AIDS, 1995 Dec, 9(12), 1307 - 11
Increasing genotypic and phenotypic selection from the original genomic RNA populations of HIV-1 strains LAI and MN (NM) by peripheral blood mononuclear cell culture, B-cell-line propagation and T-cell-line adaptation; Lukashov VV et al.; OBJECTIVE: To address the question of whether T-cell-line adaptation of the original LAI and MN (NM) HIV-1 populations biased the interpretation of the intraindividual and population-wide virus distributions . PATIENTS AND METHODS: HIV-1 genomic RNA coding for the gp120 C2V3 region was obtained from serum samples of patients LAI and MN and compared to the proviral DNA derived from simultaneously sampled peripheral blood mononuclear cells (PBMC) as well as B-and T-cell lines . RESULTS: Two (10%) of 20 clones of HIV-1 LAI RNA and none of 16 clones of the HIV-1 MN RNA carried syncytium-inducing (SI)-determining amino-acid changes . HIV-1 LAI RNA formed on SI and two non-SI (NSI) phylogenetic clusters . The HIV-1 LAI DNA in PBMC included both SI and NSI clones but lacked one NSI cluster and contributed NSI clones to the SI cluster as well as an SI clone to the NSI cluster, indicating the existence of an intermediate SI/NSI genotype . In vitro culture using either primary cells or B-cell lines yielded only SI clones, distributed over two SI/NSI mixed clusters . Long-term propagation in T-cell lines further restricted the clonality and yielded SI clones belonging to only one cluster . On the population level, HIV-1 LAI, MN and BRU sequences all clustered according to the individual host and apart from each other and separate from the epidemiological controls without notable influence of SI/NSI distinction or cell-culture adaptation . CONCLUSION: Our data demonstrate a selection bias during the cell-line adaptation of HIV-1 strains LAI and MN with more impact on phenotypic than on genotypic characteristics.

Mol Biol Cell, 1995 Dec, 6(12), 1743 - 53
Cartilage matrix protein forms a type II collagen-independent filamentous network: analysis in primary cell cultures with a retrovirus expression system; Chen Q et al.; Cartilage matrix protein (CMP) is expressed specifically in mature cartilage and consists of two von Willebrand factor A domains (CMP-A1 and CMP-A2) that are separated by an epidermal growth factor-like domain, and a coiled-coil tail domain at the carboxyl terminal end . We have shown previously that CMP interacts with type II collagen-containing fibrils in cartilage . In this study, we describe a type II collagen-independent CMP filament and we analyze the structural requirement for the formation of this type of filament . Recombinant wild-type CMP and two mutant forms were expressed in chick primary cell cultures using a retrovirus expression system . In chondrocytes, the wild-type virally encoded CMP is able to form disulfide bonded trimers and to assemble into filaments . Filaments also form with CMP whose Cys455 and Cys457 in the tail domain were mutagenized to prevent interchain disulfide bond formation . Therefore, intermolecular disulfide bonds are not necessary for the assembly of CMP into filaments . Both the wild-type and the double cysteine mutant also form filaments in fibroblasts, indicating that chondrocyte-specific factors are not required for filament formation . A truncated form of CMP that consists only of the CMP-A2 domain and the tail domain can form trimers but fails to form filaments, indicating that the deleted CMP-A1 domain and/or the epidermal growth factor domain are necessary for filament assembly but not for trimer formation . Furthermore, the expression of the virally encoded truncated CMP in chondrocyte culture disrupts endogenous CMP filament formation . Together these data suggest a role for CMP in cartilage matrix assembly by forming filamentous networks that require participation and coordination of individual domains of CMP.

Phytochemistry, 1995 Dec, 40(6), 1681 - 9
Biological activities of synthetic analogues of Alternaria alternata toxin (AAL-toxin) and fumonisin in plant and mammalian cell cultures; Abbas HK et al.; In a search for an analogue of AAL-toxin with high phytotoxicity and low mammalian toxicity, aminopentols {(AP1), hexacetyl AP1 and N-acetyl AP1}, and nine analogues (1-9), were tested for toxicity to duckweed (Lemna pausicostata), susceptible tomato (asc/asc) leaf discs, black nightshade leaf discs and mammalian cell lines, including dog kidney (MDCK), rat liver hepatoma (H4TG) and mouse fibroblasts (NIH3T3) . These were compared with AAL-toxin and fumonisin B1 (FB1) . Analogue 9 at 10 microM increased cellular leakage and chlorophyll loss from both tomato and black nightshade leaf discs . The diester 9 was the most active in the duckweed bioassay, but it was much less toxic to MDCK and H4TG cells with an IC50 of 200 microM compared to 10 microM for FB1 . Analogue 9 and FB1 showed similar low toxicities (IC50 = 150 microM) to NIH3T3 cells . Among the substances tested, only analogue 9 had significant phytotoxicity and low mammalian toxicity, indicating some potential for development of safe and effective natural herbicides.

Thromb Res, 1995 Dec 1, 80(5), 399 - 411
Basal tissue factor expression in endothelial cell cultures is caused by contaminating smooth muscle cells . Reduction by using chymotrypsin instead of collagenase; Mulder AB et al.; A discrepancy exists between basal tissue factor (TF) expression found in endothelial cell cultures and the failure to detect TF in unpertubated endothelial cells in vivo . We demonstrated that basal TF expression in endothelial cell cultures originated from contaminating cells . These cells were ultrastructurally and flowcytometrically identified as smooth muscle cells . The cell cultures had been obtained from collagenase-treated human umbilical cord vessels . Histologic studies revealed that after collagenase treatment the basement membrane was digested and underlying structures were disrupted at some areas of the vein . We selected chymotrypsin as an alternative for the isolation of endothelial cells . Using chymotrypsin, the endothelial lining was selectively lost leaving the basement membrane undisturbed . Furthermore, use of chymotrypsin instead of collagenase minimized the level of basal TF activity . Taken together, we demonstrated that basal TF expression in endothelial cell cultures is caused by contaminating smooth muscle cells . This contamination can strongly be reduced using chymotrypsin instead of collagenase for isolation of endothelial cells.

J Photochem Photobiol B, 1995 Dec, 31(3), 97 - 100
Gene expression in plant cell cultures under continuous blue light irradiation of high intensity; Kaldenhoff R; Plant cell cultures cultivated in darkness and subsequently irradiated with continuous blue light have the capacity to develop functional chloroplasts from leucoplast like precursors . This process is accompanied by chlorophyll-synthesis and expression of plastid structural genes . The nature of regulatory factors related to the specific gene activation is uncertain and approaches for their evaluation are described . Exemplarily, events connected to the regulation of a blue light induced gene are introduced . The significance as well as a possible interaction of light and phytohormone signal transduction pathways is discussed.

Acta Virol, 1994 Dec, 38(6), 333 - 7
Increased yields of Japanese encephalitis virus in heat shocked cell cultures; Paranjape SP et al.; Monolayers of porcine stable kidney (PS) and baby hamster kidney (BHK-21) cell lines were exposed to 43 degrees C and 41 degrees C, respectively, for 4 hrs and infected with Japanese encephalitis virus (JEV) Nakayama strain at low and high multiplicity of infection . Virus yields were increased by 0.2-2.5 log PFU/ml in heat shocked cultures compared to control cultures at 37 degrees C . This phenomenon was detected in the late phase of replication after 16 hrs post infection . The progeny virus obtained from heat shocked cultures was more thermostable.

Clin Immunol Immunopathol, 1995 Dec, 77(3), 358 - 65
Tumor necrosis factor-alpha mediates the release of bioactive transforming growth factor-beta in murine microglial cell cultures; Chao CC et al.; Tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta produced by glial cells have been proposed bo play a role in various neurodenegerative diseases . The interaction of these two cytokines, however, is unknown . We tested the hypothesis that the TNF-alpha released from lipopolysaccharide (LPS)-treated murine microglial cells would stimulate the release of TGF-beta, which in turn would control TNF-alpha production . Treatment of murine microglial cell cultures with LPS resulted in an acute release of TNF-alpha (peak by 8 hr) followed by delayed release of bioactive TGF-beta (peak by 48 hr) . Anti-TNF-alpha antibody significantly inhibited LPS-stimulated TGF-beta production, suggesting the involvement of TNF-alpha in TGF-beta production . Also, exogenous TNF-alpha induced in a dose-dependent fashion microglial cell expression of TGF-beta 1 mRNA and release of TGF-beta . Exogenous TGF-beta, on the other hand, suppressed LPS-stimulated TNF-alpha release . These findings suggest an autoregulation of microglial cell TNF-alpha production by TGF-beta which may limit inflammation-associated brain injury.

Circ Res, 1995 Dec, 77(6), 1107 - 13
Decreased elastin synthesis in normal development and in long-term aortic organ and cell cultures is related to rapid and selective destabilization of mRNA for elastin; Johnson DJ et al.; We have previously shown that aortic organ cultures from 1- to 3-day-old chickens initially mimic the high levels of elastin production seen in vivo . However, more prolonged incubation of these tissues results in decreased synthesis of elastin . In the present study, we demonstrate that decreased production of elastin in these aortic organ cultures is selective for elastin compared with collagen and is correlated with decreased steady state levels of mRNA for elastin . These decreases in steady state levels of elastin mRNA are due at least in part to a rapid and selective destabilization of mRNA for elastin, the half-life of which falls from approximately 25 hours in fresh aortic tissues to approximately 15 hours after incubation for only 8 hours . Destabilization of elastin mRNA can be prevented by incubation in the presence of blockers of DNA transcription (5,6-dichlorobenzimidazole riboside and actinomycin D) and mRNA translation (cycloheximide) . Furthermore, the half-life of aortic elastin mRNA decreases from approximately 25 hours in the 1-day-old chicken to approximately 7 hours in the 8-week-old chicken, demonstrating that destabilization of mRNA is an important contributing factor in the decline in production of aortic elastin taking place during normal postnatal growth.

Cell Immunol, 1995 Dec, 166(2), 261 - 74
HIV-induced syncytia in peripheral blood cell cultures crawl by extending giant pseudopods; Shutt DC et al.; It was previously demonstrated that HIV-induced syncytia of the immortalized T cell line SupT1 reorganize their cytoskeleton and form a spherical supernuclear complex, thus mimicking the organization, polarity, and morphology of a single SupT1 cell . Then, through extension of a single, giant pseudopod, these syncytia, which grow to more than 100 times the volume of a single SupT1 cell, translocate along a substratum . To verify that syncytium motility is not peculiar to the SupT1 cell line, we have analyzed the cytoskeletal organization and motile capabilities of HIV-induced syncytia formed in peripheral blood cell cultures containing more than 90% CD4-positive cells . The results demonstrate that although peripheral blood T cells differ from SupT1 cells in size and morphology, they are continuously motile and translocate along a substratum in a manner quite similar to that of SupT1 cells, and peripheral blood T cell syncytia induced by HIV-1LAI as well as two additional clinical isolates translocate by the extension of a giant anterior pseudopod in a fashion indistinguishable from that of HIV-induced SupT1 syncytia . Together, these results support the generalization that HIV-induced T cell syncytia are motile.

Ann Acad Med Singapore, 1995 Nov, 24(6), 851 - 5
Mesangial cell culture: its role in the understanding of the pathogenesis of glomerular disease; Lee GS; Many forms of renal disease that progress to renal failure are characterised histologically by mesangial cell proliferation and accumulation of mesangial matrix . These diseases include IgA nephropathy, lupus nephritis and diabetic nephropathy . In recent years the introduction of mesangial cell culture techniques has allowed investigators to characterise the mesangial cell and its functions and examine the role of the mesangial cell in the pathogenesis of glomerular disease . The properties of the mesangial cell include the ability to contract, certain macrophage functions, and the ability to synthesize matrix and produce growth factors and cytokines . Factors that control mesangial cell function include cytokines and growth mediators, matrix components including integrins, and interactions with other cells such as the endothelial and epithelial cells . When exposed to an injurious stimulus, the mesangial cell responds by cellular proliferation and matrix synthesis leading to an increase in mesangial cells and matrix accumulation that ultimately progresses to the development of glomerulosclerosis . A better understanding of this disease process will allow future investigators the opportunity to formulate therapeutic interventions.

Mycopathologia, 1995 Nov, 132(2), 111 - 6
Comparative cytotoxicity of fumonisin B1 and moniliformin in chicken primary cell cultures; Wu W et al.; Two water-soluble Fusarium metabolites, fumonisin B1 (FB1) and moniliformin (MN) were compared for their cytotoxicity in a variety of chicken primary cell cultures . Cardiac and skeletal myocytes and hepatocytes derived from embryos, and splenocytes, macrophages, and chondrocytes derived from 3- to 4-week old chickens were cultured in media containing either FB1 or MN (0 to 1 mM) for 48 hr . The colorimetric tetrazolium cleavage assay was then used for measuring cell survival . FB1 was not toxic to macrophages, hepatocytes, cardiac and skeletal myocytes but toxic to splenocytes and chondrocytes . MN was not toxic to chondrocytes and macrophages, but toxic to splenocytes, cardiac and skeletal myocytes . Median effective concentration (EC50) of MN in skeletal myocytes was 42 mu M (fiducial limits: 33 to 50 mu M) and in cardiac myocytes was 95 mu M (fiducial limits: 84 to 122 mu M) . Estimated EC50 of FB1 in chondrocytes and splenocytes and EC50 of MN in splenocytes were all greater than 200 mu M.

Vopr Virusol, 1995 Nov-Dec, 40(6), 247 - 51
{Expression of human immunodeficiency virus gag antigens and formation of virus-like particles in a cell culture, infected with recombinant vaccinia virus strains (an electron-microscopic study)}; Grigor'ev VB et al.; The synthesis of gag antigens of human immunodeficiency virus (HIV) by recombinant vaccinia virus strains containing the expressed genes gag and gag-pol and the capacity of these proteins to formation of virus-like particles during infection of various cell cultures were studied . The recombinant strain containing the truncated gag gene (p48gag) was shown to effectively synthesize gag polypeptides and to form immature virus-like particles during infection of all the cell cultures tested (CV-1, Hep-2, HT-29) . The morphogenesis of mature virus-like particles was detected by electron microscopy only in infection of Hep-2 cells with a strain containing a complete gag-pol sequence of HIV.

Eur J Clin Chem Clin Biochem, 1995 Nov, 33(11), 813 - 23
The MTT tetrazolium salt assay scrutinized: how to use this assay reliably to measure metabolic activity of cell cultures in vitro for the assessment of growth characteristics, IC50-values and cell survival; Sieuwerts AM et al.; The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay is widely used for in vitro measurement of the metabolic viability of cell cultures subjected to different culture conditions . This convenient assay, which is based on the ability of viable cells to produce formazan, can be affected significantly by a number of conditions . These conditions can be roughly divided into groups, firstly influences which affect the spectrum of the produced formazan and secondly influences which affect the amount of formazan produced per cell . We studied the various chemical and biochemical aspects involved in the MTT assay . Our data indicated that microscopical viewing of cell cultures before and after performing the assay, a medium renewal with a well-defined MTT-incubation medium at the end of the culture period and regular cell counting are essential steps to ensure a reliable performance of the MTT assay . In conclusion, providing the necessary precautions are taken, the MTT assay can be used reliably to measure metabolic activity of cell cultures in vitro for the assessment of growth characteristics, IC50-values and cell survival.

Prenat Diagn, 1995 Nov, 15(11), 1067 - 9
The preserving of chorionic villi before establishing long-term cell cultures for cytogenetic analysis; Hansson K et al.; The possibility of preserving intact chorionic villi in culture medium for up to 7 days before establishing long-term cultures for prenatal chromosome analysis is demonstrated . The preserving itself had no negative effect on the growth capacity of the mesenchymal cells.

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