J Hyg (Lond), 1984 Oct, 93(2), 213 - 23 Arginine metabolism in infected cell cultures as a marker character for the differentiation of orthopoxviruses; Osborn JG et al.; Arginine has been shown to be essential for the replication of several orthopoxviruses in mouse sarcoma 180 cells and in chick embryo fibroblast cultures . Both host systems are characterized by their inabilities to utilize citrulline for the biosynthesis of arginine due to deficiencies in the requisite cellular enzymes and cell multiplication is absolutely dependent on the availability of exogenous arginine . Virus replication in such cells maintained with citrulline results from the induction of virus-specific enzymes . Significant virus yields in the absence of exogenous arginine or citrulline can arise from the replenishment of intracellular amino acid pools by increased utilization of arginyl residues in cellular proteins . The extent of the phenotypic expression of these characters in infected cells permitted significant discrimination between the viruses examined . Distinctions could be drawn between rabbitpox, ectromelia, cowpox, buffalopox and vaccinia strains . However, cowpox could not be distinguished from other viruses isolated from diseased animals in European zoos. J Clin Endocrinol Metab, 1984 Oct, 59(4), 643 - 51 Characterization of steroidogenesis in cell cultures of the human fetal adrenal cortex: comparison of definitive zone and fetal zone cells; Simonian MH et al.; The steroidogenic capacity of cells cultured from the definitive zone and fetal zone of the human fetal adrenal cortex was compared, using a serum-free medium without lipoproteins . Comparison of {3H}pregnenolone and {3H}progesterone metabolism in cultures from each zone incubated without ACTH indicated that only 3 beta-hydroxysteroid dehydrogenase, delta 4,5-isomerase (3 beta-HSD) activity was deficient in fetal zone cultures . Basal 3 beta-HSD activity was 3- to 5-fold lower in fetal zone cultures than in definitive zone cultures assayed after 3 or 10 days in culture . Although basal hydroxysteroid sulfotransferase activity was 2- to 4-fold greater in 3-day-old fetal zone cultures than in definitive zone cultures, this difference was not found in 10-day-old cultures due to a 3-fold decrease in fetal zone basal hydroxysteroid sulfotransferase activity . However, older cultures of fetal zone cells did maintain the characteristic high delta 5-steroid sulfate and low delta 4,3-ketosteroid basal production from {3H} pregnenolone as compared to definitive zone cultures . ACTH treatment for 48 h in serum-free medium increased the steroidogenic capacity of cell cultures from both zones and stimulated delta 4,3-ketosteroid production from {3H}pregnenolone and 3 beta-HSD activity in fetal zone cultures to levels characteristic of the definitive zone . These studies show that in the absence of ACTH the difference in steroidogenic capacity between the fetal zone and the definitive zone (due to the lower 3 beta-HSD activity in fetal zone cells) was maintained in cell cultures for a period up to 10 days. J Cell Physiol, 1984 Oct, 121(1), 51 - 63 Regulation of Na+,K+-ATPase activity in MDCK kidney epithelial cell cultures: role of growth state, cyclic AMP, and chemical inducers of dome formation and differentiation; Kennedy BG et al.; Na+,K+-ATPase activity was monitored in MDCK kidney epithelial cell monolayers and in cell extracts as a function of cell density, cAMP elevation, and exposure to hexamethylene bisacetamide (HMBA) and dimethylsulfoxide (Me2SO) . Ouabain-sensitive Na+,K+-ATPase and 86Rb+ uptake activities, and the number of {3H}-ouabain binding sites were maximal in subconfluent cultures and decreased accompanying the development of a confluent monolayer . A sodium pump density of 8 X 10(7) pumps/cell was estimated for subconfluent cultures, declining to 9 X 10(5) pumps/cell at confluence . Previous studies have shown that dibutyryl cyclic AMP (Bt2cAMP), 1-methyl-3-isobutylxanthine (IBMX), or the differentiation inducers HMBA and Me2SO, which also caused cAMP elevation, all stimulated dome formation, a visible manifestation of active transepithelial Na+ and water transport (Lever, 1979) . In the present study, all of these inducers were found to elevate intracellular Na+ content, implicating this variable in control of induction of dome formation . Operationally, inducers could be divided into two classes . HMBA and Me2SO partially inhibited ouabain-sensitive 86Rb+ influx . Ouabain, at a concentration that caused partial sodium pump inhibition and increased intracellular Na+ content, was also effective as an inducer . The second class, exemplified by IBMX and Bt2cAMP caused a furosemide-sensitive increase in intracellular Na+ content . This class of inducers stimulated ouabain-sensitive 86Rb+ uptake, presumably by substrate effects due to increased Na+ levels . The Na+ or ATP activation of Na+,K+-ATPase activity assayed in cell-free extracts, the affinity of the transport system for Rb+ in intact cells and intracellular ATP levels were unchanged by inducer treatment . Elevation of intracellular Na+ concentration, either by cAMP-stimulated, furosemide-sensitive mechanisms or by partial inhibition of the sodium pump may stimulate the induction of dome formation in MDCK cells. Brain Res, 1984 Sep 17, 310(1), 99 - 105 Diazepam and its anomalous p-chloro-derivative Ro 5-4864: comparative effects on mouse neurons in cell culture; Skerritt JH et al.; The actions of diazepam and its p-chloro-derivative Ro 5-4864 were compared on mouse spinal cord and dorsal root ganglion neurons in cell culture . Diazepam enhanced but Ro 5-4864 reduced iontophoretic GABA responses in a concentration-dependent manner . Both diazepam and Ro 5-4864 limited sustained, high frequency repetitive firing of spinal cord neurons but diazepam was more potent . Ro 5-4864 was, however, more potent than diazepam in inhibiting spontaneous neuronal activity of spinal cord neurons and reducing the duration of calcium-dependent action potentials of dorsal root ganglion neurons . The differing actions of diazepam and Ro 5-4864 may account for the contrasting pharmacological spectra of the two benzodiazepines. Brain Res, 1984 Sep 17, 310(1), 142 - 8 Electrophysiological study of mammalian neurons from ventral mesencephalon grown in primary dissociated cell culture; Heyer EJ; Mammalian neurons from ventral mesencephalon were grown in primary dissociated cell culture . While dopaminergic neurons were found in culture and used as a marker for this area of the nervous system, our study did not segregate the neurons in terms of their dopaminergic nature . Indeed all of the results were probably from nondopaminergic neurons . Action potentials dependent on sodium and potassium could be elicited from neurons placed in a balanced salt solution . Following blockage of sodium current by tetrodotoxin, and reduction of potassium current by tetraethylammonium and 3-aminopyridine, long-duration action potentials could be elicited by intracellular stimulation . These action potentials were most probably calcium-dependent since they could not be elicited in bathing solution containing manganese, a calcium-conductance blocker. Neurosci Lett, 1984 Sep 7, 50(1-3), 133 - 7 Up-regulation of opiate receptors by opiate antagonists in neuroblastoma-glioma cell culture: the possibility of interaction with guanosine triphosphate-binding proteins; Barg J et al.; Neuroblastoma-glioma NG108-15 cells that were cultured for 48 h with the opiate antagonist, naloxone, respond to the guanosine 5'-triphosphate (GTP) analogue guanosine 5'-{beta, gamma-imido}-triphosphate (GMP-PNP) in the binding assay as the control, non-treated, cells . This was observed when the guanyl nucleotide was tested in the presence or absence of sodium chloride and also after subcellular fractionation of the membranes on a sucrose gradient which separated between two receptor-containing fractions . The findings suggest that the increase in delta type enkephalin receptors in naloxone-treated NG108-15 cells does not reflect an alteration in the interaction between the receptor and the adenylate cyclase-GTP-binding protein system. J Neurochem, 1984 Sep, 43(3), 716 - 23 Angiotensin II synthesis studies in dissociated brain cell cultures; Weyhenmeyer JA et al.; The biosynthesis of angiotensin II-like peptide was measured in primary cultured brain cells from the fetal rat . Cells from the whole brains of 20-day gestational age Sprague-Dawley rats were dissociated by mild trypsinization and grown for 5 days in supplemented (serum-free) medium prior to experimental analysis . The time-dependent incorporation of {3H}proline into newly synthesized angiotensin II-like peptide was measured by radioimmunoassay using specific angiotensin II antisera . Analysis of radioimmunoassay data revealed an increase in the amount of tritium-labeled angiotensin II in the crude extract of the brain cells during the first 24 h in culture . The chromatographic character of the angiotensin II-like peptide was further identified by high pressure liquid chromatography . Elution profiles for newly synthesized angiotensin II were identical to those profiles generated for {3H}angiotensin II and nonradiolabeled angiotensin II standards . To determine the bioactivity of the angiotensin II-like peptide, fractions of the column-purified peptide were injected into the region of the rat lateral ventricle via an indwelling cannula . Systolic pressure increased up to 20 mm Hg depending upon the amount of peptide injected . These data clearly support the existence of an endogenous renin-angiotensin system is dissociated brain cell cultures from the fetal rat, and provide an experimental model for further analysis of the regulatory mechanisms of angiotensin II synthesis. Vopr Virusol, 1984 Sep-Oct, 29(5), 536 - 9 {Persistent influenza virus infection in a MDCK cell culture}; Medvedeva MN et al.; A model of persistent influenza infection (PII) induced by influenza A/Victoria/35/72 (H3N2) virus in MDCK cell culture has been constructed . The model was observed for 165 days . It was characterized by the lack of visible signs of virus reproduction, a low number of antigen-containing cells (0.02-0.05%), irregular virus isolation (at 20, 28, 32, 44, 52, 62, 92, 135, 148, 158 days after primary inoculation) which was possible only with special methods . Interferon and DIP were found not to be the factors responsible for maintenance of PII in MDCK culture. Tsitologiia, 1984 Sep, 26(9), 996 - 1001 {Structural changes in the mitotic apparatus of metaphase cells exposed to 2-mercaptoethanol in a porcine embryonic kidney cell culture . A light microscopy study}; Bystrevskaia VB et al.; The sequence of reconstruction of the metaphase mitotic apparatus of the KEPV cells under the action of 2-mercaptoethanol (0.001 M) and after moving off the agent has been studied . In the presence of 2-mercaptoethanol, a destruction of the metaphase plate and that of the mitotic spindle proceed independently . The destruction of the mitotic spindle does not involve an immediate disorganization of the metaphase plate, and vice versa . In the course of resumption of the normal division after moving off the agent, the restoration of the mitotic apparatus structure proceeds within a definite temporal sequence: at first, the mitotic spindle reestablishes, and after that the metaphase plate forms . Distinctions between the 2-mercaptoethanol action and the action of other antimitotic agents are discussed. In Vitro, 1984 Sep, 20(9), 699 - 706 Comparative use of fructose and glucose in human liver and fibroblastic cell cultures; Delhotal B et al.; The effect of fructose as a substitute for glucose in cell culture media was investigated in human skin fibroblast and liver cell cultures . Cells were grown for between 2 and 10 days in identical flasks in four different media, containing 5.5 mmol X 1-1 and 27.5 mmol X 1-1 glucose and fructose, respectively . In the presence of fructose, cell growth was stimulated, but less in liver cells than fibroblasts . At Day 6, increases were observed in {3H}thymidine incorporation, protein levels, and amino acid consumption, and a reduction was noted in ATP levels . In media containing 5.5 mmol X 1-1 glucose or fructose, consumption of fructose was four times lower than that of glucose at Day 3 and did not rise until Day 6 . In fructose media, the lactate production was very low (four to five times less than that of glucose) and the pH values were always higher . Some findings were different for the fibroblasts and liver cells, owing to the specific characteristics of these two cell types in culture; this applied especially to the effects of glucose and fructose concentrations of 27.5 mmol X 1-1 . Several possible explanations for the stimulation of cell growth in fructose medium were discussed. Farmakol Toksikol, 1984 Sep-Oct, 47(5), 117 - 20 {Use of the human lymphoid Raji-line cell culture in tests of the toxicity of perfluorocarbon emulsions and their separate components}; Arkhipov VV et al.; The possibility has been shown of utilizing the culture of human lymphoid Raji cells as a highly sensitive object for toxicity testing of perfluorocarbon emulsions and their components . It has been disclosed that cytotoxicity of perfluorocarbon emulsions is primarily determined by its stabilizers--proxanoles . Theoretically, the cultures of animal cells are considered to be useful for toxicological studies. Anal Biochem, 1984 Sep, 141(2), 538 - 44 Sources of error in estimating radioactivity in protein from cell cultures by liquid scintillation counting; Silverman JA et al.; In this study, several common sample preparation techniques for liquid scintillation counting were critically reviewed . It has been shown that techniques such as NaOH or formic acid solubilization of trichloroacetic acid (TCA)-precipitated proteins led to underestimation of the radioactivity by 20-40%; this loss was not corrected by either internal or external standardization . Hydrolysis of the proteins with 6 N HCl or Pronase significantly increased the recovery of the labeled proteins . Also, 10% of the labeled cell proteins remained on the dish when cells were scraped into buffer; these labelled proteins could be recovered either by in situ hydrolysis with Pronase or by solubilization and scraping in 0.3 N NaOH . These techniques increased the recovered radioactivity by 50-60%, allowing quantitative measurements to be made over a 3-day chase period . A possible mechanism and the implications of this observation were discussed. Brain Res, 1984 Sep, 318(1), 119 - 34 Specificity of histiotypic organization and synaptogenesis in reaggregating cell cultures of mouse cerebellum; Orkand PM et al.; The fine structure of reaggregating cultures of cells from 6- to 7-day-old mouse cerebellum was studied at intervals between 3 and 21 days in vitro (DIV) . The resulting aggregates consisted mainly of small neurons (granule, stellate and basket cells), neuroglial cells and their processes . Large neurons were rarely present . By 7 DIV the previously loosely packed components had tightened into a more compact mass . A peripheral plexiform layer had formed which had many fine axons arranged into fascicles of parallel fibers . Deep to this zone was a cellular region containing clusters of neurons interspersed with small areas of neuropil . Axosomatic synapses appeared on neurons which resembled stellate or basket cells but not on granule cells . Axo-dendritic synapses formed in the neuropil of the cellular zone and, less frequently, in the outer plexiform layer . After 3 weeks glial cell processes had increased in volume at the expense of neurons . When cerebellar cells were cultured with cells from pons and medulla, which are normal sources of mossy fiber input, aggregates formed in which synaptic glomeruli were found . They were not seen in aggregates containing cells from retina and olfactory bulb cultured with cerebellum . Our observations suggest: that natural histogenetic mechanisms persist after dissociation and reaggregation of cerebellar cells resulting in a separation of an outer, 'molecular'-like layer from an inner granule cell layer and that neurons retain specificity of their synaptogenic capabilities both with regard to appropriate cell types and the morphological form that the synapses take. J Cereb Blood Flow Metab, 1984 Sep, 4(3), 415 - 24 Monoamine oxidase activity in the cerebral vasculature: comparison between fresh microvessels from different structures and cell cultures derived from microvessels; Sercombe R et al.; Monoamine oxidase (MAO) activity was studied in various preparations of porcine brain microvessels to explore further the role of this enzyme in the blood-brain barrier to catecholamines . No difference was noted (Vm and Km) between microvessels isolated from three structures (caudate nucleus, thalamus, and cerebral cortex) in which the responses to circulating catecholamines in vivo are markedly different . Large and small microvessels from the caudate nucleus and the thalamus presented the same specific activity . Cell cultures obtained from small microvessels were rich in endothelial cells as identified by the presence of Factor VIII-related antigen . These preparations displayed an MAO activity about ninefold less than freshly isolated microvessels, although their prostaglandin synthetase activity appeared normal . These results suggest that MAO activity is not the main factor determining the regional differences in the cerebrovascular reactions to catecholamines, that MAO is not specifically localized in the endothelium but must be also present in the smooth muscle, and that the MAO activity is greatly decreased during cell culture. J Clin Microbiol, 1984 Sep, 20(3), 387 - 90 Detection of Semliki Forest virus in cell culture by use of an enzyme immunoassay with peroxidase-labeled monoclonal antibodies specific for glycoproteins E1 and E2; van Tiel FH et al.; Four noncompeting monoclonal antibodies (MA) directed against either the E1 (UM 8.64 and 8.139) or E2 (UM 8.55 and 8.73) glycoprotein of Semliki Forest virus were purified and labeled with horseradish peroxidase . Each enzyme-labeled MA was tested alone and in combination with others for its sensitivity to detect virus-infected cells . Semliki Forest virus-infected L cells seeded as monolayers in 96-well plates were screened for the virus after incubation with enzyme-labeled MA and a substrate . In this system single enzyme-labeled MA even at high dilution (10(3.0) to 10(4.5} were able to detect virus-infected cells . The sensitivity of the test could be enhanced by combining two noncompeting MA (10(4.5) to 10(5.0} . Combinations of three and four MA were less effective, due to high absorbance values for noninfected cells . The threshold of virus defection was between 10(5) and 10(6) PFU/ml . This test is sensitive and specific and therefore may be useful for diagnostic purposes. Isr J Med Sci, 1984 Sep, 20(9), 882 - 5 Induction of interleukin-2 and colony-stimulating factors in lymphoid cell cultures activated by mitogenic mycoplasmas; Gershon H et al.; Mycoplasmal infections often result in chronic inflammatory diseases that involve both local cell proliferation and chemotactic phenomena . To clarify the mechanisms responsible for these inflammatory reactions, we have examined whether nonspecific activation of host lymphocytes by mitogenic mycoplasmas triggers the production of lymphokines, which influence differentiation and proliferation of lymphocytes, macrophages and granulocytes . Mycoplasma pulmonis and M . neurolyticum were tested for their ability to induce production of interleukin-2 (IL-2) and colony-stimulating factors (CSF) by activated lymphoid cells . Mitogenic doses of membranes purified from M . pulmonis, as well as concanavalin A (ConA), induce rat lymphocytes to produce IL-2 . Stimulation with the T cell mitogen ConA induces twice the level of IL-2 than does stimulation with M . pulmonis, which activates both rat B- and T-lymphocytes . Unlike these T cell mitogens, M . neurolyticum, which stimulates rat and mouse B-lymphocytes, lacks the ability to induce production of IL-2 . Nevertheless, M . neurolyticum stimulates mouse spleen cells to produce in vitro factors capable of stimulating differentiation of bone marrow cells into colonies of macrophages and granulocytes. Proc Soc Exp Biol Med, 1984 Sep, 176(4), 414 - 8 Effect of temperature on suppressor cells in chicken spleen cell cultures; Bhogal BS et al.; The plaque forming cell (PFC) response of in vivo primed chicken spleen cells to sheep erythrocytes (SRBC) at 37 degrees C in vitro was much higher when antigen was added on Day 2 of culture than on Day 0, at the initiation of the incubation period . No such difference was observed when the cells were cultured at 40 degrees C, in which case both responses were low . As shown previously the effect of delayed exposure to SRBC at 37 degrees C was due to a disappearance of suppressor T cells in the absence of antigen, which did not occur at 40 degrees C . Addition of concanavalin A on Day 0 could, even in the absence of SRBC, maintain the suppressor cell activity at 37 degrees C . The results suggest that suppressor cell activity is very temperature dependent. Ital J Neurol Sci, 1984 Sep, 5(3), 311 - 6 Rat CNS cell culture . Enhancement of neuronal survival and delay of glial proliferation by serum from patients with multiple sclerosis . A morphological study; Savettieri G et al.; The addition of serum from multiple sclerosis (MS) patients to the culture medium of dissociated cells from cerebral hemispheres of rat embryos caused a delay in glial proliferation and an enhancement of neuronal survival . Sera from normal individuals and patients with other neurological diseases failed to show this effect . These morphological observations are interpreted as the outcome of inhibition of in vitro gliogenesis. Vopr Virusol, 1984 Sep-Oct, 29(5), 589 - 92 {Immunofluorescent study of the reproduction of human rotavirus in cell culture}; Ivannikova TA et al.; The mechanism of rotavirus antigen amplification in cell culture was studied by immunofluorescence . Reproduction of human rotavirus in cell culture was shown to begin 6 h after inoculation and to be accompanied by a regular increase in the number of antigen-containing cells . A good correlation with the results of electron microscopic studies was demonstrated . The possibility of reliable detection of rotavirus antigen in cell culture by the fluorescent antibody technique at relatively early intervals after infection has been established. Diagn Microbiol Infect Dis, 1984 Sep, 2(4), 343 - 5 Comparison of cell culture with an immunoperoxidase kit for rapid diagnosis of herpes simplex virus infections; Salmon VC et al.; Cell culture technique using continuous mink lung cells was compared to an immunoperoxidase kit for rapidity and sensitivity of herpes simplex virus (HSV) detection . Forty-eight hours after inoculation, 26 (65%) of 40 clinical specimens were positive for HSV by cell culture compared with 19 (48%) by immunoperoxidase staining (p less than 0.001, Fisher's Exact Test). Tumori, 1984 Aug 31, 70(4), 301 - 6 Cooperative effect of exogenous heparin-like compounds and secreted glucocorticoid-induced inhibitor on plasminogen activator in 3T3 cell cultures; Chiarugi VP et al.; Balb/c 3T3 cultures grown in the absence of serum release both plasminogen activator and plasminogen activator inhibitor in the culture medium . Cellular transformation with SV-40 virus increased the level of the activator, whereas dexamethasone increased the level of the inhibitor . Heparin added to the medium potentiated the glucocorticoid-induced inhibitory activity, strongly decreasing or completely abolishing the activity of plasminogen activator . Heparin sulfate showed similar effects to heparin, although at higher concentrations . It is suggested that heparin-like compounds are involved in the regulation of plasminogen activator, acting as inhibitory cofactors. J Biol Chem, 1984 Aug 25, 259(16), 10270 - 83 Metabolism of proteoglycans in rat ovarian granulosa cell culture . Multiple intracellular degradative pathways and the effect of chloroquine; Yanagishita M et al.; The metabolism of endogenously labeled proteoglycans was studied in rat ovarian granulosa cell cultures by a series of pulse-chase experiments using {35S}sulfate as a precursor . More than 90% of the newly synthesized proteoglycans are transported to the cell surface (trypsin-accessible compartment) with a median transit time of 13 min . The membrane-bound heparan sulfate-proteoglycan (HS-PG) is lost from the cell surface either by release into the medium (30%, with t1/2 of 4 h) or by internalization (70%, with t1/2 of 4 h) . Internalized HS-PG, which does not recycle to the cell surface, is degraded by two major pathways . In pathway 1, 60% of the internalized HS-PG migrates to lysosomes with a relatively short t1/2 of 30 min, where it is rapidly degraded, releasing free {35S}sulfate without detectable intermediate products . Chloroquine treatment inhibited degradation, resulting in the accumulation of intact proteoglycans inside the cell . In pathway 2, 40% of the internalized HS-PG is first subjected to extensive proteolysis and limited endoglycosidic degradation yielding single HS chains about 1/3 of their original size (t1/2 of 30 min) . Chloroquine did not inhibit this step . The partially degraded HS is then degraded further by limited endoglycosidic activity to about 1/4-1/5 the original size (t1/2 of 30-60 min) . This step is inhibited by chloroquine . These smaller fragments have a relatively long t1/2 of 3-4 h before rapid degradation in the lysosomes, releasing free {35S}sulfate . Approximately 7% of the newly synthesized HS-PG that is not transported to the cell surface is degraded directly by pathway 2 . The larger dermatan sulfate proteoglycan (DS-I) is transported to the cell surface from which it is quantitatively released into the medium with a t1/2 of 4-6 h . The smaller DS-PG (DS-II) is metabolized similarly to the HS-PG . Most (greater than 90%) is transported to the cell surface from which it is lost either by release into the medium (40%) or by internalization (60%) . About 60% of the internalized DS-II is degraded by pathway 1 (t1/2 of 30 min), while the remainder appears to be degraded by pathway 2 with an overall t1/2 of 4 h . However, in contrast to the degradation of HS-PG by pathway 2, no endoglycosidic degradation of the DS chains occurred. J Biol Chem, 1984 Aug 25, 259(16), 10159 - 67 Characterization of the acute stimulation of steroidogenesis in primary bovine adrenal cortical cell cultures; DiBartolomeis MJ et al.; The early events of steroidogenesis, following adrenocorticotropin (ACTH) stimulation, were investigated in primary cultures of bovine adrenal cortical cells . Steroidogenesis was elevated 4-fold within 5 min of exposure to 10(-7) M ACTH and increased linearly for 12 h and declined thereafter . Cholesterol side-chain cleavage (SCC) activity was increased 2.5-fold in mitochondria isolated from cells exposed for 2 h to ACTH and 0.5 mM aminoglutethimide, even though cytochrome P-450scc only increases after 12 h . Mitochondrial free cholesterol levels increased during the same time period (16.5 to 25 micrograms/mg of protein), but then both cholesterol levels and SCC activity declined in parallel . It is concluded that early ACTH-induced effects on cellular steroidogenesis result from these changes in mitochondrial free cholesterol . The maximum rate of cholesterol transport to mitochondria in aminoglutethimide-blocked cells (8.6 micrograms/mg of protein/h) was consistent with both the maximum rate of mitochondrial cholesterol SCC and cellular steroidogenesis (6.0 micrograms of pregnenolone/mg/h and 5.5 micrograms of steroid/mg of mitochondria/h, respectively) . Cycloheximide (0.2 mM) rapidly blocked (less than 10 min) cellular steroidogenesis, cholesterol SCC activity, and access of cholesterol to cytochrome P-450scc without affecting mitochondrial free cholesterol . The distribution of steroid products fell into three distinct phases during a 24-h period following ACTH stimulation: an initial increase in SCC activity (0-4 h), elevation of androstenedione in place of corticosterone (4-12 h), and then in place of cortisol (12-24 h) . The changes from 4 to 24 h result from a progressive stimulation by ACTH of 17 alpha-hydroxylase activity (but not 21-hydroxylase or C17:20 lyase activities) that is maintained even when stimulation of total steroidogenesis has stopped. Anal Biochem, 1984 Aug 15, 141(1), 83 - 93 Quantitation of types I and III collagens in human tissue samples and cell culture by cyanogen bromide peptide analysis; Hill RJ et al.; A procedure for the quantitation of types I and III collagens by cyanogen bromide peptide analysis was developed with the aim of eliminating certain problems associated with this method . Ion-exchange chromatography reduced high background levels on gel scans used to quantitate the peptides; reduction with beta-mercaptoethanol substantially increased the efficiency of the cyanogen bromide cleavage; use of a concave gradient in acrylamide from 8 to 20% improved the resolution of cyanogen bromide peptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and a normalization procedure eliminated variations due to differences in the amount of material loaded on the gel system . This method of quantitation was applied to human aorta samples and to collagen secreted by human skin fibroblasts . Metachromasy of type I and type III collagen cyanogen bromide peptides stained with Coomassie blue R-250 was established and this was used as an index of the purity of the cyanogen bromide peptide preparations . Type I and III collagens were prepared from human placental tissue, and these purified collagens were used to construct calibration curves to determine the relationship between the quantity of diagnostic cyanogen bromide peptides present and the composition of the sample in terms of types I and III collagens. Endocrinology, 1984 Aug, 115(2), 833 - 5 Effect of estradiol and progesterone on human endometrial aromatase activity in primary cell culture; Tseng L; Isolated human endometrial stromal cells were cultured in RPMI 1640 medium containing insulin, antibiotics and 10% fetal calf serum . After the stromal cells developed a confluent monolayer, estradiol (E2), progesterone (P), or both were separately added to the culture medium and cells were continuously cultured for various periods of time . Aromatase activity was measured in control and hormone-treated cells by incubating the intact cells with {3H}testosterone and isolating the estrogens at the end of incubation . E2 alone did not change the aromatase activity . P caused an 8- to greater than 40-fold increase over the control values (1 to 7 fmol/mg protein X h), and the activity was further increased in the presence of E2 (20- to 100-fold) . The stimulation of aromatase activity by P was found to be both time- and dose-dependent and blocked by actinomycin D . Maximal stimulation was reached after the stromal cells were treated with 300 nM P for 3 days . At 30 nM P, a concentration similar to the plasma level during the luteal phase of the menstrual cycle, 80% of maximal stimulation was noted . These results indicate that P stimulates aromatase in endometrial stromal cells and E2 potentiates this stimulation . Much smaller effects of E2 and P on the aromatase activity were noted in endometrial epithelial glands. Endocrinology, 1984 Aug, 115(2), 762 - 9 Estrogen receptors in rat uterine cell cultures: effects of medium on receptor concentration; Kassis JA et al.; Both the estrogen responsiveness and -binding capacity of cultured rat uterine cells were decreased dramatically when the medium was not changed at 24-h intervals . Treatment of cells for 24 h with 1 nM 17 beta-estradiol in fresh medium led to a 3-fold increase in progesterone receptor concentration, but without fresh medium, no increase in progesterone receptors was observed . When the medium was changed on cells with a low estrogen-binding capacity (depleted cells), a 6- to 10-fold increase in estrogen-binding capacity (to in vivo levels) occurred within 24 h (fed cells), and total protein was increased 2-fold . The high and low affinity binding characteristics of fed and depleted cells were identical . Recovery of the estrogen-binding capacity of depleted cells was relatively slow, increasing after a 6-h lag and reaching maximal levels by 24 h . While 6 h of 10(-5) M cycloheximide treatment (protein synthesis inhibited greater than 95%) had little effect on control estrogen binding levels, it completely inhibited the increase in the estrogen-binding capacity induced by changing the medium on depleted cells . These results indicate that estrogen-binding activity can be varied in cultured rat uterine cells by changing medium conditions and suggest that these changes are due to differences in receptor protein levels and not to a receptor activation-inactivation phenomenon. Biull Eksp Biol Med, 1984 Aug, 98(8), 213 - 5 {Stimulant of antibody producers isolated from the supernatants of cell cultures of normal human bone marrow and in various diseases}; Petrov RV et al.; The biological activity of a stimulant of antibody producers (SAP) isolated from normal human bone marrow was studied and compared with that of a stimulant of antibody producers from the bone marrow of patients with acute myeloblastic leukemia, acute lymphoblastic leukemia, lymphosarcoma, and multiple myeloma . The activity of the SAP from human bone marrow in health was similar to that of analogous transmitters from the bone marrow of other species of the mammals and birds . The activity of the SAP in patients with multiple myeloma was elevated, whereas in patients with acute myeloblastic leukemia, it was lowered. J Protozool, 1984 Aug, 31(3), 398 - 402 Development of Caryospora simplex (Apicomplexa: Eimeriidae) from sporozoites to oocysts in human embryonic lung cell culture; Upton SJ et al.; Leighton tubes containing monolayers of human embryonic lung cells were inoculated with 70,000 or 30,000 sporozoites of the viperid coccidium Caryospora simplex and examined at 1, 2, 4, 6, 8, 10, 12, 14, 16, and 18 days post-inoculation (PI) . By day 1 PI, sporozoites had penetrated cells and were within parasitophorous vacuoles . Most sporozoites became spherical and then underwent karyokinesis several times between days 2 and 6 PI . Mature Type I meronts were found on days 6-16 PI and contained 8 to 22 short, stout merozoites . Mature Type II meronts were present on days 10-18 PI and contained 8 to 22 long, slender merozoites . Developing gamonts (undifferentiated sexual stages) were observed on days 14 and 16 PI . Mature micro- and macrogametes and thin-walled unsporulated oocysts were present on days 16 and 18 PI . Attempts to sporulate oocysts in tissue culture medium or in a 2.5% (w/v) aqueous solution of K2Cr2O7 at 25 degrees C and 37 degrees C were unsuccessful; only a few oocysts developed to the contracted sporont stage . Four Swiss-Webster mice injected intraperitoneally with merozoites obtained from Leighton tubes on day 10 PI did not acquire infections . This is the second coccidium reported to complete its entire development, from sporozoite to oocyst, in cell culture. J Parasitol, 1984 Aug, 70(4), 542 - 4 The effect of halofuginone, Wellcome 993 C, oxytetracycline, and diminazene diaceturate on Babesia equi-infected lymphoblastoid cell cultures; Zweygarth E et al.; The efficacy of halofuginone (DL-trans-7-bromo-6-chloro-3,3-(3-hydroxy-2-piperidyl) acetonyl-4-(3H) quinazolinone), Wellcome 993 C (2-hydroxy-3-cyclohexyl-1,4-naphthoquinone), and oxytetracycline, all of which have been shown to have a schizonticidal effect in the treatment of bovine theileriosis, and the babesicidal drug diminazene diaceturate, were tested against the schizont stages of Babesia equi in cell culture . The in vitro test system measured DNA synthesis in treated and untreated cell lines . Halofuginone (0.02 microgram/ml) and Wellcome 993 C (5 micrograms/ml) suppressed the incorporation of tritiated thymidine by more than 80% . Oxytetracycline was less effective, while diminazene diaceturate showed no notable effect . The insufficient schizonticidal activity may explain the failure of diminazene diaceturate to cure B . equi infections . Schizonticidal drugs either alone, or in combination with known babesicidal drugs, should be tested in the chemotherapy of B . equi infections. J Virol Methods, 1984 Aug, 9(1), 27 - 33 Comparison of mice and cell cultures for the isolation of tick-borne viruses; Nuttall PA et al.; Three methods of isolating viruses from 10 tick pools were compared; none of the methods produced all 13 of the viruses isolated (7 viruses of the bunyaviridae and 6 orbiviruses) . Inoculation of homogenised ticks into various cell lines was the most successful, yielding 11 virus isolations . Only 2 tick homogenates induced overt signs of infection following intra-cerebral inoculation of 2-day-old mice . However, when inoculated mouse brain was passaged in various cell lines, 8 of 12 isolations were made . The rates of success of the 3 methods of virus isolation appeared to vary according to the type and titre of the virus in the tick pool. In Vitro, 1984 Aug, 20(8), 635 - 41 Differentiation-arrested rat fetal lung in primary monolayer cell culture . IV . Paradoxical effect of a "fetal" pO2 on precursor incorporation into phospholipids and hormone responsiveness; Tanswell AK et al.; Differentiation-arrested lung cell cultures were developed from fetal rats of various gestational ages . In contrast to previously published observations with cultures in a pO2 of approximately 142 mm Hg, cultures developed in a pO2 of approximately 30 mm Hg, close to the normal fetal arterial pO2, have improved plating efficiency and a slightly increased growth rate . They did not, however, show gestation-dependent increases of choline incorporation into phospholipids, nor did immature lung cell cultures respond to dexamethasone or triiodothyronine, singly or in combination, by increased choline incorporation into saturated lecithin . The incorporation of choline and glycerol into lipids suggested a mature rate of lipid synthesis by immature cultures at a pO2 approximately 30 mm Hg, despite preservation of an immature morphology . Electron microscope observations revealed no gross differences between immature cultures developed at either pO2 . The cellular mechanisms underlying these differences are unclear but suggest that oxygen tension may significantly influence results obtained with in vitro studies of lipid synthesis by immature lung. In Vitro, 1984 Aug, 20(8), 629 - 34 Effects of cardioactive drugs on the beating activity of myocardial cell cultures isolated from offspring of trained and untrained pregnant rats; Butler AW et al.; Primary cultures of beating myocardial cells were obtained from 5-d-old offspring of trained (T) and untrained (UT), pregnant, Sprague-Dawley rats . The myocardial cells from the T and UT groups were evaluated for their beating responses to three cardioactive drugs: verapamil (V), isoproterenol (ISO), and propranolol (PRO) . The myocardial cell cultures from the UT group showed complete loss of beating when the calcium (Ca++) antagonist, V, was added to the cultures for 1 h or more; the T group was able to show some beating at comparable concentrations and durations of exposure with V . The beta agonist, ISO, markedly stimulated the beating rate of both the T and UT groups, but the beating rates were higher in the UT group at comparable concentrations and durations of exposure than with the T group . When the cultures were pretreated with the beta blocker, PRO, before treatment with ISO, a concentration inhibitory effect on the beating rate was observed in both groups . However, the T cultures were more sensitive to the inhibitory effects of PRO . These results demonstrate that primary cultures of rat myocardial cells isolated from the offspring of trained and untrained pregnant rats show differential beating responses to three well-known cardioactive drugs. Anal Biochem, 1984 Aug 1, 140(2), 380 - 5 A single-step gel-filtration method for the isolation of procollagens from cell culture conditioned medium; Holderbaum D et al.; Procollagens are the major proteins secreted into the conditioned medium of cultured arterial smooth muscle cells . Methods for the isolation and quantification of these macromolecules have traditionally required preliminary salt precipitation of procollagens from the conditioned medium followed by cellulose ion-exchange chromatography . The method described here exploits the elongated conformation of soluble procollagens and allows the direct recovery of procollagens from culture medium by a single gel-filtration chromatographic step under nondissociating conditions . Procollagens are isolated in high yield and show minimal processing by procollagen N- or C-terminal peptidase activity . This method results in rapid recovery of highly purified procollagens, free of most proteoglycans or other products of smooth muscle cell metabolism. Br J Radiol, 1984 Aug, 57(680), 723 - 8 Dose-rate effect between 1 and 10 Gy/min in mammalian cell culture; Ling CC et al.; A dose-rate effect is observed between 1 and 10 Gy/min for cultured Chinese hamster V-79 fibroblast cells irradiated under pO2 levels of less than 10 ppm, 100 ppm, 300 ppm and 21% . The dose for a cellular surviving fraction of 0.1% increases by 10-15% as the dose rate is changed from 10 to 1 Gy/min . This observed dose-rate effect is consistent with the repair of sublethal damage taking place during the radiation delivery . A related finding is that acute hypoxia in cell culture does not inhibit sublethal damage repair. J Immunol, 1984 Aug, 133(2), 758 - 63 Enhanced production of human gamma-interferon in nylon column-fractionated cell cultures; van Reis R et al.; The production of human gamma-interferon (HuIFN-gamma) in unfractionated and nylon wool column-fractionated leukocyte cell cultures stimulated with PMA and PHA was investigated . Production was studied with normal and reduced autologous serum protein levels in 96-hr spinner cultures . A 10- to 15-fold enhancement of production and a 50-fold increase in specific activity of crude HuIFN-gamma was demonstrated in nylon column-fractionated/reduced serum cell cultures . Kinetic analysis revealed a production rate maximum within 6 hr of induction in unprocessed cell cultures, whereas production occurred at an essentially constant rate for 48 hr in fractionated cell cultures. J Cell Physiol, 1984 Aug, 120(2), 169 - 80 Induction of cytochrome P-450 isozymes in rat hepatoma-derived cell cultures; Frey AB et al.; We have investigated the responsiveness of established rat hepatocyte cell cultures to inducers of cytochrome P-450 . One Reuber hepatoma-derived line (Fu5-C8), which under normal culture conditions produces no detectable cytochrome P-450(MC) or cytochrome P-450(PB)--the major cytochrome P-450 isozymes induced by 3-methylcholanthrene and phenobarbital, respectively--was tested for the ability to accumulate either cytochrome P-450 isozyme in response to treatment with various xenobiotics . By immune-precipitation from {35S}-methionine-labeled cell extracts, using monospecific anticytochrome P-450(MC) antibody or monoclonal anticytochrome P-450(PB) antibody, it was demonstrated that these cells possess the capability to synthesize cytochrome P-450(MC) in response to 3-methylcholanthrene treatment, while none of the drug treatments caused the synthesis of detectable quantities of cytochrome P-450(PB) . RNA extracted from Fu5-C8 cells directed the in vitro synthesis of immune-precipitable cytochrome P-450(MC) only after treatment of the cells with 3-methylcholanthrene . Kinetic analysis of the response of these cells to 3-methylcholanthrene induction revealed detectable levels of immune-precipitable cytochrome P-450(MC) 2 h after drug treatment with maximal induction occurring between 12 and 16 h of exposure . Another cell line (HF 1.5), obtained originally by hybridization of Fao X H5 variants of a Reuber H35 hepatoma, produces cytochrome P-450(MC) and also cytochrome P-450(PB) constitutively, as determined by specific immune-precipitation from labeled cell extracts . Exposure of confluent monolayers to either phenobarbital or 3-methylcholanthrene resulted in an induction of cytochrome P-450(PB) or cytochrome P-450(MC), respectively . Double-labeling immunofluorescence studies indicate that all cells in the culture produce albumin and most of the cells produce cytochrome P-450(MC), but only a subset of cells synthesize cytochrome P-450(PB) . Our results demonstrate that some continuously dividing hepatocyte cell cultures retain the capacity to respond to xenobiotics, including phenobarbital, a response which is typically exhibited by fully differentiated liver cells . Such established hepatocyte cell cultures should prove useful for investigating the mechanism of induction of cytochrome P-450(PB). J Neurochem, 1984 Aug, 43(2), 316 - 9 Cerebrovascular endothelial cell culture: metabolism and synthesis of 5-hydroxytryptamine; Maruki C et al.; The presence of 5-hydroxytryptamine was investigated in cultured and propagated cerebrovascular endothelium using immunohistochemistry and high pressure liquid chromatography . These studies demonstrate that the endothelium has the ability to take up and metabolize 5-hydroxytryptamine as well as to synthesize this amine from its precursor L-tryptophan, thus providing evidence for extraneural synthesis of 5-hydroxytryptamine in the central nervous system. Acta Med Okayama, 1984 Aug, 38(4), 349 - 55 Effect of angiotensin II, catecholamines and glucocorticoid on corticotropin releasing factor (CRF)-induced ACTH release in pituitary cell cultures; Murakami K et al.; The effects of angiotensin II, catecholamines and glucocorticoid on CRF-induced ACTH release were examined using rat anterior pituitary cells in monolayer culture . Synthetic ovine CRF induced a significant ACTH release in this system . Angiotensin II produced an additive effect on CRF-induced ACTH release . The ACTH releasing activity of CRF was potentiated by epinephrine and norepinephrine . Dopamine itself at 0.03-30 ng/ml did not show any significant effect on ACTH release, but it inhibited CRF-induced ACTH release . Corticosterone at 10(-7) and 10(-6)M inhibited CRF-induced ACTH release . These results indicate that angiotensin II, catecholamines and glucocorticoid modulate ACTH release at the pituitary level. Tohoku J Exp Med, 1984 Aug, 143(4), 441 - 9 Influence of various experimental conditions on the inhibitory effects of (E)-5-(2-bromovinyl)-2'-deoxyuridine on varicella-zoster virus replication in cell culture; Baba M et al.; Inhibition of varicella-zoster virus (VZV) replication by (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) has been examined in vitro under various experimental conditions . The 50% inhibitory dose (ID50) of BVDU for VZV replication in human embryonal fibroblast (HEF) cultures was 0.016 micrograms/ml, if the assay was based on the reduction of visible foci . If the assay was based on the reduction of immunofluorescent foci, the ID50 was 3 times higher . There was no significant difference in ID50, whether the HEF cultures were infected with cell-free or cell-associated VZV . When the HEF cells were infected with VZV at different multiplicities of infection (MOI), the ID50 of BVDU increased in parallel with the increase of MOI . BVDU was normally added immediately after virus infection . However, the addition of BVDU could be delayed until 8 hr after infection without substantial decrease of activity . On the other hand, BVDU did not cause any inhibition of focus formation when it was removed within 8 hr after VZV infection . If removed at 24 or 48 hr after infection, BVDU caused a significant reduction in focus formation . BVDU had no effect on focus formation if added to the HEF cells before virus infection . BVDU was also found to inhibit VZV focus formation in Vero cells, but only at an ID50 that was 5-10 times higher than the ID50 noted in HEF cells. Carcinogenesis, 1984 Aug, 5(8), 1065 - 9 Benzo{e}pyrene-induced alterations in the binding of benzo{a}pyrene to DNA in hamster embryo cell cultures; Smolarek TA et al.; Benzo{e}pyrene (B{e}P), a weakly carcinogenic polycyclic aromatic hydrocarbon (PAH) modifies tumor induction in mouse skin and the induction of mutation in mammalian cells by carcinogenic PAH . To determine how B{e}P alters the metabolic activation of the carcinogen benzo{a}pyrene (B{a}P), early passage Syrian hamster embryo cell cultures were exposed to {3H}B{a}P or {3H}trans-7,8-dihydro-7,8-dihydroxyB{a}P (B{a}P-7,8-diol) in the presence of various concentrations of B{e}P for 24 h . The DNA was isolated, degraded to deoxyribonucleosides and the B{a}P-deoxyribonucleoside adducts were analyzed by h.p.l.c . As the dose of B{e}P increased, the amount of B{a}P bound to DNA decreased and the ratio of anti-B{a}P-7,8-diol-9,10-epoxide (B{a}PDE)-deoxyguanosine adduct to syn-B{a}PDE-deoxyguanosine adduct decreased . B{e}P treatment inhibited the binding of B{a}P-7,8-diol to DNA to a greater extent than it inhibited the binding of B{a}P and decreased the ratio of anti- to syn-B{a}PDE-deoxyguanosine adducts formed from the 7,8-diol . These results indicate that B{e}P decreases the activation of B{a}P to DNA-binding intermediates in these cells; especially the oxidation of B{a}P-7,8-diol to a diol-epoxide . The B{e}P-induced alterations in the ratio of DNA adducts formed from the syn- and anti-isomers of B{a}PDE suggest that B{e}P selectively inhibited certain pathways of metabolic activation of B{a}P . Thus, B{e}P-induced modifications in the biological activity of PAH may result from alteration in both the amounts and the relative proportions of various isomeric forms of the ultimate carcinogenic metabolites formed from PAH. Virology, 1984 Jul 30, 136(2), 448 - 52 Dexamethasone enhances human cytomegalovirus replication in human epithelial cell cultures; Tanaka J et al.; An epithelial human hepatoma cell line (PLC/PRF/5) and a primary epithelial human baby kidney (HBK) cell culture showed restricted growth of human cytomegalovirus (HCMV) . Treatment of these two epithelial cell cultures with dexamethasone greatly enhanced their ability to support HCMV replication . Growth kinetic experiments and infectious center assay revealed that in both the hormone-treated cultures infectious progeny virus appeared earlier by 1 or 2 days and 5- or 10-fold more cells are able to produce infectious virus . There was an approximate 50- or 100-fold increase in virus yield compared to that in the untreated control cultures . Enhanced HCMV replication in the hormone-treated cultures was not due to differences in the cell growth or the virus adsorption and was supported by evidence of increased synthesis of HCMV-specific immediate early antigens and DNA. Science, 1984 Jul 27, 225(4660), 424 - 7 Sindbis virus mutants selected for rapid growth in cell culture display attenuated virulence in animals; Olmsted RA et al.; Mutants of Sindbis virus were selected for rapid growth in baby hamster kidney (BHK) cell cultures and screened for attenuation of virulence in suckling mice . Comparisons among independently isolated virulent and attenuated strains, as well as a classical reversion analysis, showed that accelerated penetration of BHK cells was correlated with attenuation in vivo . Both phenotypic changes resulted from a reorganization of virion structure as detected by monoclonal antibodies . These results suggest that mutants selected for rapid growth in cell culture may be useful as attenuated vaccines and for studies of the molecular basis of virus pathogenesis. J Pharmacol Exp Ther, 1984 Jul, 230(1), 82 - 8 Multiple actions of convulsant barbiturates on mouse neurons in cell culture; Skerritt JH et al.; The convulsant barbiturate 5-(2-cyclohexylidene-ethyl)-5-ethyl barbituric acid (CHEB) depolarized most (greater than 90%) mouse spinal cord (SC) neurons in primary dissociated cell culture in a concentration-dependent fashion with threshold effects at 10 to 50 nM . Dorsal root ganglion (DRG) neurons were also depolarized by CHEB, but only about 50% of the neurons responded . The threshold concentration for depolarization of DRG neurons was several hundred-fold higher than for SC neurons . CHEB depolarizations may be mediated by an increase in a cation conductance which is calcium-dependent because CHEB depolarizations had an extrapolated reversal potential near 0 mV, were insensitive to intracellular anion (chloride ion) injection, were absent after removal of extracellular calcium ions and were reduced by cadmium ions . In contrast, the nonconvulsant barbiturates, pentobarbital and phenobarbital, did not produce membrane depolarization . However, at concentrations of CHEB somewhat higher than those which directly depolarized cells, CHEB resembled pentobarbital and phenobarbital because it reduced the spontaneous activity of SC neurons and shortened calcium-dependent action potentials of DRG neurons . Two other convulsant barbiturates, trans-5-ethyl-5-(3'-methyl-but-2'-enyl) barbituric acid and trans-5-ethyl-5-(1',3'-dimethyl-but-1'-enyl) barbituric acid, also produced membrane depolarization, reduced spontaneous activity and shortened calcium-dependent action potentials . Another convulsant barbiturate, S(+)-1-methyl-5-phenyl-5-propyl barbituric acid, did not alter membrane potential or conductance of SC neurons, suggesting that mechanistic subclasses of convulsant barbiturates exist.(ABSTRACT TRUNCATED AT 250 WORDS) J Neurochem, 1984 Jul, 43(1), 42 - 8 Increase of choline acetyltransferase by colchicine in primary cell cultures of spinal cord; Ishida I et al.; Colchicine (5-10 microM) increased choline acetyltransferase (ChAT) activity 5-10-fold and suppressed acetylcholinesterase (AChE) and glutamate decarboxylase (GAD) activities to 30% and 50%, respectively, of the levels of control cells in mouse spinal cord cells cultured for several days . The synthesis of radiolabeled acetylcholine (ACh) from {14C}choline was also enhanced 4.6-fold, although the uptake of {14C}choline into cells was decreased to 80% of control level . Neither the incorporation of {3H}leucine into protein nor the total amount of protein was increased by colchicine . Vinblastine also increased ChAT activity while cytochalasin B was not effective . Immunochemical titration study revealed that the increase of ChAT activity by colchicine was due to the accumulation of ChAT molecules . Co-culture of spinal cord cells with skeletal muscle markedly stimulated ChAT activity, and the addition of colchicine to the cocultures showed greater than additive effect . These observations indicate that colchicine increases ChAT molecules in a specific manner, that the stimulatory effect of colchicine on ChAT activity is possibly mediated via the interaction with microtubules, and that the increase of ChAT activity is based on a mechanism different from that of co-cultures with skeletal muscle cells. Proc Natl Acad Sci U S A, 1984 Jul, 81(13), 4075 - 9 Teratogens induce a subset of small heat shock proteins in Drosophila primary embryonic cell cultures; Buzin CH et al.; Drosophila embryonic cells placed into culture just after gastrulation differentiate in vitro over the next 24 hr . A number of drugs that are teratogenic in mammalian systems have been found to inhibit muscle or neuron differentiation (or both) in these developing cultures . We have examined, by two-dimensional gel electrophoresis, the effects of these drugs on protein synthesis in embryonic cells . For nine teratogens tested, cells treated for 20 hr with the drug show a dramatic induction of three proteins of about 20 kilodaltons, in addition to the normal proteins synthesized by untreated cells . Three teratogens as well as all eight nonteratogens tested did not show this induction . The induced proteins appear to be identical to three of the heat shock proteins (hsp 23, 22a, and 22b), as shown by electrophoretic mobilities and peptide mapping by partial proteolysis . A 37 degrees C heat shock of the embryonic cells produces the full complement of heat shock proteins, whereas drug-treated cells induce only the subset hsp 23, 22a, and 22b but not hsp 26 or 27 . beta-Ecdysterone, the Drosophila molting hormone, also inhibits embryonic differentiation and induces hsp 23, 22a, and 22b, a partial subset of the heat shock proteins (hsp 22, 23, 26, and 27) induced by the hormone in imaginal discs and some Drosophila continuous cell lines . Dose-response studies of several drugs show a correlation between the degree of inhibition of differentiation and the level of induction of hsp 23, 22a, and 22b . The induction of heat shock proteins by drugs may reflect specific types of stress that can also give rise to teratogenesis. Anal Biochem, 1984 Jul, 140(1), 104 - 7 A semi-automated protein assay for cell cultures; Shopsis C et al.; A semi-automated modification of the protein determination procedure of O . H . Lowry, N . J . Rosebrough, A . L . Farr, and R . J . Randall (1951, J . Biol . Chem . 193, 265-275) is described . The assay is well suited to the analysis of the protein of adherent cultured cells . The procedure is carried out in 96-well microtest plates on protein solutions of 50 microliter or less, and can detect less than 0.5 micrograms of protein (equivalent to about 10(3) cultured cells) . Optical densities are read and printed by an automatic microplate reader capable of processing 96 samples in less than 2 min. Vopr Virusol, 1984 Jul-Aug, 29(4), 497 - 502 {Isolation of strains of the virus of hemorrhagic fever with renal syndrome in cell culture}; Bashkirtsev VN et al.; Four strains of hemorrhagic fever with renal syndrome (HFRS) virus were isolated from the lungs of Cl . glareolus caught in natural foci of HFRS in the European USSR . Preliminary experiments using enzyme-immunoassay (EIA) established the appurtenance of HFRS antigen in lung suspensions to the "western" serotype . Four strains were isolated in Vero-E6 cell culture after several blind passages and identified as the "western" serotype of HFRS virus . One stram originally isolated in Wistar rats and passaged in them 4 times and then adapted to Vero-E6 cells showed a closer antigenic relationship with the "eastern" HFRS virus serotype. Cornell Vet, 1984 Jul, 74(3), 208 - 17 Cultivation of a porcine adenovirus in porcine thyroid cell cultures; Dea S et al.; The porcine adenovirus type 4 was adapted to grow in porcine thyroid cell cultures . A readily recognizable cytopathic effect appeared in these cells as soon as the first passage of the virus and complete degeneration of the monolayers was obtained after only 72 hours post-infection at the fourth passage . A viral yield of 10(6.0) TCID50/ml was calculated after the third passage . The virus was purified by CsCl density gradient centrifugation and was shown to possess a buoyant density of 1.33 g/ml . A specific antiserum was prepared from two specific-pathogen-free piglets and used for indirect immunofluorescent staining . The fluorescence was observed in the nucleus of infected cells at 24 to 72 hours post-inoculation . The use of TP cells is suggested for routine porcine adenovirus diagnosis. J Neurochem, 1984 Jul, 43(1), 49 - 57 Parvalbumin, a neuronal protein in brain cell cultures; Pfyffer GE et al.; Dissociated brain cell cultures were derived from 14-day-old embryonic as well as from newborn mice . The cells were grown in a medium containing 10% fetal calf serum . Indirect immunofluorescence was performed using antisera directed against the Ca2+-binding protein parvalbumin (Mr 12,000) . In embryonic cultures a large proportion of cells was intensely stained by antiparvalbumin . In double-labelling experiments involving the simultaneous application of antisera against parvalbumin and the neuron-specific enolase, the enolase-containing cells were also parvalbumin-positive and both antisera revealed identical intracellular staining patterns . Conversely, almost no parvalbumin- and enolase-positive cells were present in cultures derived from newborn mice . However, in these cultures many cells were immunoreactive toward the myelin basic protein, an accepted marker for oligodendrocytes . The presence of parvalbumin within the embryonic brain cell cultures was confirmed by analyses of the culture extracts (4 mM EDTA, pH 7.5) by HPLC on reverse-phase supports, two-dimensional polyacrylamide gel electrophoresis, and immunoblotting . The present study suggests that in mouse brain cell cultures, parvalbumin is localized in neurons. Neurochem Res, 1984 Jul, 9(7), 871 - 86 Polyamines and the development of isolated neurons in cell culture; Seiler N et al.; The possible role of polyamines in the development of isolated neuroblasts from the cerebral cortex of embryonic chick brain was studied by means of three enzyme activated irreversible inhibitors of ornithine decarboxylase . alpha-Difluoromethylornithine (MDL 71782) showed no effects on development at doses which depleted dramatically neuronal putrescine and spermidine levels . In contrast, the two other inhibitors, (E)-alpha-(fluoromethyl)dehydroputrescine (MDL 72197) and 6-heptyne-2,5-diamine (MDL 72175) blocked the formation of neuronal outgrowths completely at 100 microM and higher concentrations . Their effects on neuronal polyamines differed at this concentration considerably . The growth inhibitory effect of the ornithine decarboxylase inhibitors was in all cases reversible: cells which were grown after 3 days of exposure to the drugs in normal medium produced neuronal networks . The presence of putrescine at 10 microM concentration in the culture medium prevented the growth inhibitory effect of 100 microM concentrations of the drugs . This concentration of putrescine was not only capable of preventing, but also of reversing growth inhibition by the ornithine decarboxylase inhibitors . Although the cellular polyamine levels were not correlated with the morphological development of chick embryo cortical neurons, the present study leaves no doubt that putrescine plays an essential role in neuronal differentiation. Stain Technol, 1984 Jul, 59(4), 187 - 92 A simplified procedure for the observation in situ of chromosome aberrations or sister chromatid exchanges and the estimation of the mitotic index in mammalian monolayer cell cultures; Sideris EG et al.; Chinese hamster V-79 cells are widely used in short term screening for potential physical or chemical mutagens of the environment . A simplified version of the standard Giemsa protocol of Moorhead and the Feulgen plus Giemsa protocol of Wolff and Perry is given which permits the observations in situ of chromosome aberrations or sister chromatid exchanges and the estimation of the mitotic index in the Petri dishes for the culture of the V-79 cells. J Pharm Pharmacol, 1984 Jul, 36(7), 473 - 5 'Superactivation' of alkaline phosphatase activity by cycloheximide in rat hepatoma cell cultures; Sorimachi K et al.; We have found that rat hepatoma cells (R-Y121B) retain alkaline phosphatase activity, and that this enzyme activity is increased by cycloheximide . Actinomycin D also increased the enzyme activity . This increase due to actinomycin D was partially inhibited by cycloheximide . The characteristics of alkaline phosphatase of the cells treated or untreated with cycloheximide or actinomycin D were similar to each other; they were heat labile and the enzyme reaction was strongly inhibited by L-homoarginine, but weakly by L-phenylalanine . The increase in alkaline phosphatase activity with cycloheximide has been termed a 'superactivation' of alkaline phosphatase. Biofizika, 1984 Jul-Aug, 29(4), 633 - 6 {Iron-sulfur centers in the mitochondrial respiratory chain at different stages of cell culture growth of the hamster Cricetulus griceus}; Burbaev DSh et al.; The dramatic diminishing of the concentration of the N-2 iron-sulfur centre of NADH-dehydrogenase of mitochondria during the growth of cell culture of hamster fibroblasts with the subsequent recovery of concentration to the initial level was discovered by means of low-temperature ESR spectroscopy . It was concluded that the results obtained are due mainly to the decrease of the number of respiratory chains, but not to the change of the electron transport chain structure. Acta Cytol, 1984 Jul-Aug, 28(4), 509 - 13 A new device and method for rapid processing of cytologic smears, histologic sections and cell cultures for electron microscopy; Federman Q et al.; A device and technique are described by means of which samples may be selectively lifted from flat surfaces, such as microscope slides or coverslips, and processed for examination with the electron microscope . The device consists of a spring clamp that holds the narrow end of an open-tipped, conical B.E.E.M . or similar capsule sealed against the surface from which the sample of interest is to be removed . All processing of the sample for electron microscopy can take place within the capsule . When completed, the capsule filled with solidified resin is removed with the area of interest of the sample embedded in its tip. Brain Res, 1984 Jun 25, 304(2), 339 - 49 Cell production and morphological pattern formation in primary brain cell cultures . I . Pattern formation within the basal layer(s); Madarasz E et al.; Spontaneous pattern formation within the basal cellular layer of primary brain cell cultures were studied . Basal cells were found to be organized into a limited number of morphologically well distinguishable types of cell-arrangements . Phase contrast microscopy was used to characterize the different cell-assemblies morphologically . Under standard culture conditions the time of appearance and specific changes of relative frequency in time were also characteristic of different patterns . The distribution of mitotically active cells among the different morphological patterns within the basal layer was also investigated by recording {3H}thymidine uptake of cells . Reduction of mitotic activities of cells in the basal layer was found in the vicinity of overlying cells . In a given area of cultures the reduction of mitotic activity was proportional to the number of overlying cells . The influence of cell proliferation on the morphological pattern formation in primary cultures is discussed. J Biol Chem, 1984 Jun 25, 259(12), 7382 - 90 The development of physiologic responsiveness to muscarinic agonists in chick embryo heart cell cultures . Role of high affinity receptors and sensitivity to guanine nucleotides; Galper JB et al.; Prior to ingrowth of the vagus nerve (4-5 days in ovo), embryonic chick hearts are relatively unresponsive to muscarinic stimulation ( Pappano , A . J . (1977) Pharmacol . Rev . 29, 3-33) . We studied the correlation between the development of physiologic responsiveness in the embryonic chick heart and changes in the properties of muscarinic receptors . In cultures from hearts 10 days in ovo, muscarinic agonists decreased beating rate by 15% and increased the rate of K+ efflux by 35% . In cultures of embryonic hearts 3 1/2 days in ovo, muscarinic receptors had no effect on beating rate and mediated only an 11% increase in the rate of K+ efflux . We previously demonstrated that in cells cultured from hearts 10 days in ovo, 26% of receptors bound agonist with a high affinity (RH) and that incubation with guanine nucleotides mediated the conversion of RH to a low affinity form (RL ( Galper , J . B., Dziekan , L . C., O' Hara , D . S., and Smith, T . W . (1982) J . Biol . Chem . 237, 10344-10356} . In cultures of hearts 3 1/2 days in ovo, RH constituted 52% of total {3H} quinuclidinyl benzilate-binding sites, and guanine nucleotides had no effect on the conversion of RH to RL . Growth of cells cultured from hearts 3 1/2 days in ovo in medium supplemented with specific lots of serum resulted in a 3-fold increase (from 11 to 30%) in the ability of muscarinic agonists to increase K+ permeability . This increased sensitivity to muscarinic stimulation was accompanied by a 20% increase in RH and sensitivity of 75% of RH to guanine nucleotides . Thus, enhanced number of RH and development of guanine nucleotide responsiveness are associated with the development of a physiologic response . The relationship of these developmental changes to the appearance of a guanine nucleotide regulatory protein is discussed. Biull Eksp Biol Med, 1984 Jun, 97(6), 764 - 6 {Floating cell cultures of the fetal pancreas}; Bliumkin VN et al.; A simple method for preparing human and animal fetal pancreatic cell cultures has been developed . It is based on enzymatic treatment of the fetal pancreatic tissue with collalitine in combination with microdissection . As a result of subsequent cultivation there form floating cytotypic and organotypic cultures consisting mainly of B cells in different phases of the secretory cycle . The floating cultures prepared by the above-described method produce insulin and can be successfully used in experimental and clinical transplantology. J Hyg (Lond), 1984 Jun, 92(3), 285 - 96 The duration of immunity in cattle following inoculation of rinderpest cell culture vaccine; Plowright W; The duration of immunity following a single administration of rinderpest cell culture vaccine, of 90 or more monolayer passages, was studied in E . African zebu (Boran) and grade (cross-bred European) cattle . All animals were kept for periods of 6-11 years in rinderpest-free environments; groups of them (in all 23 Borans and 10 grades) were then challenged by parenteral or intranasal inoculation of virulent virus or by contact exposure to reacting cattle . Nasal excretion of virus was studied daily over the 10-to 14-day period following challenge, and simultaneous attempts were made to detect viraemia . The neutralizing antibody response was followed at 6-month intervals over the whole post-vaccination period and then daily for 10 days and at longer intervals to 3 weeks after challenge . All 33 animals which were exposed by various routes failed to react clinically and a rinderpest viraemia was never detected . No transmission of virus from the vaccinates to susceptible in-contact controls occurred within 14 or more days, from the 20 animals which could be so tested . Clearcut serological responses to challenge were seen in six cattle (four Borans and two grades) which were challenged after 7 years or more; these reactions were all delayed to the 9th or 10th days, i.e . they were not typically 'anamnestic' . These results are discussed in relation to mass vaccination campaigns for the control of rinderpest and from the comparative viewpoint of measles vaccination in man. J Gen Virol, 1984 Jun, 65 ( Pt 6), 1123 - 6 Propagation of human candidate calicivirus in cell culture; Cubitt WD et al.; Evidence is presented for the first time that a human candidate calicivirus (HCV) replicates in human embryo kidney cells when trypsin is incorporated in the culture medium . The virus multiplies in the presence of actinomycin D and radiolabelling experiments with {3H}uridine indicate that it has an RNA genome . These observations provide further support for the view that HCV should be tentatively classified as a member of the Caliciviridae . Exp Cell Res, 1984 Jun, 152(2), 565 - 70 Elimination of mycoplasmas from cell cultures and establishment of mycoplasma-free cell lines; Schmidt J et al.; Several antibiotics were examined for their potential to eliminate mycoplasmas from contaminated cell cultures . Acholeplasma laidlawii, Mycoplasma arginini, Mycoplasma hyorhinis and Mycoplasma orale were effectively eliminated from experimentally contaminated mouse fibroblasts and mink epithelial cells by the use of the antibiotics minocycline and tiamutin . An elimination procedure was established, which involved the consecutive treatment of the cultures over a period of 3 weeks, followed by cell cloning . This procedure was effective when applied to cell lines which had been contaminated with unidentified and partially non-cultivable strains of mycoplasmas. Ophthalmology, 1984 Jun, 91(6), 580 - 95 Trabecular meshwork cell culture in glaucoma research: evaluation of biological activity and structural properties of human trabecular cells in vitro; Polansky JR et al.; The propagation of human trabecular cells in culture allows the study of the structural and functional properties of this distinct cell type under reproducible experimental conditions . Human trabecular cells can be effectively grown from dissected explants of trabecullar tissue, and the cultured cells can maintain the distinctive ultrastructural features of uncultured trabecular cells through at least five passages in vitro . The trabecular cell possesses a wide range of biochemical and structural properties that may be important for the maintenance of the aqueous outflow pathway . These properties include the growth of trabecular cells as an endothelial monolayer with a nonthrombogenic cell surface, the production of plasminogen activator, avid phagocytosis, and the ability to synthesize glycosaminoglycans, collagen, fibronectin, and other connective tissue elements . The presence of hyaluronidase and other lysosomal enzymes emphasizes that human trabecular cells are capable of metabolizing hyaluronic acid and other extracellular materials . Potential mechanisms of trabecular cell damage in vitro are examined by evaluating the effects of extended passage, peroxide exposure, and laser treatment on cellular morphology. Brain Res, 1984 Jun, 316(2), 263 - 70 Blockers of calcium permeability inhibit neurite extension and formation of neuromuscular synapses in cell culture; Suarez-Isla BA et al.; Growth of neurites from trypsin-dissociated retinal neurons from chick embryos is very sensitive to the extracellular calcium concentration . Blockers of calcium permeability such as Co2+, Mn2+, La3+ and nitrendipine, when added to dissociated neurons, decrease the fraction of cells that extend neurites and the rate of neurite growth without influencing cell-substratum adhesion or survival capacity . Inhibition is concentration dependent, is related to the age of the donor chick embryo and can be prevented by increasing the extracellular calcium concentration . 50% inhibition in 8-day neurons is produced by 120 microM Co2+, 250 microM Mn2+, 50 microM La3+ and 10 microM nitrendipine in medium containing 1.8 mM Ca2+ . Inhibition of neurite extension is accompanied by a concentration dependent inhibition of synapse formation between retinal neurons and muscle cells in culture, as determined by intracellular recording . 50% inhibition in the fraction of innervated myotubes is produced by 0.4 mM Co2+ and 4 mM Mn2+ . These results suggest that: (1) a voltage-dependent calcium flux is a signal not only for growth cone expansion but also for neurite extension in primary dissociated neurons; and (2) that neurite extension is a prerequisite for synaptogenesis between neurons and muscle cells in culture. Endocrinol Exp, 1984 Jun, 18(2), 101 - 7 Action of inhibitory LH-RH analogs in rat pituitary and luteal cell cultures; Spona J et al.; The effect of LH-RH antagonists on LH-RH stimulated LH release was studied in primary rat pituitary cell culture and the inhibition by LH-RH antagonists of HCG provoked progesterone production was investigated in primary rat luteal cell culture . Antagonists used in this study were Ac-D-Trp1, D-Phe(Cl)2, D-Lys6, D-Ala10-LH-RH(I1) and the respective D-Lys6 isophthaloyl dimer (I2) . LH-RH activity was noted to be reduced to 1/12 by I1 and to 1/23 by I2 in the pituitary cell culture system . The LH-RH inhibitors did not possess intrinsic LH releasing activity up to 10(-6) mol 1(-1) . Incubation of rat luteal cells with HCG in the presence of LH-RH, I1 or I2 resulted in a smaller progesterone release than that observed in the absence of the peptides . ED50 of HCG was noted to be 5.7 X 10(-13) mol 1(-1) and 10(-8) mol 1(-1) LH-RH, I1 or I2 caused a shift of ED50 to 6.3 X 10(-12) mol 1(-1), 1.2 X 10(-12) mol 1(-1) and 7.9 X 10(-13) mol 1(-1), respectively . The present investigation is the first demonstration of reduction by LH-RH antagonists of gonadal steroid production . The present results suggest the use of such inhibitory LH-RH analogs in the treatment of hormone dependent tumors such as prostatic carcinoma and would not cause a transient rise of gonadal steroids as seen by the use of LH-RH agonists. J Clin Microbiol, 1984 Jun, 19(6), 920 - 2 Comparison of Cultureset and primary rabbit kidney cell culture for the detection of herpes simplex virus; Rubin SJ et al.; Herpes simplex virus isolation by conventional cell culture in primary rabbit kidney cells (RK) was compared with Cultureset (CS) . At 24 h CS was more sensitive (31 versus 25 of 55 isolates detected) than RK, but at 48 h there was little difference (43 detected by CS, 46 by RK) . CS stained at 48 h was significantly less sensitive (78.2%) than RK observed for 1 week . CS stained at 24 or 48 h was not an acceptable substitute for RK observed for 1 week. Biochem Biophys Res Commun, 1984 May 16, 120(3), 741 - 6 A transient species of poly(A)+ RNA detected by a myosin heavy chain cDNA probe in muscle cell culture during terminal differentiation; Liang R; Primary cell cultures were prepared from breast muscles of 11 day 4 hour-embryonic chicks . Cytoplasmic RNAs were isolated from the cultured cells at various time intervals from day 3 to day 8 . A {P32} DNA probe complementary to messenger RNA of myosin heavy chain was used to hybridize with the RNAs after gel electrophoresis . A transient species of polyadenylated RNA with a decreased mobility in electrophoresis was detected during a period of time when contractions of syncytial fibers were first observed. Science, 1984 May 11, 224(4649), 603 - 5 Complete development of Cryptosporidium in cell culture; Current WL et al.; Protozoan parasites of the genus Cryptosporidium cause a short-term, flu-like, gastrointestinal illness in immunocompetent persons and severe, persistent, life-threatening diarrhea in immunodeficient individuals . No effective therapy is available for the treatment of cryptosporidiosis in the immunodeficient host . Complete development (from sporozoite to sporulated oocyst) of a human isolate of Cryptosporidium was achieved in cultured human fetal lung cells and primary chicken kidney and porcine kidney cells . The growth of this newly recognized zoonotic agent in cell culture now provides a means of studying its behavior, development, and metabolism, and a mechanism for evaluation of potentially useful therapeutic agents. J Chromatogr, 1984 May 11, 307(2), 251 - 60 Quantitative analysis of prostaglandins in cell culture medium by high-resolution gas chromatography with electron-capture detection; Berens ME et al.; Prostaglandins have been shown to be important modulators of haemostatis , immune responses, and growth of normal and neoplastic cells . In order to investigate the cell origin and metabolic profile of the endogenous prostaglandins in human tumours, a convenient extraction and gas chromatographic method for measuring the various classes of prostaglandins was developed . Infiltrating macrophages from human tumours were isolated using adherence to plastic . Macrophage-enriched and macrophage-depleted cell populations were then cultured in vitro and the media supernatant was studied for the presence of prostaglandins E1, E2, F2 alpha, and 6-keto-F1 alpha (the spontaneous breakdown product of prostacyclin, PGI2) . Routinely, 1 ml of medium containing 10(6) cells was studied . The eicosanoids were extracted using commercially available octadecylsilyl silica reversed-phase columns prior to derivatization . Standards and samples were prepared as pentafluorobenzyl ester (methoxime) trimethylsilyl ether derivatives for analysis on an OV-101 (25 m X 0.2 mm) fused-silica capillary column . Recovery of standards ranged from 93% to 37%, with linear recovery in all instances (regression coefficients greater than 0.98) . Detection limits were 20 pg for each of the prostaglandins . Analysis of cell subpopulations from six human tumours revealed that infiltrating macrophages produce various prostaglandin profiles and are largely responsible for the prostaglandin production in human cancer . The described analytical method is the first application of high-resolution gas chromatography with electron-capture detection to the quantitative profiling of prostaglandins from human cell culture. J Immunol Methods, 1984 May 11, 70(1), 101 - 9 Rapid immunofluorescent screening procedure using primary cell cultures or tissue sections; Furst A et al.; We describe a rapid procedure for immunofluorescent screening of hybridoma supernatants . Use of primary cell cultures as substrates allows immediate detection and partial characterization of antibodies binding selectively to specific cell types . Sulfonated tissue culture cluster lids are used as a single substrate upon which cells are cultured and all stages of the antibody binding assay are performed, including microscopic observation . They may also be used for mounting cryostat sections of tissues . Condensation rings on the lids isolate individual groups of cells for each assay . Cells may be live or fixed, as desired . Very small volumes of culture supernatant are required for each assay, and nearly all steps are performed in bulk. Natl Cancer Inst Monogr, 1984 May, 65, 175 - 8 Primary cell cultures from the teleost, Cyprinodon variegatus: culture establishment and application in carcinogen exposure studies; Martin BJ et al.; Methods were developed for aseptic maintenance of Cyprinodon variegatus fry for extended periods . Preliminary studies indicated that under optimum conditions sterile embryos develop normally for a sufficient time to function as carcinogen-teratogen assay systems . An embryo-primary cell culture technique was developed that incorporates, in a single system, certain characteristics of both intact embryos and primary cell cultures and allows simultaneous observation of the effects of carcinogens on the whole organism and primary cell monolayers . The effective use of these systems provides one the opportunity to study the effects of carcinogens on teleosts at the cellular and organismic level. J Neurol Sci, 1984 May, 64(2), 149 - 60 Comparison between the growth pattern of cell cultures from normal and Duchenne dystrophy muscle; Delaporte C et al.; The growth "in vitro" of muscle cells from 12 patients with Duchenne muscular dystrophy (DMD) was compared with that of muscle cells from 20 age-matched controls . In the DMD explants, the lag phase (3 days) was shorter than in controls (6 days) . In dissociated cells, plating efficiency (20%) and doubling time (30 h) were identical in DMD and controls . In cultures from three DMD patients, cell clusters were occasionally observed . Myotube morphometry showed significant abnormalities in DMD cultures: the number of myotubes per field was 8.2 +/- 0.8 and 26.7 +/- 0.6 in controls, P less than 0.001; myotube length (151 +/- 20 micron) and diameter (8.2 +/- 0.9 micron) in DMD cultures were half the control values (312 +/- 46 micron and 15.6 +/- 1.2 micron, respectively, P less than 0.001) . The number of nuclei per myotube in DMD was one-quarter of that in control muscle (4.0 +/- 0.2 vs 15.8 +/- 2.2, P less than 0.001) . It is concluded that DMD cultures show cellular heterogeneity with the presence of fibroblasts and non-fusing myoblasts; furthermore they show delayed myoblast fusion and poor myotube differentiation. J Neurosci, 1984 May, 4(5), 1398 - 404 Production of "ectopic" vasoactive intestinal peptide-like and neurotensin-like immunoreactivity in human pheochromocytoma cell cultures; Tischler AS et al.; Neoplastic chromaffin cells from human pheochromocytomas can exhibit extensive spontaneous and nerve growth factor (NGF)-induced outgrowth of neurite-like processes in vitro, despite the absence of such processes in vivo . To determine whether acquisition of neuron-like features by human pheochromocytoma cells in culture is accompanied by functional alterations, process outgrowth, vasoactive intestinal peptide-like immunoreactivity ( VIPLI ), neurotensin-like immunoreactivity (NTLI), and catecholamine content were studied in freshly dissociated cells and in 21-day-old cultures from six human pheochromocytomas . All of the cultures produced VIPLI and exhibited spontaneous process outgrowth . NGF stimulated process outgrowth and enhanced production of VIPLI . Dexamethasone inhibited process outgrowth and tended to decrease production of VIPLI . NTLI was detected in cells from only one of the tumors, and its production appeared to be regulated comparably to that of VIPLI . Catecholamine content decreased markedly in all of the cultures and was not regulated in parallel with either VIPLI or NTLI . The findings suggest that human pheochromocytoma cultures may help to elucidate cellular and molecular mechanisms regulating ectopic and normal VIP production. In Vitro, 1984 May, 20(5), 404 - 8 Detection of mycoplasmas infecting cell cultures by DNA hybridization; Razin S et al.; Infection of cell cultures by mycoplasmas can be detected and the mycoplasma identified by Southern blot hybridization of the Eco RI-digested DNA of the suspected cell cultures with a nick-translated probe consisting of cloned ribosomal RNA genes of Mycoplasma capricolum . The probe does not hybridize with eukaryotic DNA . The hybridization pattern with mycoplasmal DNA is species specific, enabling the identification of the four most prevalent mycoplasma contaminants, Mycoplasma orale, Mycoplasma hyorhinis, Mycoplasma arginini, and Acholeplasma laidlawii . The test is also very sensitive and can detect as little as 1 ng of mycoplasmal DNA, roughly equivalent to the DNA content of 10(5) mycoplasmas. In Vitro, 1984 May, 20(5), 369 - 75 Inhibition of growth of mammalian cell cultures by extracts of arginine-utilizing mycoplasmas; Sasaki T et al.; Mechanisms of the inhibition of growth of mammalian cell cultures caused by mycoplasmal infection were investigated by using cell-free extracts of 14 species of mycoplasmas . In four mammalian cell lines tested, the growth of two cell lines, FM3A and MDCK, was inhibited by the extracts of arginine-utilizing mycoplasmas, whereas that of the other two cell lines, Vero and LLC-MK2, was not inhibited by extracts of either arginine- or glucose-utilizing mycoplasmas . These results suggest that there are two types of cell cultures, one susceptible and the other insusceptible to arginine-utilizing mycoplasmas . In a series of experiments using FM3A cells, it was found that the growth inhibition caused by the extracts of arginine-utilizing mycoplasmas was due to removal of arginine from the medium by the action of arginine deiminase present in the extracts and that none of the metabolic products of arginine had any effect on the growth . A highly positive correlation (r = 0.96, P less than 0.01) was observed between the activity of arginine deiminase and the growth-inhibiting activity of extracts of arginine-utilizing mycoplasmas. Br J Haematol, 1984 May, 57(1), 61 - 70 Long-term haemopoiesis in human fetal liver cell cultures; Cappellini MD et al.; Haemopoiesis in human fetal liver is almost entirely restricted to the erythroid series but when fetal liver cells were cultured under conditions established for the long-term maintenance of adult marrow haemopoiesis, a rapid switch to granulopoiesis was observed . Erythroid progenitor cells (BFU-E) rapidly disappeared, even though no humoral or cellular inhibitors of erythropoiesis could be detected, while myeloid progenitors (CFU-GM) increased in number . When the fetal liver cells were seeded onto stromal layers derived from adult marrow, in which endogenous haemopoiesis had ceased, granulopoiesis was established and maintained for more than a year, considerably longer than has previously been achieved with human haemopoietic cells. Am J Clin Pathol, 1984 May, 81(5), 586 - 90 Pemphigus and pemphigoid antibody production by pokeweed mitogen-stimulated peripheral blood mononuclear cell cultures in vitro; Fitzmaurice M et al.; Peripheral blood mononuclear cells from pemphigus vulgaris and bullous pemphigoid patients produced antiepithelial antibodies in a pokeweed mitogen-stimulated in vitro cell culture system . Antiintercellular substance (ICS) antibodies were detected in supernatants of cell cultures from four of four pemphigus vulgaris patients by indirect immunofluorescence . Similarly, antibasement membrane (BM) antibodies were detected in supernatants of cell cultures from three of four bullous pemphigoid patients . No anti-ICS or anti-BM antibodies were detected in supernatants of cell cultures of six normal controls, although antinuclear antibodies were detected occasionally . This pokeweed mitogen-stimulated in vitro cell culture system is suggested as a model to study the role of defects in immunoregulation in the pathogenesis of pemphigus vulgaris and bullous pemphigoid. Gann, 1984 May, 75(5), 436 - 41 Generation of human cytotoxic T lymphocytes against fresh autologous and allogeneic solid tumors by mixed lymphocyte tumor cell culture with T cell growth factor; Ichino Y et al.; Autologous mixed lymphocyte tumor cell culture (MLTC) induced cytotoxic lymphocytes against autologous tumor cells in 4 out of 14 donors (29%) . Further cultivation of MLTC-activated lymphocytes with T cell growth factor (TCGF) resulted not only in the propagation of cytotoxic T lymphocytes (CTL) and an increase of cytotoxicity against autologous tumors, but also in the induction of killer cells against allogeneic tumor cells . Briefly, the results of crisscross tests using fresh tumor cells from 17 donors and lymphocytes from 10 donors indicate that cultivation of MLTC-activated lymphocytes with TCGF generated killer cells against allogeneic tumor cells in most cases, but not against autologous or allogeneic phytohemagglutinin-induced lymphoblasts . These effector cells were mainly OKT3+, OKT8+, OKM1- and Leu7- . Further, the results of cold target inhibition tests undertaken in an autologous tumor killing system suggest that at least 2 different subsets, specifically cytotoxic for autologous tumors and cytotoxic for both autologous and allogeneic tumors, were developed in the CTL induced by autologous MLTC followed by cultivation with TCGF. Res Vet Sci, 1984 May, 36(3), 270 - 5 Morphological and immunocytochemical characterisation of mixed glial cell cultures derived from neonatal canine brain; Zurbriggen A et al.; Three different regions from neonatal dog brain were mechanically dissociated and cultured . At seven, 10, 14 and 19 days, cultures were harvested and studied with immunocytochemical techniques for the demonstration of astrocytes, oligodendrocytes and fibroblasts . The canine brain cultures were confluent at seven days and contained fibroblasts, both types of glial cells and several small fragments of undissociated brain tissue . Many cells were myelin associated glycoprotein (MAG) positive . In the older cultures increasing numbers of myelin basic protein (MBP) positive oligodendrocytes were also demonstrated . Double labelling studies demonstrated that MBP and MAG were produced by the same cells . MAG positive cells were therefore identified as oligodendrocytes . Most intensive oligodendroglial growth occurred in the vicinity of undissociated tissue fragments . The results were discussed in respect to glial differentiation and the question of whether the canine cultures may contain neurons. Acta Virol, 1984 May, 28(3), 185 - 90 Electron microscopic investigations of rotavirus morphogenesis in cell cultures; Schulze P et al.; The replication of simian rotavirus SA11 in GMK cells and of bovine rotavirus in calf kidney cells was studied by electron microscopy . By 30 min post-inoculation (p.i.) SA11 virus was absorbed to the cell membrane in the absence of trypsin and became engulfed into the cell; clusters of viral particles were internalized also into cytoplasmic vacuoles . At 2 hr p.i., viral particles were seen in lysosomes and 6 hr p.i., the first progeny virus was found in the cisternae of endoplasmic reticulum (ER) . Precursor virus particles budded from viroplasm into the cisternae of endoplasmic reticulum, where they became enveloped reaching a diameter of 80-90 nm . There was no difference between SA11 virus and bovine rotavirus . Although the budding of rotavirus particles is essential for acquiring of glycoproteins, the envelopment of capsids was transient . After stripping off the envelope, mature particles were formed 65-70 nm in diameter, consisting of either smooth or rough capsids . The final cytocidal stage of cell infection is described. Life Sci, 1984 Apr 30, 34(18), 1769 - 74 EGF receptor binding studies in endometrial cell culture; Sorrentino JM et al.; Guinea pig endometrial cells were isolated and maintained in cell culture . Our investigation of these cultures for the presence of epidermal growth factor (EGF) binding activity demonstrated that these cells possess EGF receptors . Upon the addition of EGF to growing cell cultures, an increase in the rate of cell growth resulted along with a higher saturation density . The binding of EGF to these cells is saturable at 40-50 ng/ml., and the receptors demonstrate "down-regulation" in response to hormonal challenge at 37 degrees C. Life Sci, 1984 Apr 23, 34(17), 1651 - 8 Stimulation of spontaneous and dopamine-inhibited prolactin release from anterior pituitary reaggregate cell cultures by angiotensin peptides; Schramme C et al.; In superfused anterior pituitary reaggregate cell cultures angiotensin II (AII) stimulated both spontaneous and dopamine-inhibited prolactin (PRL) release from subnanomolar concentrations . Angiotensin I (AI) and angiotensin III (AIII) also stimulated PRL release . The magnitude and rate of response to AI was equal to or only slightly lower than that to AII . However, the angiotensin converting enzyme (ACE) inhibitors captopril and teprotide (1 microM) completely abolished the PRL response to 0.1 nM AI and strongly reduced that to 1 nM AI . The intrinsic activity of AIII was lower than that of AII but could be enhanced by adding 2 microM of the aminopeptidase inhibitor amastatin to the superfusion medium . After withdrawal of AIII, PRL secretion rate rapidly returned to baseline levels, whereas after withdrawal of AI or AII, secretion fell to a level remaining significantly higher than basal release . The present findings indicate that stimulation of PRL release by AI is weak unless it is converted into AII by ACE and that aminopeptidase may be important in determining the magnitude and termination of the PRL response . Furthermore, the active peptides induce a different pattern of response. Neurosci Lett, 1984 Apr 20, 46(1), 25 - 9 Angiotensin stimulates beta-endorphin release from anterior pituitary gland cell cultures of rats; Kraft K et al.; The effect of exogenous and locally generated angiotensin II (ANG II) on the release of beta-endorphin (beta-END) from anterior pituitary cell cultures of rats was studied . Angiotensin I (ANG I) and ANG II stimulated the release of beta-END, the ANG I effects being inhibited by addition of the converting enzyme inhibitor captopril . Renin and angiotensinogen had no effect when given separately, but their combination increased beta-END release . Thus ANG II causes the release of beta-END, but the putative pituitary renin system cannot be stimulated by exogenous renin or angiotensinogen; converting enzyme, however, acts locally to produce biologically active ANG II from ANG I. Toxicology, 1984 Apr 2, 30(3), 227 - 41 Screening of potential reproductive toxicants by use of porcine granulosa cell cultures; Haney AF et al.; While approximately 60 000 chemicals are in widespread use with 1000 new chemicals introduced into the environment each year, the biologic effects of these agents are poorly understood . With the specific goal of testing for potential reproductive toxicity, we have established methodology for the screening of compounds in vitro by measuring effects on progesterone production by porcine granulosa cells in culture . Granulosa cells were harvested by mechanical agitation, cryopreserved, and cells with known progesterone production capacity utilized for culture . Agents to be tested were added to cultures of 10(5) cells and the media assayed for progesterone by radioimmunoassay . Estradiol suppression of progesterone production was easily demonstrated in this system and utilized as a verification of responsiveness . The pesticide o,p-DDT and its isomer p,p-DDT produced dramatic suppression of progesterone production apparently with equal potencies to estradiol . By contrast, the pesticides malathion, parathion and dieldrin and the fungicide hexachlorobenzene were without effect in this test system. Eur J Obstet Gynecol Reprod Biol, 1984 Apr, 17(1), 43 - 51 Applications of a human tumour clonogenic cell culture system in gynaecological oncology: review and personal experience; Verheijen RH et al.; Current and future clinical applications of the Human Tumour Clonogenic Cell Culture (HTC3) system are presented . Advanced gynaecological cancers, especially ovarian, endometrial and cervical carcinomas, require extensive systemic chemotherapy . The HTC3 system seems to be an adequate instrument for individualized chemosensitivity testing in order to choose proper cytostatic treatments; that is, maximum tumour cell kill with minimal side-effects . Furthermore, this system helps in assessing the response after treatment by detection of remaining clonogenic, and thus viable, tumour cells . Other clinical applications of the system include grading of the tumour and recognition of the histologic type . Thus the HTC3 provides a potential tool in diagnosis, treatment and follow-up of gynaecologic malignancies. J Submicrosc Cytol, 1984 Apr, 16(2), 227 - 35 Scanning electron microscopic study of nerve-muscle junctions in embryonic rat cell cultures; Rouche A et al.; The morphology of neuromuscular junctions between embryonic rat spinal cord and muscle cells grown in vitro has been studied by scanning electron microscopy (SEM) . Histochemical detection of acetylcholinesterase (AChE) spots and autoradiographic detection of acetylcholine receptor protein (AChR) were performed in parallel . At the scanning EM level the contacts exhibit a marked polymorphism; the nervous endings may present as a bulbous swelling, or often as a plexiform network on the surface of the myotubes . Many neuromuscular contacts seem to occur 'passing by'; in other places, neurites processes slide along myotubes without any differentiated contacts . The muscle cell surface does not look substantially modified in the contact area . The distribution of the contacts between nerve and muscle cells is at the same time convergent and divergent. J Histochem Cytochem, 1984 Apr, 32(4), 444 - 6 A simple and reliable method for the localization in cell culture of single identifiable cells for ultrastructural analyses; Morrison-Graham K et al.; A simple method for relocating single cells in monolayer cultures for subsequent morphological or ultrastructural analysis is reported . This consists of producing, on the culture dish surface, a nontoxic carbon grid that is preserved during processing for either transmission (TEM) or scanning (SEM) electron microscopy . For TEM studies these grids are readily transferred along with the cells into the embedding plastic, and thus individual grid squares containing a cell(s) of interest can be quickly located, remounted, and sectioned . These grids may be useful for ultrastructural analyses of single cells previously studied electrophysiologically or after microinjection of macromolecules. Arch Ophthalmol, 1984 Apr, 102(4), 598 - 604 Selection of therapeutic agents for intraocular proliferative disease . Cell culture evaluation; Blumenkranz MS et al.; A variety of antimetabolites, steroids, and nonsteroidal anti-inflammatory agents were tested for their ability to inhibit rabbit dermal and conjunctival fibroblast proliferation in cell culture . Doxorubicin hydrochloride and fluorouracil produced notable inhibition in concentrations of less than 1 mg/L . Meclofenamate sodium and indomethacin produced notable inhibition at concentrations of 11 and 40 mg/L, respectively . Dexamethasone sodium phosphate and triamcinolone acetonide produced inhibition at 200 and 150 mg/L, respectively, but paradoxically increased proliferation almost two-fold at concentrations ranging from 1 to 30 mg/L under identical culture conditions . Methotrexate sodium demonstrated only limited effectiveness . This assay system may be a useful approach to drug selection in the treatment of a variety of ocular proliferative disorders . Fluorouracil may prove to be of significant value in the treatment of intraocular proliferative disorders. Cancer Genet Cytogenet, 1984 Apr, 11(4), 425 - 8 Indirect stimulation of B-cell proliferation in vitro by T cells, as evidenced by cytogenetic analysis of PHA-stimulated cell cultures of B-cell lymphomas; Vermaelen K et al.; In a retrospective study of non-Hodgkin's lymphomas, the 14q+ marker was found in at least one of the samples examined from 17 patients with B-cell lymphoproliferative diseases (LPD) . In the PHA-stimulated cultures, the marker was found in each sample in 10%-100% of the cells . An indirect stimulation, as indicated by a 3H-thymidine incorporation and IG secretion, of normal B cells by a T-cell mitogen, such as PHA, has been recently documented . This phenomenon is confirmed by our chromosome analysis, which demonstrated characteristic chromosome changes in PHA-stimulated cultures of patients with B-cell malignancies and indicated that the phenomenon can be observed not only in normal B cells but also in malignant B cells. Br J Vener Dis, 1984 Apr, 60(2), 99 - 105 Pathogenic Trichomonas vaginalis cytotoxicity to cell culture monolayers; Alderete JF et al.; Exposure of monolayer cultures of human urogenital and vaginal (HeLa), human epithelial (HEp-2), normal baboon testicular (NBT), and monkey kidney (Vero) cells to live pathogenic Trichomonas vaginalis resulted in extensive disruption of monolayers . Trypan blue was taken up by all host cells released from cell monolayers, which indicated irreversible damage of these cell types by trichomonads . Time and dose related data on cytotoxicity kinetics were obtained using increasing ratios of parasites to cells . All cell types were most sensitive to trichomonads at a multiplicity of infection of one . Release of tritiated thymidine (3H-thymidine) of the deoxyribonucleic acid (DNA) of prelabelled host cells after incubation with T vaginalis corroborated that extensive cytotoxicity was caused by pathogenic trichomonads in man . Only living parasites were cytotoxic, and no trichomonal toxic products were implicated in disruption of the cell monolayer cultures . A pathogenic bovine trichomonad, Tritrichomonas foetus KV-1, produced half as much cell damage as did T vaginalis . Trichomonas tenax, a non-pathogenic member of the normal flora of the oral cavity in man, produced no measurable cytotoxicity to HeLa cells when compared with the pathogenic human trichomonads. Blood, 1984 Apr, 63(4), 912 - 6 A new method for the detection of Lesch-Nyhan heterozygotes by peripheral blood T cell culture using T cell growth factor; Kamatani N et al.; Thioguanine-resistant T lymphoblast populations were selectively amplified using T cell growth factor in the cultures of peripheral blood T cells from four Lesch-Nyhan heterozygotes . Although Lesch-Nyhan T lymphoblasts were all thioguanine-resistant, none of the cultures from 13 control subjects yielded the growth of such defective cell populations . These data provide direct evidence for the existence of a small percentage (5%-40%) of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient T cells in the heterozygotes, but not in normal individuals . Conversely, culture of the T lymphoblasts with azaserine plus hypoxanthine permitted the growth of the other part of the cell population that was enzyme positive . The low percentages of HGPRT-negative cells among T cells in heterozygotes suggest that the presence of this enzyme is beneficial for differentiation of lymphocytes of T cell linkage . Considering the ease and the reliability, culture of the peripheral T cells with thioguanine and T cell growth factor is very likely of practical use for detecting Lesch-Nyhan syndrome carriers among predisposed females. Avian Dis, 1984 Apr-Jun, 28(2), 504 - 13 Observations on the preparation and stability of infectious bronchitis virus hemagglutination antigen from virus propagated in chicken embryos and chicken kidney cell cultures; King DJ; A total of 166 infectious bronchitis virus (IBV) hemagglutination (HA) antigen preparations were made during a 30-month period from allanto-amnionic fluid (AAF) from chicken embryos inoculated with 10 different IBV strains (Mass 41, Conn 46, H52, Florida 18288, Ark 99, JMK, T, Holte, EF, SE17) . Antigens were prepared by inoculating 9- or 10-day-old embryos with 10(5.0) to 10(6.5) EID50 IBV, harvesting AAF after a 30-hour-postinoculation incubation, and phospholipase C (PLC) treatment of virus concentrated by pelleting from the AAF . Longer (48 hr) incubation times were tried, but production of H52 HA antigen was successful only from AAF harvested after 30 hours of incubation . AAF from JMK-infected embryos had lower infectivity titers and frequently yielded lower HA antigen titers than the other strains . The treatment of AAF with fluorocarbon did not enhance or diminish HA activity but did yield clearer antigens by removing extraneous material . Polyethylene glycol precipitation of virus was an acceptable alternative to pelleting virus at 39,000 X g . Inactivation of IBV with 0.1% betapropiolactone did not affect HA activity, whereas inactivation with 0.1% formalin caused a marked reduction in HA titer . Different buffer formulations including phosphate, tris, or HEPES were tried to optimize the conditions for PLC treatment of virus concentrate, but there were no apparent differences in the antigens prepared in the different buffers . The HA antigen preparations were stored and were stable at 4 C . Antigen titers of greater than or equal to 64 after storage for 20 months or longer were not uncommon . Addition of merthiolate as a preservative had no deleterious effect on HA activity . Antigen stability appeared to be enhanced by incorporating EDTA in buffer for virus pellet recovery and during enzyme treatment . Attempts to produce HA antigens from cell-culture-adapted virus propagated in chicken kidney cells were less satisfactory . An acceptable HA antigen was prepared from only two (Mass 41, SE17) of the seven strains that were tried . Virus propagation in chicken embryos is the better method of the two for IBV HA antigen production . Aside from the need to concentrate virus and treat the concentrate with PLC, there appeared to be considerable latitude in the procedures that can be used to make acceptable IBV HA antigens. Antimicrob Agents Chemother, 1984 Apr, 25(4), 522 - 3 Evaluation of lithium as an inhibitory agent of herpes simplex virus in cell cultures and during reactivation of latent infection in rabbits; Trousdale MD et al.; Lithium carbonate inhibited plaque formation of herpes simplex virus types 1 and 2 in rabbit kidney and Vero cells (50% effective dose, 435.5 to 490 micrograms/ml) . Plasma lithium levels of 67 to 134 micrograms/ml were achieved by oral therapy in rabbits . However, neither ocular virus shedding nor virus-positive trigeminal ganglia were reduced after intentional reactivation of latent herpes simplex virus infection. Biol Reprod, 1984 Apr, 30(3), 603 - 8 Effects of partially and more highly purified platelet-derived growth factor preparations on luteinizing hormone receptor induction in granulosa cell cultures; Mondschein JS et al.; The effects of partially and more highly purified platelet-derived growth factor (PDGF) preparations on luteinizing hormone (LH) receptor induction by follicle-stimulating hormone (FSH) and cholera toxin (CTX) were studied in cultured granulosa cells from immature, diethylstilbestrol-primed rats . Partially purified PDGF, prepared by carboxymethyl Sephadex C-50 chromatography (CMS-PDGF), and more highly purified PDGF, further purified by Cibacron Blue Sepharose chromatography (BS-PDGF), enhance FSH-dependent LH receptor induction in serum-free and in serum-containing medium . BS-PDGF is more potent than CMS-PDGF, and is relatively more efficacious in serum-free and less efficacious in serum-containing medium than CMS-PDGF . CMS-PDGF and BS-PDGF enhance LH receptor induction by CTX in serum-containing medium, but levels achieved are significantly less than those attained with FSH and CMS- or BS-PDGF . BS-PDGF does not enhance induction by CTX in serum-free medium . The results suggest that the action of PDGF to enhance LH receptor induction is complex and may represent actions of several components of PDGF preparations . These findings may also provide indirect evidence for a component of FSH action which is independent of cAMP. J Clin Microbiol, 1984 Apr, 19(4), 563 - 5 Rapid detection of herpes simplex virus in clinical specimens with human embryonic lung fibroblast and primary rabbit kidney cell cultures; Callihan DR et al.; The performance of a culture system for isolation of herpes simplex virus, consisting of one tube each of human embryonic lung fibroblasts and primary rabbit kidney cells, was evaluated . Cultures were incubated at 37 degrees C on a roller drum and observed daily for characteristic cytopathic effect for 5 days . During 1982, a positive isolation rate of 28.1% was seen among 3,154 specimens submitted . Cultures from genital sources were positive more frequently from males (43.8%) than from females (25.5%) . Oral lesion cultures were positive as often from males (34.6%) as from females (38.4%) . Although detection of herpes simplex virus occurred significantly earlier in rabbit kidney cells on days 1 and 2 of incubation, by day 3 the number of positive cultures was nearly the same in both cell types . By day 4 of incubation, 99.5% of t