Cell cultures

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 the positive cultures were detected . These results demonstrate that cell culture can be a rapid and sensitive method for detecting herpes simplex virus.

Chem Biol Interact, 1984 Apr, 49(1-2), 209 - 24
Characterization of DNA lesions produced by HgCl2 in cell culture systems; Cantoni O et al.; HgCl2 is extremely cytotoxic to Chinese hamster ovary (CHO) cells in culture since a 1-h exposure to a 75- microM concentration of this compound reduced cell plating efficiency to 0 and cell growth was completely inhibited at 7.5 microM . The level of HgCl2 toxicity depended upon the culture incubation medium and has previously been shown to be inversely proportional to the extracellular concentration of metal chelating amino acids such as cysteine . Thus, HgCl2 toxicity in a minimal salts/glucose maintenance medium was about 10-fold greater than the toxicity in McCoy's culture medium . The HgCl2 toxicity in the latter medium was 3-fold greater than that in alpha-MEM which contains more of the metal chelating amino acids . When cells were exposed to HgCl2 there was a rapid and pronounced induction of single strand breaks in the DNA at time intervals and concentrations that paralleled the cellular toxicity . The DNA damage was shown to be true single strand breaks and not alkaline sensitive sites or double strand breaks by a variety of techniques . Consistent with the toxicity of HgCl2, the DNA damage under an equivalent exposure situation was more pronounced in the salts/glucose than in the McCoy's medium and more striking in the latter medium than in alpha-MEM . Most of the single strand breaks occurred within 1 h of exposure to the metal . We believe that the DNA damage caused by HgCl2 leads to cell death because the DNA single strand breaks are not readily repaired . DNA repair activity measured by CsCl density gradient techniques was elevated above the untreated levels at HgCl2 concentrations that produced little measurable binding of the metal to DNA or few single strand breaks assessed by the alkaline elution procedure . DNA repair activity decreased at HgCl2 concentrations that produced measurable DNA binding and single strand breaks . These irreversible interactions of HgCl2 with DNA may be responsible for its cytotoxic action in cells.

Cell Immunol, 1984 Apr 1, 84(2), 299 - 310
Spontaneous proliferation in unfractionated spleen cell cultures: autologous mixed-lymphocyte reactions (AMLR) which can be differentially regulated by prostaglandins and lymphokines; Zuberi RI et al.; The present studies were undertaken to define the contribution of the autologous or syngeneic mixed-leukocyte reactions (AMLR/SMLR) to the cellular proliferation observed in unfractionated spleen cell cultures . Proliferation was studied in whole, untreated 6-day murine spleen cell cultures supplemented with syngeneic serum . These cultures exhibited relatively low but significant levels of cellular proliferation as measured by uptake of radioactive thymidine ({3H}TdR) . Treatment of spleen cells with monoclonal anti-Thy 1.2 antibody and complement before culture, the addition of specific anti-I-A monoclonal antibodies to the cultures or removal of Ia+ adherent cells before initiation of culture all inhibited the proliferative response significantly . Thus, the autologous proliferation of untreated and unfractionated spleen cells manifests the main characteristics of the AMLR/SMLR, namely, its dependence on T (responder) and Ia+ (stimulator) cells and specific inhibition by anti-I-A antibodies . A marked augmentation in cellular proliferation was observed in unfractionated spleen cell cultures treated for the initial 24 hr of culture with 5 X 10(-6) M indomethacin, an inhibitor of prostaglandin synthesis . Conversely, the addition of 7 X 10(-9) M prostaglandin E1 (PGE1) to these cultures depressed cellular proliferation . This suppression of autologous splenic cell proliferation induced by PGE1 could be partially reversed by the addition of concanavalin A-induced lymphokine (LK) preparations early in the culture . These findings indicate that (a) the proliferation of unfractionated spleen cell cultures occurring in the absence of exogenous stimulatory signals is due largely to an ongoing AMLR, and (b) biologically active mediators with opposing influences, namely, prostaglandins and immunostimulatory LK, participate in the regulation of the AMLR.

J Immunol, 1984 Apr, 132(4), 2135 - 42
Induction of NKCF-like activity in mixed lymphocyte-tumor cell culture: direct involvement of mycoplasma infection of tumor cells; Wayner EA et al.; Co-culture of CBA/J spleen cells and certain lines of YAC-1 stimulators resulted in the appearance of NKCF-like activity in 24- to 48-hr supernatants . Numerous other in vitro cell lines were effective stimulators of this splenic cytotoxic factor (SCF) . The cells participating in SCF production were absent from normal thymocytes and were present in BALB/c nu/nu spleen, were nonadherent, asialo GM1+, and bore low levels of Thy-1.2 . SCF could mediate lysis of certain NK-sensitive tumor targets in an 18-hr 51Cr-release assay . However, the induction of SCF was not correlated with the ability of a particular cell line to be lysed by NK cells, but showed an absolute correlation with the presence of mycoplasma contamination in cultured tumor cell lines . Mycoplasma negative cell lines, including an uninfected but NK-sensitive subline of YAC-1, were unable to induce SCF . Decontamination of mycoplasma-infected lines with antibiotics or by passage through syngeneic mice abrogated the ability of infected tumor cells to stimulate SCF . The ability to induce SCF could be restored by reinfection with mycoplasma . Tumor cell-free supernatants from contaminated cultures were mitogenic for CBA spleen cells and could themselves induce SCF activity in spleen cell supernatants . SCF production and the agent responsible could be removed by passing such supernatants through 0.1-micron filters . The organism apparently responsible for SCF induction from CBA spleen cells was typed and found to be Mycoplasma orale, a nonfermentative, arginine-dependent, common tissue culture contaminant . About 50 to 60% of SCF activity could be removed by 0.1-micron filters, suggesting that SCF is composed of two components: mycoplasma organisms themselves and a soluble cytotoxic factor produced in response to mycoplasma.

J Histochem Cytochem, 1984 Apr, 32(4), 347 - 57
Proteoglycans in arterial smooth muscle cell cultures: an ultrastructural histochemical analysis; Chen K et al.; The extracellular matrix in cultures of arterial smooth muscle cells has been examined by ultrastructural histochemistry using each of the following cationic dyes: ruthenium red, Alcian blue, acridine orange, and safranin O . All dyes exhibited an affinity for a structural component that was either preserved as a granule with ruthenium red or Alcian blue, or as an extended filament or bottlebrush structure with acridine orange or safranin O . Both granules and filaments were removed when the cultures were pretreated with chondroitinase ABC, an enzyme that degrades the glycosaminoglycan moiety of some proteoglycans . These structural components of the extracellular matrix were not observed when cultures were prepared in the absence of the cationic dyes . Labeling experiments (35S-sulfate) revealed that approximately 40% of the total labeled proteoglycans were lost during routine processing for electron microscopy (i.e., fixation through dehydration) . Inclusion of any one of the cationic dyes during fixation reduced the losses to less than 1% . The extended filamentous structure preserved by safranin O and acridine orange resembled the structure of purified proteoglycans prepared from the same cultures and spread on cytochrome c monolayer films . These observations suggest that proteoglycans exist as extended bottlebrush structures within the extracellular matrix, and support the interpretation that the granular deposits observed in the ruthenium red and Alcian blue preparations most likely represent individual proteoglycan monomers that have undergone molecular collapse during processing . In addition, the dyes also exhibited an affinity for chords of fine fibrils that contained small granules and/or filaments . Both the fibrillar material and the associated granular and filamentous structures enmeshed in the fibrils resisted digestion with chondroitinase ABC.

Boll Soc Ital Biol Sper, 1984 Mar 30, 60(3), 473 - 8
{Endothelial cell culture as a model for the study of wound healing}; Mancianti ML et al.; Endothelial cells culture can be considered a reliable method for investigating about granulation tissue production in wound healing and for evaluating the pharmacological action of some chemicals on granulation tissue development . Endothelial cells have been obtained from human umbilical cords after trypsin treatment and their endothelial origin has been demonstrated by light microscopy, by immunofluorescence against factor VIII associated protein and by the platelet adhesion assay . The influence of fibronectin as substratum and of hyaluronic acid as soluble factor on adhesion and growth of endothelial cells has been investigated . Both these substances, but especially hyaluronate, determine a better attachment and an increase in the growth rate when compared with control cultures plated on plastic substratum and without hyaluronic acid in the culture medium.

FEBS Lett, 1984 Mar 26, 168(2), 299 - 302
Proinsulin-like material in mouse foetal brain cell cultures; Birch NP et al.; Two main forms of immunoreactive insulin have been identified in cultures of foetal mouse brain using HPLC and gel filtration . The major component which resembled proinsulin was converted by trypsin to the minor form which was similar to authentic pancreatic insulin in chromatographic behaviour . Both components showed immunological properties comparable to insulin and proinsulin including sensitivity of the former to reduction and alkylation.

J Biol Chem, 1984 Mar 25, 259(6), 3818 - 24
Effect of p-nitrophenyl-beta-D-xyloside on proteoglycan synthesis and extracellular matrix formation by bovine corneal endothelial cell cultures; Robinson J et al.; The effect of p-nitrophenyl-beta-D-xyloside on proteoglycan synthesis and extracellular matrix (ECM) formation by cultured bovine corneal endothelial (BCE) cells was investigated . BCE cells actively proliferating on plastic dishes produced in the absence of xyloside an ECM containing various proteoglycans . Heparan sulfate was the main 35S-labeled glycosaminoglycan component (83%) . Dermatan sulfate (14%) and chondroitin sulfate (3%) were also present . Exposure of actively proliferating BCE cells to xyloside totally inhibited synthesis of proteoglycans containing dermatan sulfate or chondroitin sulfate and caused an 86% inhibition of heparan sulfate proteoglycan synthesis . The heparan sulfate proteoglycans that were extracted from the ECM produced by BCE cells exposed to xyloside had a smaller size and a reduced charge density compared to their counterparts extracted from the ECM of cultures not exposed to xyloside . In contrast to the inhibitory effect of the xyloside on proteoglycan synthesis, exposure of actively proliferating BCE cells to xyloside stimulated synthesis of free chondroitin sulfate and heparan sulfate chains . All of the xyloside-initiated glycosaminoglycan chains were secreted into the culture medium . The proteoglycan-depleted matrices produced by BCE cells exposed to xyloside were used to study the effect of these matrices on proteoglycan synthesis by BCE cells . BCE cells growing on proteoglycan-depleted ECM showed a considerable increase in the rate of proteoglycan synthesis compared to BCE cells growing on normal ECM . Moreover, the pattern of glycosaminoglycan synthesis by BCE cells growing on proteoglycan-depleted ECM was changed to one which resembled that of BCE cells actively proliferating on plastic dishes . It is postulated that BCE cells are able to recognize when an ECM is depleted of proteoglycan and to respond to it by increasing their rate of proteoglycan synthesis and incorporation into the ECM.

Brain Res, 1984 Mar 12, 295(1), 184 - 9
Neuron generation in dissociated cell cultures from fetal rat cerebral cortex; Kriegstein A et al.; Examination of dissociated cell cultures of rat fetal cortex revealed that mature cultures contained more neurons (identified by phase contrast morphology and tetanus toxin staining) than the number of cells initially adhering to the coverslips . Labeling with {3H}thymidine confirmed that neuronal precursors were undergoing mitosis throughout at least the first 10 days in vitro.

J Cell Sci, 1984 Mar, 66, 343 - 51
Primary cell cultures from human embryonic corneas; Hyldahl L; This study shows that primary cell cultures can be established from corneas obtained from 8-week-old human embryos . Such corneas, even though they were obtained at an early stage of the embryonic development, are completely differentiated into three layers as in the adult cornea . Both corneal endothelial cells and stromal cells were found to attach and proliferate in vitro . Furthermore, it was possible to obtain pure uncontaminated cultures of stromal cells after five passages in vitro . It was also possible, using feeder cells as the substratum, to obtain pure endothelial cell cultures from embryonic corneas.

Prenat Diagn, 1984 Mar-Apr, 4(2), 99 - 108
Mosaicism or pseudomosaicism: the problem of hypermodal cells in amniotic fluid cell culture; Zhang YJ et al.; A series of 2029 consecutive amniotic fluid specimens studied for prenatal genetic diagnosis were reviewed and reassessed so as to evaluate the frequency and clinical significance of hypermodal cells in amniotic fluid cell cultures . Hypermodal cells were defined as those with more than 46 chromosomes, and were characterized by an additional structurally normal or structurally abnormal chromosome . Of 2029 specimens, 47 (2.31 per cent) contained a total of 167 hypermodal cells . True fetal mosaicism was detected in three cases (0.14 per cent) . All had hypermodal cells in more than one culture flask or colony which contained the same aberrant chromosome complement . In all but one case the babies were normal when only one cell was hypermodal, or when several cells were hypermodal but present in only one colony or one culture vessel . One case had an extra No . 20 chromosome in one cell . Although the child had multiple anomalies, they were not characteristic of trisomy 20, and subsequent chromosomal study on the baby postnatally revealed a 46,XX karyotype . The in situ coverslip technique is recommended as the preferred method for prenatal diagnosis, and it is useful as an aid in differentiating true mosaicism from pseudomosaicism.

Prenat Diagn, 1984 Mar-Apr, 4(2), 151 - 3
Maternal cell contamination of amniotic fluid cell cultures from two consecutive pregnancies complicated by fibroids; Benn PA et al.; The detection of maternal cells in amniocyte cultures is thought to be due to the outgrowth of cells from small fragments of maternal tissue removed by the amniocentesis needle . An unusual case is reported in which maternal cell contamination (MCC) was found in the cell cultures from a woman in two different amniocenteses from two consecutive pregnancies . Both pregnancies were complicated by the presence of fibroids and the fibroid tissue may have been the source of the maternal cells . A history of an amniocentesis in which there was MCC of cell cultures, or the detection of fibroids, may pose an additional risk for MCC attributable misdiagnosis in prenatal genetic studies.

Tohoku J Exp Med, 1984 Mar, 142(3), 349 - 50
Decrease in saturation density of mammalian carcinoma cell culture during exposure to bestatin, a clinically applicable agent; Okuyama S et al.; Bestatin, one of the small molecular products of Streptomyces olivoreticuli , and a potential immunostimulator, was shown to decrease the saturation density of murine carcinoma cell culture . The finding may indicate a possibility of direct action of bestatin upon cancer cells presumably resulting in cancer cell redifferention .

Ann Microbiol (Paris), 1984 Mar-Apr, 135A(2), 249 - 54
Rabbit lens cell cultures in the characterization of Spiroplasma mirum pathogenicity; McGarrity GJ et al.; Spiroplasma mirum grew to high titres, 10(8) colour-changing units per ml of supernatant medium, and produced cytopathology which consisted of vacuolization, granulation and polynucleation . S . mirum did not grow in cell culture medium (Dulbecco's MEM+10% foetal bovine serum), thereby indicating the need for cultured cells or a cell culture product . Growth was also obtained from cell-free supernatants from AG-4676 cultures . S . mirum propagated in AG-4676 produced cataracts and death in suckling Wistar rats.

Eur J Biochem, 1984 Mar 1, 139(2), 303 - 13
Synthesis, modification and structural binding of heat-shock proteins in tomato cell cultures; Nover L et al.; Synthesis of about 30 acidic and 18 basic heat-shock proteins (hsps) is induced in suspension cultures of tomato (Lycopersicon peruvianum) if subjected to supraoptimal temperature conditions (35-40 degrees C) . A characteristic aspect of the plant heat-shock response is the formation of cytoplasmic granular aggregates, heat-shock granules, containing distinct heat-shock proteins as major structural components and, in addition, several hitherto undetected minor acidic and basic heat-shock proteins . Structural binding of heat-shock proteins, i.e . assembly of heat-shock granules, is dependent on the persistance of supraoptimal temperature conditions . Despite the ongoing synthesis also at 25 degrees C, e.g . in pulse heat-shocked cultures, these proteins are accumulated exclusively in soluble form . Individual heat-shock proteins are characterized by their kinetics of synthesis and are classified by their compartmentation behaviour into class A proteins (exclusively found in soluble form, e.g . hsps 95 and 80), class B proteins (5-10% bound to heat-shock granules, e.g . hsps 70, 68), class C proteins (30-80% bound to heat-shock granules, e.g . hsps 21, 17, 15) and class D proteins, which are minor heat-shock proteins only detected in structure-bound form . Major representatives are modified proteins, i.e . hsps 95, 80, 70 and 68 are phosphorylated and hsps 80, 74, 70 and 17 are methylated proteins (numbers 70, 80 etc . refer to 10(-3) Mr) . Under heat-shock conditions synthesis of the proteins detected in control cells (25 degrees C proteins) exhibits two patterns . There are proteins with continued and proteins with discontinued synthesis . Synthesis of most of the latter proteins is resumed very rapidly after shift-down to 25 degrees C, even in the presence of actinomycin D . We conclude that reversible segregation of distinct mRNA species from the translation apparatus contributes to the heat-shock-specific pattern of protein synthesis in plants also.

Brain Res Bull, 1984 Mar, 12(3), 307 - 13
Vasopressin in reaggregated cell cultures of the developing hypothalamus; Notter MF et al.; A microsystem for rotation-mediated aggregate cell culture studies has been devised to examine vasopressin (VP) biosynthesis of developing rat hypothalamus . Trypsin-dispersed hypothalamic tissue was placed into 24 well tissue culture dishes and VP content of culture medium and cells was measured over time by a radioimmunoassay . Reaggregates formed within 4 hr when rotated at 70 rpm in a humid CO2 incubator . Nineteen days post coitus (dpc) hypothalamic reaggregates had 336 pg VP/10(6) cells while the medium showed 260 pg VP/ml after four days . Measurable VP was seen in fetal tissue after ten days while comparable amounts of VP were present in one day neonatal hypothalamus over this same period . Morphological examination of reaggregates indicated the presence of viable cells throughout the cell mass after ten days of culture . Co-cultivation studies with dispersed posterior pituitary indicated that reaggregates from one day neonate hypothalamus had significantly increased VP levels when co-cultured with one day neonatal posterior pituitary; however, this effect was not seen with 19 dpc co-cultures . These data demonstrate that development of neurosecretory activity of discrete regions of the hypothalamus can be examined early in vitro in a reaggregate cell culture system.

Int J Radiat Oncol Biol Phys, 1984 Mar, 10(3), 375 - 8
Radiation induced secretion of surfactant from cell cultures of type II pneumocytes: an in vitro model of radiation toxicity; Shapiro DL et al.; The pathogenesis of pneumonitis and fibrosis secondary to lung irradiation is incompletely understood . The role of the type II alveolar epithelial pneumocyte in these processes has been under investigation . The type II pneumocyte has been shown in vivo to respond to radiation induced injury with release of pulmonary surfactant . The effect of irradiation on cell cultures of type II pneumocytes was studied to determine if this could be reproduced in vitro . Type II pneumocytes were found to release surfactant material with a threshold of radiation dose between 1000 and 1500 rad . This is similar to the dosage range over which the same effect has been demonstrated in vivo . Experimental results support the concept that the release of surfactant is not due to either cell disruption or non-specific release of phospholipid from cell membranes . Irradiation appears to trigger membrane receptor mediated surfactant release . In addition, irradiation abolishes the ability of cells to subsequently respond to a physiologic agonist, suggesting radiation induced damage to the secretory mechanism . These studies establish that surfactant release in response to irradiation in vivo is a direct effect on type II pneumocytes . Cell cultures of type II pneumocytes can serve as a laboratory model of lung cell radiation toxicity.

Cell Struct Funct, 1984 Mar, 9(1), 25 - 35
Phenylthiourea enhances Cu cytotoxicity in cell cultures: its mode of action; Masuda A et al.; PTU markedly enhanced the cytotoxic effects of CuCl2 on chick embryonic PECs cultured in vitro . We investigated this newly discovered effect of PTU and its analogues in relation to the toxic effects of Cu ion . Most PECs maintained in medium containing 0.5 mM PTU were lysed within 4 h by the addition of 0.1 mM CuCl2, which addition killed no PECs in the absence of PTU . The effect of PTU was not specific to PECs . All the cell lines tested, KB, N-18, N-115 and B-16, reacted against exogenous Cu in the presence of PTU as did the PECs . Analogues of PTU had effects on PECs similar to those on PTU in the presence of Cu ion . ANTU had a greater effect than PTU . MTU and TU had less effect than PTU . PTU did not affect the cytolysis induced by the addition of the divalent cations Mn, Co and Zn . About 6-fold the 64Cu-uptake by PECs was scored in the presence of PTU . The relation between this cytotoxic-enhancing effect and other biological activities of PTU are discussed.

Exp Cell Res, 1984 Mar, 151(1), 134 - 47
Effects of D-valine on pulmonary artery endothelial cell morphology and function in cell culture; Picciano PT et al.; The effects of D-valine on the cell culture of bovine pulmonary artery endothelial cells were studied using D-valine-modified Minimal Essential Medium (MEM) . D-Valine-treated cultures (46-920 mg/l) were compared with replicate cells grown in L-valine (46 mg/l)-MEM . All media were supplemented with 15% fetal bovine serum (FBS) . Endothelial cells were grown for 14 passages with split ratios varying from 1:3 to 1:6 . Unlike cells grown in L-valine MEM, cells grown in D-valine MEM did not become contaminated by the growth fibroblasts in primary cultures . D-Valine-treated cells were found to grow in cobblestone array, exhibit contact inhibition and strongly express factor-VIII antigen (F-VIII) . D-Valine-grown cells produced PGI2 in greater proportion to PGE2, both constitutively and when stimulated by bradykinin, on comparison with cells grown in L-valine . In addition, cells grown in L-valine, although able to express factor VIII, were not comparable to D-valine cells with respect to other parameters assayed (morphology and growth as a monolayer).

J Neurosci Methods, 1984 Mar, 10(3), 229 - 35
A simple method for the separation of retinal sublayers from the entire retina with special reference to application for cell culture; Shiosaka S et al.; We developed a simple method for the separation of rat retinal sublayers . The rat retina was carefully removed from the sclera and treated in trypsin solution . After this treatment, the retinal sublayers could be easily separated by using Millipore filter paper into an outer nuclear layer, an inner nuclear layer, an inner plexiform layer, and a ganglion cell layer + nerve fiber layer . The viable cells of each of the sublayers could be cultured on a poly-L-lysine coated chamber for at least several weeks in Eagle's minimum essential medium supplemented with calf serum and glucose.

Tsitologiia, 1984 Mar, 26(3), 340 - 3
{Immunomorphological study of fibronectin localization in a primary human aorta cell culture}; Andreeva ER et al.; Using indirect immunofluorescence, fibronectin localization in primary culture of human aortic cells was studied . Intimal and medical cultures obtained from normal and atherosclerotic zones were investigated . Four morphological cell types, previously described elsewhere, were found in these cultures . All the cell types were able to synthesise fibronectin and to form the extracellular matrix in culture . But the pattern and quantity of this matrix differs in different cell types . Elongate cells formed a fine network of fibronectin on their surface, but stellate, polygonal and asymmetric cells contained only several fibers of extracellular fibronectin . There was difference in the fibronectin localization pattern of the surface of cells, isolated from intima and media as well as from normal and atherosclerotic zones.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1984 Mar, 256(3), 356 - 66
Toxoplasmacidal activity of Obioactin derived from hydrolyzed Toxoplasma immune bovine serum in heterologous cell cultures; Suzuki N et al.; Serum samples were collected from Toxoplasma-infected cattle after challenge and 24 h after Toxoplasma lysate injection . They were hydrolyzed with proteinase, HCl and NaOH . Then substances 3,000 to 5,000 in molecular weight were obtained from fractionation by chromatography . Native serum was collected from cattle immune only to Toxoplasma was affected in homologous bovine monocytes . Of the hydrolyzed fractions, one termed Obioactin inhibited the growth of Toxoplasma in heterologous cells, such as mouse macrophages and kidney cells, canine monocytes, and human heart and brain cells . Its toxoplasmacidal activity was presumed to be derived from its non-specific cell potentiating effects . In mice immunized to Toxoplasma peritoneal macrophages released about 10 times as much superoxide (O-2) and hydrogen peroxide (H2O2) as glycogen-elicited macrophages in normal mice . The release of O-2 and H2O2 from glycogen-elicited and resident macrophages in normal mice showed a tendency to increase up to 72 h of incubation with 0.5% Obioactin . No changes in the O-2 and H2O2 release were found in kidney cells during the period of incubation with Obioactin . In those immunized mice activated macrophages and kidney cells inhibited the intracellular multiplication of Toxoplasma parasites remarkably . It was suggested that the increase in O-2 and H2O2 generating activity in the macrophages might be associated with the enhancement of antitoxoplasmatic activity . The mechanism of inhibition of Obioactin , however, might be different from that of mouse macrophages on the multiplication of Toxoplasma in mouse kidney cells.

Neuroendocrinology, 1984 Mar, 38(3), 176 - 81
Melatonin inhibits beta-adrenoceptor-stimulated cyclic AMP accumulation in rat astroglial cell cultures; Vacas MI et al.; We investigated whether astroglial cells are a site of action for the effect of melatonin on brain cyclic AMP content . Rat astroglial cell subcultures, identified according to morphological and immunochemical criteria, were used . Addition of melatonin to the cultures did not result in changes of cyclic AMP content . However, melatonin at 0.1-1 microM concentrations was able to impair the cyclic AMP increase elicited by 1 microM norepinephrine or isoproterenol in astroglial cultures . This melatonin effect was also shared by its biologically active analogues 5-methoxytryptophol and 6-chloromelatonin . Serotonin was only effective at a 100-fold greater concentration, while the biologically inactive melatonin metabolite 6-hydroxymelatonin was devoid of activity at any concentration used . These results suggest that methoxyindoles modulate negatively beta-adrenoceptor-induced cyclic AMP accumulation in cultured rat astroglial cells.

Br J Dermatol, 1984 Mar, 110(3), 257 - 63
Dynamics of Langerhans cells in genetically defined murine epidermal cell culture; Cohen RL et al.; Unlike keratinocytes, Langerhans cells express both surface ATPase activity and Ia (HLA-DR) antigens . A well-characterized in vitro system containing Langerhans cells would be of great use in elucidating their functions . Thus, epidermal cell cultures derived from neonatal Balb/c mice were examined for the presence of Langerhans cells . Twenty-four hours after initiation of culture, ATPase- and Ia-positive cells were seen to be associated with cell aggregates . By day 3, Langerhans cells migrated on to the substratum and, as the cultures matured and stratified, were seen both in groups and as single cells for the duration of the cultures (day 14) . During culture, although the total number of cells increased, the percentage of cells expressing Ia antigen and ATPase activity remained constant, suggesting that Langerhans cells increase in number during cell culture . Such a situation could arise from actual division of Langerhans cells during culture or from latent expression of Ia antigen and ATPase activity by pre-existing cells . This is the first study of the dynamics of Langerhans cells in a cell culture system and shows that Langerhans cells are present throughout the lifespan of the cultures.

Tsitologiia, 1984 Mar, 26(3), 299 - 306
{Morphological and metabolic changes in a HeLa cell culture exposed to fluorine}; Strochkova LS et al.; Fluoride in subtoxic (1.5 mcg/ml) and toxic (12 mcg/ml) concentrations induced characteristic changes in the mitotic regime of HeLa cell culture . Fluctuations of the mitotic index, variations in the duration of division phases and in the spectrum of pathologic mitosis were noticed . It was shown that fluoride inhibited the genome reduplication . In lower concentrations the halogen produced the most expressed effect on transcription and translation, which corresponds to a so called paradoxial action of this trace element . Definite shifts in the ultrastructure of the cell surface and cell organelles were registered, which confirms a connection between morphological and functional changes in the culture during incubation with fluoride.

Onderstepoort J Vet Res, 1984 Mar, 51(1), 89 - 90
Acid production by flavivirus-infected VERO and CER cell cultures; Barnard BJ; VERO and CER cell cultures infected with flaviviruses produce more acid than non-infected control cultures . Acid production is dependent on the presence of glucose . This phenomenon can be utilized for the titration of flaviviruses.

Exp Cell Res, 1984 Feb, 150(2), 338 - 46
Limb bud chondrogenesis in cell culture, with particular reference to serum concentration in the culture medium; Hattori T et al.; When limb bud mesodermal cells of stages 23-24 chick embryos were plated at low cell density (2 X 10(5) cells/cm2) and cultured in medium containing 10% fetal calf serum (FCS) (serum-rich medium), all cells became fibroblastic and no chondrocyte differentiation occurred in the culture . However, when cells of the same origin were cultured in a medium containing only 0.1% FCS (serum-poor medium), almost all the cells formed aggregates which developed further to form cartilage nodules . The loss of chondrogenic activity in serum-rich medium culture was irreversible: cultivation of the limb bud cells in serum-rich medium for 12 h abolished chondrogenic activity completely and these cells could not resume activity on re-cultivation in serum-poor medium . Calf, horse and chick serum at a concentration of 10% also induced the loss of chondrogenic activity in low cell density culture . Failure of chondrogenesis in serum-rich medium culture seemed to be due to the commitment of bipotential limb bud mesodermal cells to fibroblastic cells rather than to selective detachment of pre-committed chondroblasts.

Can J Biochem Cell Biol, 1984 Feb-Mar, 62(2-3), 143 - 9
Effects of erythropoietin on uridine metabolism in cell cultures of fetal calf liver; Congote LF; The effect of sheep plasma erythropoietin preparations on the incorporation of {5(-3)H}uridine into erythroid cells has been studied using cells of fetal calf liver cultured in serum-free medium . The cells were incubated for 20 h with the hormone, followed by a 1-h incubation with {3H}uridine . Erythropoietin caused a 2.5-fold increase in the incorporation of uridine into cold trichloroacetic-acid-insoluble cell extracts and a 70% increase in the incorporation of uridine into the cold acid-soluble cell extracts . The phosphorylated metabolites of labeled uridine present in the cold acid-soluble fraction were analyzed by anion-exchange high performance liquid chromatography (HPLC) . Erythropoietin increased the amounts of labeled UDP and UTP per cell . However, the specific activity of UTP and the labeled amounts of UDP-glucose in erythropoietin-treated cells were not significantly different from those in control cell cultures . After chromatography of the crude erythropoietin preparations on reversed-phase and gel-permeation HPLC, there was a perfect coincidence of the fractions stimulating uridine incorporation into acid-soluble and acid-insoluble cell extracts . The protein fractions from crude erythropoietin which stimulated uridine incorporation after purification by reversed-phased HPLC were also able to stimulate globin chain synthesis in fetal calf liver cells . These experiments suggest that the multiple effects on uridine metabolism described above are due to erythropoietin, rather than other proteins contaminating the crude hormone preparations.

Exp Parasitol, 1984 Feb, 57(1), 81 - 5
Toxoplasma gondii: growth in ovine fetal kidney cell cultures; Chang GN et al.; Serial, in vitro passage of Toxoplasma gondii (Rh strain) was successfully performed in a cell line derived from ovine fetal kidney cells . Invasion of this parasite into the kidney cells was easily discernible 1 hr after inoculation . The subsequent proliferation of the parasite was followed in the cytoplasm of the kidney cells . Very active endodyogeny and rosette formations, as many as 13 in a cell, were observed in the cytoplasm of the kidney cells 48 hr postinoculation . After 96 hr of incubation, the parasite population had increased about 132-fold . The virulence of T . gondii against mice was not attenuated after 2 years of in vitro growth which represented 100 serial passages through the kidney cell cultures . Although no "exotoxin" was produced by T . gondii grown in vitro, a Toxoplasma sp . agar gel immunodiffusion test antigen was isolated from the cell-free supernatant fluid of the kidney cell cultures which was identical to an antigen isolated from "toxogenic" organisms harvested from infected mice.

Am J Physiol, 1984 Feb, 246(2 Pt 1), E145 - 52
LH responses to LHRH in perifused pituitary cell culture: sex differences in the rat; Loughlin JS et al.; The present experiments were designed to study male and female luteinizing hormone (LH) secretory patterns following pulsatile or continuous LH-releasing hormone (LHRH) administration using a perifused dispersed rat anterior pituitary cell culture system . In male cells, consistent LH responses were elicited by hourly LHRH pulses (30 pmol), whereas in the female cells (proestrus or diestrus I), a statistically significant (P less than or equal to 0.01) decrease in the LH secretion occurred with successive LHRH pulses . Proestrous cells secreted significantly (P less than or equal to 0.01) more LH than diestrous cells during the first 24 h but not after 48 h in culture . When exposed to a 6-h continuous infusion of LHRH (10 nM), male cells released LH in a single phase and female cells secreted LH in a biphasic pattern . These data suggest that significant sex differences exist in the rat LH secretory pattern elicited by LHRH in vitro.

J Med Microbiol, 1984 Feb, 17(1), 23 - 30
Detection of immunoperoxidase labelled mycoplasmas in cell culture by light microscopy and electronmicroscopy; Chasey D et al.; A McCoy cell line persistently infected with Mycoplasma orale was examined by light microscopy and electronmicroscopy after specific labelling with a direct immunoperoxidase conjugate . Mycoplasmas were readily detected in monolayer cultures in bright-field conditions and these were related to labelled organisms observed by electronmicroscopy in thin sections of similar cells . The specificity of the conjugate for M . orale was demonstrated by blocking with unconjugated antiserum, and by its inability to detect M . bovirhinis . Non-specific background labelling was consistently absent.

J Virol Methods, 1984 Feb, 8(1-2), 47 - 55
Parameters for the characterization of antiviral compounds in cell culture experiments; Horvath S; The antiviral activity of seven compounds against the replication of herpes simplex and rubella viruses in cell culture was examined using different parameters as follows: cytotoxicity (CT) of compounds giving 50 and 0% (CT50, CT0) reduction in cell growth; the slope of the curve of CT; decrease in virus infectivity titer in the presence of CT50 and CT0 concentrations of the compound (D); maximum amount of the compound giving zero inhibition of virus replication (I0); therapeutic index (TI); further decrease in the virus titer after withdrawal of the compound from the medium (DS); and the 'shadow' showing the degree of virus replication after withdrawal of the compound from the medium . The higher the values of D, DS, TI, and the lower the values of I0 and the 'shadow', the more valuable the antiviral compound . These parameters characterize the CT, the potency and the specificity of the antiviral compound and they may be used in antiviral tests to select new and more specific antiviral agents.

Brain Res, 1984 Feb, 314(2), 159 - 65
Synapse repression in cell culture; Schaffner AE et al.; The phenomenon of synapse repression was investigated in cell culture . It was found that the number of synapses between ciliary ganglion neurons and myotubes was reduced by the concomitant presence of ventral spinal cord neurons . Neurons from dorsal root ganglia, dorsal spinal cord or cortex did not cause a reduction . Conditioned medium from ventral spinal cord-myotube co-cultures was without effect suggesting the absence of soluble 'repression factors' . The addition of D-tubocurarine partially reversed the repression, indicating that the phenomenon has both activity-dependent and activity-independent aspects.

Cardiovasc Res, 1984 Feb, 18(2), 99 - 106
Release of compartment-specific enzymes from neonatal rat heart cell cultures during anoxia and reoxygenation; Altona JC et al.; The effects of anoxia and substrate deprivation on the extent of the release of several intracellular marker enzymes and DNA were studied using monolayer cultures of neonatal rat heart cells . After varying periods of anoxia, in a number of experiments followed by 1 h of reoxygenation and substrate repletion, the cells were analysed for cytoplasmic, lysosomal, and mitochondrial enzymes, and for nuclear DNA . Cellular enzyme activity and DNA content at any time during incubation were compared with the remaining activity of alpha-hydroxybutyrate dehydrogenase (HBDH), a cytoplasmic enzyme . The study demonstrates that the ultimate extent of enzyme depletion induced by anoxia with or without reoxygenation, is high (greater than 80%) for cytoplasmic enzymes, is intermediate for sarcolemmal and lysosomal enzymes, and enzyme associated with the mitochondrial outer membrane (about 50%) and small (less than 10%) for enzymes associated with the mitochondrial inner membrane, and for nuclear DNA.

Proc Soc Exp Biol Med, 1984 Feb, 175(2), 179 - 84
Effects of various secretagogues on gonadotropin release by rat anterior pituitary cell cultures; Grotjan HE Jr et al.; Rat anterior pituitary cell cultures were utilized to compare LH and FSH release induced by LHRH, cAMP derivatives, cGMP derivatives, the calcium ionophore A23187, and elevated K+ . Of the cyclic nucleotide derivatives tested, only 8-Br-cAMP released significant quantities of both LH and FSH . A23187 and elevated K+ released significant quantities of LH and FSH but supramaximal concentrations of these stimuli liberated only approximately 50% of the gonadotropins released by LHRH . Thus, none of the secretagogues tested was as efficacious as LHRH in releasing either LH or FSH.

J Clin Endocrinol Metab, 1984 Feb, 58(2), 344 - 52
Characterization and regulation of epidermal growth factor receptors in human placental cell cultures; Lai WH et al.; We have confirmed that cultured human placental cells rapidly release hCG . Preincubation with epidermal growth factor (EGF) for 24 h significantly increased the amount of hCG released and also increased human placental lactogen release by these cells . To better understand the mechanisms of action of EGF on the feto-placental unit, we studied EGF receptor binding and regulation by examining the characteristics and specificity of EGF receptors in human placental syncytiotrophoblast cultures . Maximal {125I}EGF binding occurred at pH 7.5 and 4 C, and exhibited a high degree of specificity . In the presence or absence of Bacitracin at 4 C, specific binding values were similar, and labeled EGF was physically intact, as assessed by trichloroacetic acid precipitation or rebinding to human placental membranes . The percent specific binding was proportional to cell and ligand concentrations and was significantly increased in term (52.9 +/- 1.2%; n = 11) compared to early gestation placental cells (22.7 +/- 3.4%; n = 7; P less than 0.001) . Both term and midterm EGF displacement curves generated curvilinear Scatchard plots, suggesting receptor heterogeneity . Pretreatment of cells with EGF resulted in a dose and time-dependent decrease in specific binding, which was maximal (80%) at 200 ng/ml EGF . This loss of binding was due to decreases in the number of both high and low affinity receptor sites, with no significant change in the apparent affinity . The induction of EGF receptor loss by EGF was a specific effect on the EGF receptor . Preincubation of these same cells with insulin caused a decrease in the number of insulin receptors, while the number of EGF receptors remained unaltered . Conversely, preincubation with EGF, in a dose that down-regulated EGF receptors, did not alter insulin receptor number or affinity . Down-regulation of EGF receptors was reversible, with 50% recovery by 16 h . However, cycloheximide (10 micrograms/ml) blocked EGF-induced down-regulation and receptor recovery . The presence of EGF receptors in human placental cells and the ontogenic changes found suggest that EGF may be involved in the regulation of fetal growth and development . These studies indicate the feasibility of using human placental cells in culture as a model system to probe hormone-cell interaction in the fetoplacental unit.

Infect Immun, 1984 Feb, 43(2), 722 - 9
Serotyping of cell culture-adapted subgroup 2 human rotavirus strains by neutralization; Gerna G et al.; Nine human rotavirus strains from stools of infants with gastroenteritis were serially propagated in MA-104 cell cultures . All strains were identified as subgroup 2 rotaviruses by RNA gel electrophoresis, complement fixation, and enzyme-linked immunosorbent assay . The human rotavirus strains were propagated for 15 to 20 passages and then used for immunization of guinea pigs and rabbits . Animal antisera were also raised against a subgroup 1 human strain purified from stools and against the cell culture-adapted Wa strain, a reference subgroup 2 rotavirus of human origin . Cross-neutralization studies revealed the existence of two distinct serotypes within the cell culture-adapted subgroup 2 human rotaviruses: strains related and unrelated to strain Wa were classified as serotypes 1 and 3, respectively . Results with convalescent-phase sera from infants with primary rotavirus infections confirmed the existence of two serotypes within subgroup 2, and the serotypes responsible for primary subgroup 2 infections could be determined on the basis of the neutralizing reactivity of convalescent sera.

J Urol, 1984 Feb, 131(2), 223 - 6
Autologous mixed lymphocyte tumor cell culture in patients with renal cell carcinoma; Nakano E et al.; Cell-mediated immunity was studied by measurement of lymphocyte response to autologous tumor cells in 19 surgically treated patients with histologically proved (mixed lymphocyte tumor cell culture) renal cell carcinoma . Tumor stage was low in 9 patients and high in 10, while grade was low in 11 and high in 8 . Of 8 patients in whom a positive lymphocyte response was detected 6 had high and 2 had low stage tumors (p less than 0.05), while the grade of disease was low in 7 and high in 1 (p less than 0.05) . Furthermore, the more advanced and undifferentiated the tumor the more significant the decrease in lymphocyte response (p less than 0.05) . Lymphocyte response was positive in 5 of 8 patients with low stage and low grade tumors but negative in 7 with high stage and high grade disease . However, no correlation between the lymphocyte response and the degree of microscopic lymphocytic infiltration in and around the tumor was found . This study confirms that the specific immunological defense mechanism of patients with renal cell carcinoma against the tumors remains well at an earlier stage of tumor development, especially in cases with well differentiated malignancy, and showed attenuation in parallel with pathological spread or in poorly differentiated tumors.

J Virol Methods, 1984 Feb, 8(1-2), 63 - 71
A microcarrier cell culture system for large scale production of hepatitis A virus; Widell A et al.; Hepatitis A virus (HAV) was isolated from human faeces using a fetal rhesus monkey kidney cell line (Frhk-4) . Infectious medium from passage 12 was used to inoculate a large (5000 cm2) microcarrier cell culture maintained in suspension . The microcarriers used were swollen, collagen-coated dextran beads on which it was easy to propagate Frhk-4 cells . Intra- and extra-cellular virus levels were assayed and compared with conventional cultures in 25 cm2 plastic flasks . The results show that virus production per cell was similar in both systems . The number of cells per area unit in confluent cultures was initially lower in the microcarrier culture but subsequently increased . Two to three weeks post inoculation the virus yield per area unit in the microcarrier system was half of that of the conventional culture . The lower cell density per area unit in the microcarrier system was compensated by the large growth area that could be maintained in a single vessel and the total production of virus was substantial . Weekly harvests of medium with HAV antigen titres around 10(-2) contained antigenic material sufficient for several thousands of anti-HAV IgM tests . Propagation of HAV in microcarrier cell cultures thus seems a safe and simple way to produce large amounts of HAV.

Biochim Biophys Acta, 1984 Jan 24, 797(1), 71 - 5
Effect of varying amounts of ascorbate on collagen, elastin and lysyl oxidase synthesis in aortic smooth muscle cell cultures; Faris B et al.; In the presence of ascorbate, there is an increase in collagen synthesis with a concomitant decrease in insoluble elastin and lysyl oxidase activity in cultured rabbit aortic smooth muscle cells . While the addition of 0.5 micrograms ascorbate per ml of medium enhances collagen synthesis and accumulation, detectable insoluble elastin and lysyl oxidase activity remain essentially unchanged . However, at 2 micrograms ascorbate per ml, the integrity of the insoluble elastin is lost and lysyl oxidase activity is decreased . These studies suggest that by modifying the levels of ascorbate in the culture medium one can alter the nature of the extracellular matrix produced by the smooth muscle cells.

Thromb Res, 1984 Jan 15, 33(2), 145 - 53
Quantitation of tissue-type plasminogen activator in human endothelial cell cultures by use of an enzyme immunoassay; Rijken DC et al.; Endothelial cells from human umbilical cord were cultured to study plasminogen activator synthesis and secretion . Since simultaneous production of plasminogen activator inhibitor(s) prevented detection of plasminogen activators by use of fibrinolytic assays, an enzyme immunoassay for tissue-type plasminogen activator (t-PA) was developed . In this assay, t-PA in test samples was adsorbed onto microtiter plates coated with rabbit antibody against t-PA and then quantitated by successive incubation with goat antibody against t-PA and enzyme labeled rabbit antibody against goat IgG . The sensitivity of the assay was found to be 1 ng t-PA/ml . In the absence of serum, arterial and venous endothelial cells continuously produce t-PA antigen during a 24 h period, reaching a level of 5.1 +/- 2.5 ng t-PA/ml (n = 8) . In serum containing (20%) medium, 9.3 +/- 6.0 ng t-PA/ml (n = 17) was produced during this period (0.1 - 0.2 ml medium per cm2 confluent cells) . It is concluded that the enzyme immunoassay is a useful method for quantitating t-PA secretion by endothelial cells in the presence of proteinase inhibitors.

Biochem Biophys Res Commun, 1984 Jan 13, 118(1), 206 - 11
Gonadotropin receptor complexes and free receptors in porcine Leydig cell cultures during recovery from hCG stimulation; Mombrial FC et al.; Free and occupied gonadotropin receptors were studied in vitro in porcine Leydig cells culture maintained in chemically defined medium . Free receptors were evaluated by the binding capacity for 125I-hCG . hCG bound molecules (or hCG receptor complexes) were evaluated using immunocytochemical visualization on fixed cells . Exposure to hCG for 16 hours (.5 to 50 ng/ml) induced the disappearance of free receptors . After removal of the hormone, the return to control levels was observed at 48 and 72 hours . Visualization of hCG bound at the cell surface indicates that, following continuous exposure to gonadotropins for 48 hours, hCG molecules are still present on the cell . Following short-time exposure (1 h) to hCG and 48 hrs washing the number of stained cells is very close to the initial value suggesting that the occupied sites (at 48 hours) represent the initial hormone receptor complexes . These results indicate that, during prolonged incubation, hCG binding is not reversible, that the half-life of some of the complexes at the cell surface is very long and that the receptors recovery is slow and is probably the result of a de novo synthesis.

Brain Res, 1984 Jan 9, 290(2), 321 - 32
Physiology and pharmacology of olfactory bulb neurons in dissociated cell culture; Frosch MP et al.; Cells from olfactory bulbs of embryonic rats were grown in dissociated cell culture for up to 5 weeks . Both neurons and non-neuronal cells grew in these cultures, with a variety of neuronal populations appearing . A population of 20-25% of the neurons were GABAergic by the criterion of {3H}GABA uptake . Electrophysiologic measurements were made of the baseline activity of the cultured neurons . Cells showed a mean resting potential of 60.1 +/- 1.2 mV and a mean input resistance of 87.6 +/- 9.5 M omega . All cells were sensitive to microperfusion of GABA with half-maximal effect occurring at about 20 microM . Glutamate was universally excitatory but with variations in degree . Carnosine (beta-Ala-L-His), tested over the concentration range of 10 nM to 100 microM, had no effect on input resistance, resting potential, action potential shape, on-going synaptic activity or the responsiveness to either GABA or glutamate . These results are further evidence against a role for carnosine as the excitatory transmitter of the primary olfactory afferents.

Brain Res, 1984 Jan 2, 290(1), 77 - 86
Autoradiographic localization and depolarization-induced release of acidic amino acids in differentiating cerebellar granule cell cultures; Levi G et al.; Granule cells from 8-day-old rat cerebella were grown in basal Eagle's medium with 10% fetal calf serum, for 2,5,8 or 12 days in vitro (DIV), in conditions giving a purity greater than 90% . The results obtained can be summarized as follows: (1) Light microscopic autoradiography showed that cultured granule cells and their processes can accumulate the glutamate analog {3H}D-aspartate once they have reached an advanced degree of morphological differentiation (8 and 12 DIV), but, even then, only a limited number of cells was heavily labeled . In contrast, astrocytes were heavily labeled at all stages . (2) Calcium-dependent, high {K+}-induced release, or tetrodotoxin-sensitive, veratridine-induced release of {3H}D-aspartate from granule cell-enriched cultures was detectable only in cultures of 8 or 12 DIV . (3) When subject to 3 consecutive depolarizations, cultured granule cells maintained their ability to release {3H}D-aspartate and endogenous glutamate almost unchanged . (4) Newly synthesized {3H}glutamate was autoradiographically localized in both neurons and astrocytes (the latter, however, were not preferentially labeled as with {3H}D-aspartate), but was specifically released from neuronal structures (perikarya and processes) by depolarizing stimuli.

Exp Lung Res, 1984, 6(2), 149 - 58
Differentiation-arrested rat fetal lung in primary monolayer cell culture . III . Antioxidant enzyme activity; Tanswell AK et al.; Differentiation-arrested monolayer lung cell cultures were developed from day 18, 20, and 22 rat fetuses and 3-day-old neonatal rats . These cultures were examined for antioxidant enzyme activity, and the values obtained were compared with previously reported in vivo activity . All cultures were catalase deficient, and activity could be restored by the addition of 0.25 microM Fe(NO3)3 X 9H2O to the culture medium . The other measured antioxidant enzymes--copper-zinc and manganese superoxide dismutase, glutathione peroxidase, and glucose 6-phosphate dehydrogenase-demonstrate gestation-dependent increases of activity in vivo that were not evident in vitro, supporting the concept of a circulating "maturation factor" during fetal life . When cultures from fetal days 20 and 22 and from neonatal day 3 lungs were challenged with 50% oxygen in the presence of serum, antioxidant enzyme activities were unchanged, and there was no evidence of cell damage as assessed by release of lactate dehydrogenase . In the absence of serum, however, fetal day 20 (but not fetal day 22 or neonatal day 3) lung cells showed evidence of cell damage and increased antioxidant enzyme activities . It is concluded that cultured immature fetal cells are more susceptible to oxygen toxicity than those derived from mature fetal or neonatal animals . This increased susceptibility cannot be explained on the basis of the reduced antioxidant enzyme activity observed in vivo.

Adv Exp Med Biol, 1984, 174, 525 - 34
Studies of gangliosides in diverse nerve cell cultures; Dimpfel W et al.; Primary nerve cell cultures and tumor cells have been analysed with respect to synthesis of gangliosides . Neither developmental aspects of primary cultures nor nerve growth factor-induced fiber extension in PC12 or ZPH tumor cells gave any evidence of an involvement of gangliosides . Thus, it is concluded that neurite sprouting and ganglioside synthesis may not be interrelated, but that gangliosides might well serve as receptors for trophic factors.

Adv Exp Med Biol, 1984, 172, 139 - 49
A high efficiency stirrer for suspension cell culture with or without microcarriers; de Bruyne NA; The stirrer described in this paper employs "Teaspoon stirring" in which a secondary motion is superimposed on the rotation of the liquid . This secondary motion arises from the viscous drag from the wall and bottom of the flask . The culture medium is rotated by a bulb-ended rod suspended from inside the flask cap: the bulb orbits around in a circular trough formed between a central indent in the base and the rounded periphery of the base . There are no bearings and no stagnant areas . The power used to stir 4 flasks each holding 500 ml of medium is less than 2 watts . The speed is electronically controlled by a tachometer and automatically gives a smooth start and stop as well as "Interval stirring" to assist initial attachment of cells.

Avian Dis, 1984 Jan-Mar, 28(1), 216 - 23
A comparison of avian and mammalian cell cultures for the propagation of avian reovirus WVU 2937; Barta V et al.; Two avian and seven mammalian cell lines were evaluated for their application in propagating avian reovirus WVU 2937 . Cultures were compared for monolayer-formation time, support of viral replication, passages and postinfection time required for expression of cytopathic effect (CPE), type of CPE, and virus yield . CPE was observed on the first passage with infected egg yolk in primary chicken embryo kidney cells, primary through tertiary chicken embryo liver (CEL) cells, and African green monkey kidney (VERO) cells; on the third blind passage of infected supernatant in Georgia bovine kidney cells, Crandall feline kidney cells, and baby hamster cells; on the fifth blind passage in rabbit kidney cells; and on the tenth blind passage in porcine kidney cells . CPE was not observed after 10 viral passages in rabbit bone-marrow cells . Monolayer formation time and postinfection time for CPE expression occurred sooner, and virus yield was greater, with CEL and VERO cells than with other cell lines.

Res Commun Chem Pathol Pharmacol, 1984 Jan, 43(1), 43 - 54
Metabolic blocker-induced cell damage in rat cardiac tissue . Comparison of three models currently used: the isolated perfused heart, heart cell cultures and isolated myocytes; van der Laarse A et al.; In a comparative study on metabolic blockade-induced cardiac cell damage, three different heart (cell) preparations are exposed to potassium cyanide (KCN) and iodoacetamide (IAA) in fixed concentrations (5 and 0.1 mmol/l, respectively) . The preparations used are the perfused rat heart, isolated rat heart myocytes and rat heart cell cultures . Irreversible heart cell damage is reflected by the release of a cytoplasmic enzyme, LDH (lactate dehydrogenase), or HBDH (alpha-hydroxybutyrate dehydrogenase) from the heart (cell) preparations . Metabolic blockade-induced release of 50% of the originally intracellularly located LDH or HBDH is reached after 2-3 h from rat heart cell cultures, after 2-4 h from isolated rat heart myocytes and after variable (coronary flow-dependent) durations of perfusion from perfused rat hearts . It is concluded that to study the effect of toxic agents on myocytic integrity, the use of isolated cardiac myocytes and heart cell cultures is preferable to the use of isolated perfused hearts.

In Vitro, 1984 Jan, 20(1), 53 - 8
Renal cell culture using autopsy material from children with cystinosis; Pellett OL et al.; Renal cell cultures were initiated using fresh autopsy material from two individuals with cystinosis, ages 5 and 8 yr . Cells obtained from collagenase treated autopsy material were grown in a selective kidney medium containing Coon's modified F12, 2.5% fetal bovine serum, transferrin, insulin, selenium, hydrocortisone, PGE1, and fibronectin . These cells had an epithelial appearance, formed domes, and were periodic acid-Schiff positive . Both tight junctions and microvilli were seen by electron microscopy . Fibroblasts had a cloning efficiency of zero in the selective medium and grew poorly compared to their growth in Coon's F12 with 10% fetal bovine serum . The lysosomal cystine content of the renal cells was greatly elevated and comparable to that of fibroblasts from cystinotic patients . Renal cell lysosomal cystine levels were only partially reduced by exposure to either pantethine or the aminothiol, cysteamine . However, exposure to either compound effectively depleted cystinotic cultured fibroblasts of their lysosomal cystine . Study of cultured renal material may have practical significance in pharmacologic considerations.

In Vitro, 1984 Jan, 20(1), 45 - 52
Effect of D-valine and cytosine arabinoside on {3H}thymidine incorporation in rat and rabbit epididymal epithelial cell cultures; Orgebin-Crist MC et al.; Epithelial cell enriched primary cultures were established from the rat and the rabbit epididymis . Epithelial cell aggregates, obtained after pronase digestion of minced epididymis, attached to the culture dish and after 72 h in vitro spread out to form discrete patches of cells . These cells have an epithelioid morphology and form a monolayer of closely apposed polygonal cells where DNA synthesis, as judged by {3H}thymidine uptake, is very low . In L-valine medium the nonepithelial cell contamination was no more than 10% in rat and rabbit epididymal primary cultures . The labeling index of rat epididymal cells cultured in D-valine medium was significantly lower than that of cells cultured in L-valine medium . In contrast, the labeling index of rabbit epididymal cells cultured in D-valine medium was significantly higher than that of cells cultured in L-valine medium . Cytosine arabinoside decreased the number of labeled cells in both L-valine and D-valine cultures . From these results, it appears that D-valine is a selective agent for rat epididymal epithelial cells, but not for rabbit epithelial cells, and that cytosine arabinoside is a simple and effective means to control the proliferation of fibroblast-like cells in both rat and rabbit epididymal cell cultures.

Am J Physiol, 1984 Jan, 246(1 Pt 1), C106 - 13
Carotid body cell culture and selective growth of glomus cells; Fishman MC et al.; Cells of the fetal carotid body have been obtained by enzymatic digestion and maintained in culture both as single cells and as clusters for up to 2 mo . The glomus cells in culture synthesize catecholamines and adenine nucleotides, as determined by histochemical methods, and contain characteristic dense-core vesicles . Their growth requirements are different from other cells of neural crest origin in that they do not depend on nerve growth factor (as do sympathetic neurons) or corticosteroids (as do SIF cells) for survival . Neither hypoxia nor hypercarbia affects survival or relative preponderance of glomus cells in culture . Tyrosine-free medium, which selects for cells containing tyrosine hydroxylase, eliminates most of the nonadrenergic cells, thereby providing a culture dramatically enhanced in glomus cells.

Prostate, 1984, 5(1), 113 - 22
Acid phosphatase-producing androgen-independent subline of rat prostatic adenocarcinoma (Dunning R3327 tumor) in cell culture; Igarashi T et al.; Establishment of a cell line derived from the androgen-independent subline of rat prostatic adenocarcinoma (Dunning R3327 tumor) is reported . Cells of this line produced acid phosphatase . When the cultured cells were transplanted to Copenhagen rats, solid tumors were formed . Histologically, the tumor consisted of spindle-shaped, large and bizarre polygonal cells; this feature was almost identical to that of the original tumor . Chromosomes were in the triploid range with seven frequently appearing marker chromosomes.

Arch Dermatol Res, 1984, 276(5), 288 - 92
Human Langerhans cells in epidermal cell culture, in vitro skin explants and skin grafts onto "nude" mice; Czernielewski J et al.; In order to find a model system which best preserves human Langerhans cells (LC) outside of the human body, three possibilities were examined: epidermal cell culture, skin explants, and skin grafts onto "nude" mice . Using OKT-6 and anti-HLA-DR monoclonal antibodies, we quantified LC in epidermal sheets or epidermal cell cultures . All observations were carried out over a period of 4 weeks . We found that under epidermal cell culture conditions, LC rapidly disappeared, to the extent that after 10 days only rare HLA-DR-positive cells could be observed . In contrast, in the presence of intact dermis (explants and grafts), 60%-80% of the original number of LC, morphologically unchanged, dendritic and OKT-6 and HLA-DR-positive, were seen . These findings suggest that human LC are either a long-lived cell population or else can proliferate locally . The systems studied may be a useful tool for future investigation of LC function.

Br J Cancer Suppl, 1984, 6, 121 - 7
Induction and repair of X-ray damage in mammalian cell cultures treated with membrane-active drugs; Bertsche U; Cultures of Ehrlich ascites tumour cells (EATC) were treated before and after X-irradiation with the membrane active drugs chlorpromazine (CPZ) and procaine . Under hypoxic conditions of irradiation CPZ sensitized cells and was most effective at about 50 microM, whereas at higher drug concentrations the extent of sensitization was less . Sensitization was however not observed in cultures supplemented with vitamin E . Likewise, CPZ inhibited repair of potentially lethal damage (RPLD) measured by delayed plating of stationary cell cultures either using the colony forming ability or micronucleus formation as endpoints . Procaine, on the other hand, was found to sensitize cells only slightly under hypoxia and protected slightly under oxic conditions in the concentration range from 10-100 mM . Both drugs induced an increase in ATP content at these concentrations . Since it has also been observed that these drugs cause depletion of intracellular sulfhydryl groups which may serve for protection of membrane sites, it is assumed that the radiobiological effects observed arise mainly from an influence on cellular and nuclear membranes where lipid bilayer fluidity and conformational status of membrane-bound enzymes may be changed . The possible role of heterochromatin anchored or near to the nuclear membrane as a radiation sensitive compartment of the cell is discussed.

Vet Med Nauki, 1984, 21(10), 19 - 27
{Isolation, identification and typing of Newcastle disease virus isolates in a chick embryo cell culture}; Khadzhiev H; Routine methods of investigation were employed in parallel experiments for the isolation (in chick embryos), identification (serologically via HI), and typing (MDT, ICPI, IVPI, and via cloacal tests) of local isolates and standard strains of the Newcastle disease virus (NDV) . The results were the same as those obtained by the single-phase complex investigation after the plaque technique method in a monolayer cell culture of chick embryo fibroblasts under agar cover.The cultures were inoculated with tissue suspension centrifugate in multiple dilutions of trachea and lung, spleen and liver, and brain of experimentally infected birds, using local isolates (13 in all) and standard strains of NDV (6 of the various types) as well as of birds that died of Newcastle disease (3 cases) . The complex cell culture technique of investigation included plaque-forming under agar (and under agarose as well) following the direct inoculation with the infectious tissue suspensions in the presence of a normal (known negative), resp., specific hyperimmune serum against Newcastle disease . It also included a plaque-neutralization (plaque reduction) test and tests for hemadsorption and elution of erythrocytes--all made use of in a single planned programme of investigation . This technique, which is a modification of the one suggested by Beard et al . (1970) and known as single-phase complex plaque technique, provides the simultaneous isolation and identification of NDV isolates, which may well be coupled with some of the routinely practiced methods for definitive isolate typing (e . g., with the cloacal test for velogenic, and with MDT for lento- and mesogenic isolates) . Thus, time can be saved, and labour and means are reduced, i . e., the laboratory diagnostics of ND may be perfected.

Zentralbl Allg Pathol, 1984, 129(6), 499 - 505
{Growth behavior of amnion cell cultures}; Weise W et al.; Both amniotic fluid samples and amniotic fluid cell cultures of a total of 52 patients were evaluated . At 15 to 16 weeks' gestation there are 2.2 X 10(4) cells/ml amniotic fluid increasing significantly to 3.7 X 10(4) cells/ml at 19 to 20 weeks' gestation . The percentage of viable cells is about 18 . After 10 d of culture 14.4 +/- 8.2 cell colonies/culture flask are developed consisting of 61% AF-cells, 33% E-cells and 6% F-cells . After 14 d of culture 60 +/- 32 metaphases/cell culture flask were found . At the 10th day of culture 80% of cell colonies are already present indicating that reduced duration of culture appears possible . Therefore the interval to diagnosis can be shortened and a possible interruption of pregnancy can be earlier done under reduced risks.

Bioelectromagnetics, 1984, 5(4), 399 - 410
Design of a magnetic field generator for experiments on magnetic effects in cell cultures; Kinouchi Y et al.; A magnetic field generator constructed of rare earth-cobalt magnets is proposed for examining the biological effects of static magnetic fields (less than 1 T) on tissue cultures . Important quantities of a magnetic field from a biological-effects viewpoint, ie, its strength and the product of strength and gradient, are analysed . A practical procedure for designing the generator with optimum parameters is given . Also, parameters are determined which will yield a sinusoidal spatial field distribution.

J Toxicol Environ Health, 1984, 13(4-6), 865 - 77
Cellular distribution of inorganic mercury and its relation to cytotoxicity in bovine kidney cell cultures; Bracken WM et al.; A bovine kidney cell culture system was used to assess what relationship mercuric chloride (HgCl2) uptake and subcellular distribution had to cytotoxicity . Twenty-four-hour incubations with 0.05-50 micro M HgCl2 elicited a concentration-related, with 1.0 nmol/10(6) cells at the IC50 . Measurement of Hg uptake over the 24-h exposure period revealed a multiphasic process . Peak accumulation was attained by 1 h and was followed by extrusion and plateauing of intracellular Hg levels . Least-squares regression analysis of the cytotoxicity and cellular uptake data indicated a potential relationship between the Hg uptake and cytotoxicity (r = 0.91) . However, the subcellular distribution of Hg was not concentration-related . Mitochondria and soluble protein fractions accounted for greater than 65% of the cell-associated Hg at all concentrations . The remaining Hg was distributed between the microsomal (6-10%) and nuclear and cell debris (11-22%) fractions at all concentrations tested . Less than 20% of the total cell-associated Hg was bound with metallothionein-like protein.

Histochemistry, 1984, 80(5), 497 - 501
The use of ultrathin cryosections for localisation of influenza virus antigens in infected vero cell cultures; Beesley JE et al.; The localisation of influenza virus antigens in infected Vero cell monolayer cultures by post embedding immunoelectron microscopy requires both good resolution and the retention of antigenicity in the tissue sections . Ultrathin cryosections are superior to ultrathin resin sections for this purpose . The colloidal gold probe was used in conjunction with specific antibody preparations to localise three viral proteins . Antibody raised against haemagglutinin glycoproteins labelled the host cell membrane and the virus fringe without contamination of the host cell nucleus, whereas antibody raised against viral nuclear protein labelled throughout the host cell cytoplasm and nucleus . Matrix protein was localised within the nucleus and was associated with the host cell membrane of the infected cell . The appearance of all these proteins was maximal 24 h post infection.

Dev Pharmacol Ther, 1984, 7(5), 307 - 18
Preparation and electrophysiological characterization of cardiac cell cultures derived from the fetal canine ventricle; Robinson RB et al.; We have developed a primary cardiac cell culture preparation using the fetal canine ventricle . These cultures exhibited widespread and synchronous spontaneous contractile activity, were sensitive to isoproterenol and had the following transmembrane action potential characteristics (means +/- SE): maximum diastolic potential (MDP) -70.9 +/- 0.9 mV; maximum velocity of phase 0 (Vmax) 84 +/- 4 V/s (and tetrodotoxin sensitive); amplitude (AMP) 98.9 +/- 1.1 mV; duration at half amplitude (APD50) 244 +/- 10 ms at a spontaneous cycle length of 1,600 +/- 100 ms . APD50 was linearly dependent on spontaneous cycle length (CL) (slope = 67 ms/s) . The action potential of the intact fetal canine ventricle of comparable gestational age had MDP = -67.0 +/- 1.1 mV, Vmax = 127 +/- 6 V/s, AMP = 92.2 +/- 1.6 mV and APD50 = 153 +/- 2 ms at a paced CL of 500 ms . We conclude that myocardial cultures derived from the fetal dog ventricle exhibit electrophysiological characteristics similar to those of the intact canine ventricle, thereby making them a possible alternative to avian and rodent cardiac cultures.

Cell Tissue Res, 1984, 237(1), 161 - 8
Non-neuronal cells of rat hypothalamus in dissociated cell culture . Evidence that neurophysin modulates growth and DNA synthesis of non-neuronal cells; Worley RT et al.; The neurophysin that is biosynthesised in association with the neurohypophysial hormone vasopressin (vasopressin-neurophysin) affects the growth and DNA synthesis of rat hypothalamic non-neuronal cells in culture . Over a narrow range of concentrations vasopressin-neurophysin stimulated growth, as assessed by increase in cell numbers, about five-fold, in conditions where fetal calf serum concentration was limiting (0.2% fetal calf serum) . Maximum stimulation occurred in the presence of 20 to 30 ng vasopressin-neurophysin per ml of medium . DNA synthesis was increased by a factor of three in the presence of 30 ng vasopressin-neurophysin per ml of medium . At least two populations of non-neuronal hypothalamic cells were present in the cultures, and these were both affected by vasopressin-neurophysin . This study allows the suggestion that neurophysin may be acting as a growth-regulating factor at its release site, playing a part in the interactions of neurones and glial cells in the hypothalamo-neurohypophysial system.

Ann Parasitol Hum Comp, 1984, 59(3), 321 - 2
{Initial division of Isospora sporozoites from a sparrow in cell culture}; Millet P et al.; The study of the sparrow Isospora in cell culture has demonstrated that during the extra-intestine life cycle, sporozoites undergo a primary division, resulting into two similar elements, whereas, in plain intra-intestinal cycle, the sporozoite is straightforwardly transformed into the first generation schizont.

Prenat Diagn, 1984 Spring, 4 Spec No, 145 - 62
European collaborative study on prenatal diagnosis: mosaicism, pseudomosaicism and single abnormal cells in amniotic fluid cell cultures; Bui TH et al.; A report is given of the results of a European collaborative study on mosaicism, pseudomosaicism and single abnormal cells in amniotic fluid cell cultures . The mean frequency of cases with mosaicism was 0.10 per cent, with pseudomosaicism 0.64 per cent and with single abnormal cells 2.83 per cent in a series of 44 170 amniotic fluid samples . There was no significant difference between the colony (in situ) and the flask method with regard to the frequency of mosaicism . Pseudomosaicism and single abnormal cells were more frequent in cases studied with the flask method probably due to other factors than the method of cultivation of the cells . The frequency of maternal cell contamination was 0.17 per cent and the frequency of wrong sex assignment was 0.11 per cent . A more correct estimation is obtained if these frequencies are doubled . There was a considerable variation between laboratories with regard to the frequencies given above . One reason for this variation is that there are no sharp limits between mosaicism, pseudomosaicism and single abnormal cells . Thus the material contained cases diagnosed as having pseudomosaicism which turned out to be mosaics at birth and to have an abnormal phenotype . These cases were very rare but pose a definite problem in prenatal cytogenetic diagnosis.

Prenat Diagn, 1984 Spring, 4 Spec No, 131 - 44
A Canadian collaborative study of mosaicism in amniotic fluid cell cultures; Worton RG et al.; Data on chromosomal mosaicism was collected retrospectively on 12 386 amniotic fluid samples cultured over a 10 year period in 14 Canadian centres . Level I mosaicism (a single abnormal cell--excluding single cell monosomy) was encountered in 863 cases (7.1 per cent) . Level II mosaicism (multiple cells with the same abnormality in a single flask or colony) was encountered in 138 cases (1.1 per cent) . Level III mosaicism (multiple cells distributed over multiple flasks or colonies) was encountered in 34 cases (0.3 per cent) . Analysis of the details of these cases allowed five major conclusions to be drawn: (1) Single cell abnormalities should not be taken as an indication of true fetal mosaicism . Only rarely will this interpretation prove to be incorrect . (2) Mosaicism involving multiple cells confined to a single flask should not be regarded as an indication of true fetal mosaicism . Only occasionally will this interpretation prove to be incorrect . (3) Mosaicism involving multiple cells distributed over more than one flask should be regarded as a strong indication of true fetal mosaicism . Sixty per cent will be confirmed by karyotype analysis of the fetus or infant . (4) Mosaicism of the XX/XY type is usually due to maternal cell contamination . Occasionally it can be a female fetus with XY cells from an unknown source . (5) The in situ or colony method of chromosome analysis has no clear advantage over the flask method for either the detection of true fetal mosaicism or for the ability to distinguish true mosaics from false positives.

Acta Physiol Hung, 1984, 64(2), 135 - 8
Hormonal imprinting in cell culture II . Evidence of hormonal imprinting and thyrotropin (TSH) -gonadotropin (FSH) overlap in a Chinese hamster ovary (CHO) cell line; Csaba G et al.; Reexposure of cultures of the Chinese hamster ovarian cell line CHO K1 to FITC-labeled hormone 48 h after the first 24-h exposure to FSH or TSH showed that hormonal imprinting, accounting for a greater binding capacity on reexposure, also took place in in vitro conditions . TSH amplified the receptors of FSH to a greater degree than FSH itself, although the reverse effect failed to happen . TSH was able to bind the ovarian cells at first exposure, and to amplify the receptors for itself and--remarkably--to a considerably greater degree for FSH, exactly as observed earlier in in vivo systems.

Ann Microbiol (Paris), 1984 Jan-Feb, 135A(1), 47 - 53
Insect cell cultures in the study of attachment and pathogenicity of spiroplasmas and mycoplasmas; Steiner T et al.; The insect cell lines Dm-1 (Drosophila melanogaster), AS-2 (Aceratagallia sanguinolenta) and AC-20 (Agallia constricta) were infected with spiroplasmas, mycoplasmas and Acholeplasma laidlawii . In Dm-1 cultures maintained at 25 degrees C in M1A medium, all strains multiplied except M . hyorhinis and the uncultivable sex-ratio organism . Spiroplasma citri R8A2, S . floricola BNR-1 and OBMG, S . apis PPS-1 and the strains BC-3, corn stunt spiroplasma (CSS) and 277F produced cytopathogenic effects (CPE), whereas S . mirum SMCA, M . orale, M . arginini and A . laidlawii did not . Cytadsorption was found with the cultivable spiroplasmas and A . laidlawii . At 30 degrees C SMCA, M . orale, M . arginini and A . laidlawii killed the Dm-1 cultures . M . hyorhinis grew without any CPE . In AS-2 and AC-20 cultures grown at 28 degrees C in LB medium, R8A2, B88, 277F, BNR-1 and PPS-1 multiplied and reached titres of 2 X 10(8) to 4 X 10(9) CFU/ml . They produced CPE leading to culture death . CSS did not grow . R8A2 reached higher titres in AS-2 cultures than in fresh LB medium . This stimulating factor was studied by means of conditioned medium . All 6 spiroplasmas cytadsorbed to AS-2 and AC-20 cells . B88 and 277F adsorbed heavily, while the other 4 strains adsorbed only slightly . Fluorescent DNA staining with "Hoechst 33258" revealed the presence of non-helical forms inside the cells.

Ann Microbiol (Paris), 1984 Jan-Feb, 135A(1), 39 - 45
Competitive inhibition and attachment assays in cell cultures to detect pathogenic binding components of mycoplasmas: a review; Chandler DK et al.; A microattachment assay for quantitating adherence of radiolabelled Mycoplasma pneumoniae to human WiDr cell culture monolayers is described . Preincubating the WiDr cell monolayers with a protein-rich extract of M . pneumoniae inhibited the subsequent attachment of radiolabelled organisms . Competitive attachment inhibition provided a quantitative procedure to determine M . pneumoniae-binding components in the extract . The microattachment assays also measured attachment inhibition by the sialoglycoconjugates ceruloplasmin, orosomucoid and gangliosides, indicating that these reagents may be structural analogues of the mammalian cell receptor . Attachment of virulent M . pneumoniae strains to glutaraldehyde-treated monolayers was reduced approximately 60% and showed a different temperature dependence compared with untreated cells . These results suggest that maximal attachment of virulent M . pneumoniae may require two or more different receptors and binding components.

Acta Physiol Hung, 1984, 64(1), 57 - 63
Hormonal imprinting in cell culture I . Impact of a single exposure to insulin on cellular insulin binding capacity in permanent cell lines; Csaba G et al.; Three permanent cell lines showed a durable change in cellular insulin binding capacity in response to a single exposure to insulin . Initial increase in binding of FITC-labeled insulin to both cytoplasmic and nuclear membrane was followed by a lasting decrease in the case of fibroblasts (NCTC) and return to approximately the control level in epithelial (Chang liver) and tumour (HeLa) cell cultures . While the amplifying effect of a single 4-h exposure was measurable as soon as after 24 h, that of a single 24-h exposure came into display only after 72 h in the epithelial and tumour cell cultures . The latter two lines showed return of the binding capacity to the control value and a drop of it to a minimum 20 days after exposure for 4 and 24 h, respectively . These observations support the implication that although the degree and manner of insulin binding differs between cell lines, the impact of a single exposure to insulin is durably "felt" by all.

Differentiation, 1984, 26(2), 121 - 6
Growth and differentiation of chicken embryo muscle cell cultures derived from fast- and slow-growing lines . Intrinsic differences in growth characteristics and insulin response; Ridpath JF et al.; Primary myogenic cell cultures derived from 12-day embryos of genetically fast-growing chickens (fast cultures) and slow-growing chickens (slow cultures) were grown under identical conditions to examine differences in growth and differentiation at the cellular level . The two types of cultures exhibited significant (P less than 0.01) differences in proliferation, protein accumulation, response to the addition of insulin to the culture medium and the amount of insulin bound per nucleus . The fast cultures exhibited a larger number of both total nuclei and fused nuclei at 48, 72 and 96 h in culture, accumulated more protein per nucleus at 24, 48 and 72 h in culture and demonstrated a greater response to the addition of insulin to the culture medium, as reflected by increased fusion rate and protein accumulation at 24 h in culture . Maximal response to insulin in both types of cultures was obtained at 24 h to added insulin concentrations of 10(-10)-10(-9) M . Slow cultures bound more {125I}-insulin than fast cultures at 24 h in culture . These experiments suggest that different muscle growth potentials in animals of the same species are at least partly due to intrinsic cellular differences in the myogenic cells that give rise to adult muscle tissue.

Adv Exp Med Biol, 1984, 172, 471 - 88
Use of cell culture to identify human precancer; Lyko HC et al.; In the United States, colon cancer is the most common form of internal cancer in both sexes . Prevention of the disease depends on early diagnosis of polyps or pre-cancerous lesions . The response of normal human colon fibroblasts ( CRL1459 ) was used to identify individuals with clinical pre-cancer . Their plasma induced transformation associated morphology characterized by the retraction of cellular processes, cell rounding and eventual detachment from the vessel surface . Those plasma samples which induced a transformation associated morphology contained significantly increased levels of protease as shown by casein hydrolysis (Bio-Rad, CA) . We are using hyperproteinasemia as a biomarker to identify individuals with polyps who have hereditary adenomatosis of the colon and rectum (ACR) . We are currently evaluating cell cultures versus biochemical assays as a means for early detection of precancerous tumors in the general population . The findings of a tumor associated protease in clinical precancer, and its effect on cell cultures support our proposal that protease activity promotes tumor progression in ACR and may represent the gene defect in this hereditary disease.

J Microsc, 1984 Jan, 133 ( Pt 1), 79 - 81
A method differentiating between bacterial adhesiveness and invasiveness in cell culture monolayer; Bukholm G et al.; In order to differentiate between bacterial adhesiveness and invasiveness in cell culture monolayers, a method is reported which enables the same specimen to be examined by both scanning electron microscopy and Nomarski interference contrast microscopy . The source of error made by statistical variation when the two microscopical techniques are used individually is thus avoided.

Am J Trop Med Hyg, 1984 Jan, 33(1), 158 - 65
Mosquito cell cultures and specific monoclonal antibodies in surveillance for dengue viruses; Gubler DJ et al.; During the fall of 1981, a new method for the routine isolation and identification of dengue viruses in Puerto Rico was implemented utilizing C6/36 cell cultures and serotype specific antidengue monoclonal antibodies . A blind comparison of the monoclonal antibody indirect fluorescent antibody test (IFAT) and the complement fixation (CF) test for identification of 89 newly isolated dengue viruses of all four serotypes from the Caribbean, Asia and Africa showed 100% agreement . Although virus isolation rates were slightly lower than with the mosquito inoculation technique, use of the C6/36 cell culture system was much less time-consuming and allowed the processing of larger numbers of sera . Beginning in November 1981, a new virologic surveillance system was begun in Puerto Rico . Acute sera from persons with suspected dengue were selected for virus isolation attempts on the basis of geographic area of residence on the island, day after onset the blood was taken and clinical signs and symptoms . These sera were processed for virus isolation in C6/36 cell cultures, and virus isolates were identified by the IFAT using the monoclonal antibodies . Using this system, 2,702 sera were tested from November 1981 through August 1982 . Dengue virus was isolated from 518, for an isolation rate of 19.2% . Dengue 1 was the predominant virus until December 1981, when dengue 4 became dominant . The changing patterns of dengue 1 and 4 distribution by time and geographic location on Puerto Rico were followed . This system allows the dengue viruses being transmitted in an area to be monitored with a minimal amount of effort and provides the early warning capability necessary to predict epidemic dengue.

Br J Dermatol, 1984 Jan, 110(1), 17 - 27
Effect of retinoic acid and low calcium conditions on surface glycoconjugates defined by differential lectin labelling in mouse epidermal cell culture; Robinson JK et al.; The appearance of cell surface glycoconjugates (detected by fluorescein-isothiocyanate-conjugated lectins and bullous pemphigoid antibody) was serially examined in mouse epidermal cell cultures treated with trans-retinoic acid and aromatic retinoic acid (etretinate) and in cultures maintained under low calcium conditions . The changes in lectin staining occurred in concert with the process of differentiation as assessed by cell morphology and colony growth characteristics, and they correlated with the patterns observed in whole mouse skin . The keratocyte cultures treated with retinoic acid showed delayed and reduced differentiation and stratification, and this was associated with markedly reduced binding of lectins specific for N-acetyl-glucosamine and fucose . The low calcium concentration produced similar changes . Thus, the loss of surface glycoconjugates in the epidermal cell culture system was not specific for either retinoic acid or low calcium, but correlated with the degree of cell differentiation.

Leuk Res, 1984, 8(5), 791 - 800
Myelopoiesis following phorbol ester exposure in human long-term bone marrow cell culture; Gerson SL et al.; Phorbol esters have two opposing effects on bone marrow granulocyte/macrophage colony forming cells (CFC-GM): they reduce the number of CFC-GM which respond to colony-stimulating activity (CSA), and they also induce the release of CSA from accessory marrow cells . We questioned whether the direct inhibitory effect of phorbol esters on CFC-GM was reversible and whether phorbol ester-treated accessory bone marrow cells could increase the in vitro recovery of cultured CFC-GM . Normal human bone marrow cells were exposed to phorbol-12,13-dibutyrate (PDB) for 5 days in liquid cultures . Total nucleated cell counts decreased in a dose-dependent fashion to 42 +/- 6% of control at 5 X 10(-8) M PDB, and CFC-GM decreased to 20 +/- 8% of control . The cultures were then washed to remove PDB and continued for up to 8 weeks . Total cell counts and CFC-GM/ml remained reduced in PDB-pretreated cultures compared to control . To determine whether this was a direct effect of PDB on nonadherent, proliferating cells or whether PDB acted indirectly by affecting adherent, accessory cells, nonadherent bone marrow cells from control and PDB-preincubated cultures were co-cultured for 8 weeks with either control or PDB-preincubated adherent bone marrow cells . Nonadherent cells preincubated with PDB had a similar growth pattern whether or not the co-cultured adherent cells had been preincubated with PDB . In contrast, nonadherent cells preincubated in control medium and co-cultured with PDB-preincubated adherent cells displayed increased numbers of nucleated cells, CFC-GM and adherent hematopoietic islands compared to control . This was associated with increased amounts of CSA present in the culture supernatants . Thus PDB causes an irreversible decrease in CFC-GM in long term marrow cultures but enhances the ability of adherent stromal cells to support normal CFC-GM growth in culture.

Vet Med Nauki, 1984, 21(3), 20 - 8
{Immunofluorescence and electron microscopy study of bovine reovirus multiplication in cell culture}; Tunkara A et al.; The appearance and development of reovirus antigens was followed up in Vero cells . The earliest immunofluorescence finding was established at the 6th hr, consisting in the appearance of small shining granules in and around the nuclear cytoplasm . At the 24th hr in 15 per cent of the cells there was a positive immunofluorescence finding in the form of one or several objects grouped closely to the nucleus . At the 36th hr fluorescence was seen in about 50 per cent of the cells to surround the nucleus and fill the whole cytoplasm or appearing as a limited and demarkated one . By form the fluorescent objects resembled viral inclusions . The immunofluorescence finding reached its peak at the 48th hr . In cells infected with Reo 2 the appearance and cumulation of the virus antigen ran its course in the same sequence as that seen with Reo 1, however, it was manifested with higher intensity . The electron-microscopic study of Reo 1-infected cells revealed greater diversity in the stages of infection as early as the 24th hr--from the appearance of sporadic virions up to the emergence of large virus crystals the virions in which assumed square or hexagonal arrangement . There was also linear arrangement of the virions by the length of the tubular cell structures . Cells infected with Reo 1 and Reo 2 presented likewise zones with large masses of virus matrix prior to the appearance in them of new virions . There was also particular cumulation of the cell ribosomes along with the matrix formation . It is believed that the appearance of these formations is associated with the virus morphogenesis.

J Med Virol, 1984, 13(4), 377 - 83
Cultivation of human rotaviruses in cell culture; Albert MJ et al.; Sixteen specimens of faeces from children with acute diarrhoea due to rotavirus were inoculated into MA-104 cells . Rotaviruses present in six of the specimens were successfully adapted to growth after serial passage . Two of these strains had "short RNA" patterns and had caused epidemics of diarrhoea in children in Melbourne, Australia from 1977 to 1979, or in children in the Highlands of Papua New Guinea in 1979 . The remaining four strains had "long RNA" patterns . One of these four strains was of major epidemiological importance as a cause of childhood diarrhoea in Melbourne during 1981 . The other three strains appeared identical and were isolated from babies born in a Melbourne obstetric hospital during 1977 . All six strains were successfully adapted to stationary culture, but only four strains could be plaqued . Selection of strains of rotavirus for culture on the basis of their known epidemiological importance in different communities will increase information about clinically important strains throughout the world.

Vet Med Nauki, 1984, 21(2), 40 - 8
{Cytomorphological and cytochemical studies on the multiplication of bovine reovirus type 1 in a cell culture}; Tunkara A et al.; Parallel cytomorphologic and cytochemical studies were carried out on the replication of the bovine reovirus type 1 in the Vero cell line . It was found that a high multiplicity of infection M = 5 the first morphologic changes took place some 12 hours following infection, while at the 24th hour there appeared also the characteristic inclusion bodies . At the same time the appearance was noted of acidophile masses in the cytoplasm of some of the infected cells . The morphologic changes reached the peak values of their development at the 48th hour, and the cytoplasmic inclusions assumed a net-shaped structure of lower density . It was also found that following a short-term rise in the first 12 hours after infection there set in a general suppression of the RNA-containing material, which spread irregularly through-out the cell thus marking the nonpyroninophile viral inclusions . The desoxiribonucleic synthesis was not visibly affected, however, the nuclear chromatin remained with an unchanged structure . The viral inclusions were Feulgen PAS-negative . The hydrolytic enzymes alkaline and acid phosphatase and 5-nucleotidase were strongly inhibited after a short-term rise within the first 12 hours of infection.

Avian Dis, 1984 Jan-Mar, 28(1), 183 - 96
Efficacy and safety of a cell-culture live virus vaccine for hemorrhagic enteritis of turkeys: laboratory studies; Fadly AM et al.; Poults free from hemorrhagic enteritis (HE) antibody were vaccinated by gavage at 1 day or 2 weeks of age with a live HE vaccine virus that had been propagated in a Marek's disease (MD)-induced B-lymphoblastoid cell line of turkey origin . Vaccinated and unvaccinated poults were challenged with a virulent HE virus at various times postvaccination . One hundred tissue-culture-infectious doses of the vaccine virus per poult were sufficient to induce a serological response as well as to protect poults against HE lesions and mortality . Vaccinated poults were protected against the disease as early as 1 week and as late as 8 weeks PV . The vaccine was efficacious by several routes of application . The vaccine virus spread horizontally from vaccinated to contact-exposed poults, as indicated by seroconversion and resistance of contact-exposed poults to challenge . The vaccine had no detectable harmful effects on the humoral immune response to particulate antigens or on weight gain of vaccinated poults . The vaccine proved to be free from MD virus, as indicated by the absence of MD lesions and antibody in 8-week-old chickens inoculated intra-abdominally with the vaccine at hatching . These findings indicate that the cell-culture-propagated HE vaccine is efficacious and safe.

Arch Virol, 1984, 80(2-3), 231 - 7
Serological characterisation of human rotaviruses propagated in cell cultures . Brief report; Beards GM et al.; Fourteen strains of human rotaviruses were propagated in MA 104 cell culture using serum-free medium containing 10 micrograms/ml trypsin . Eleven of the strains were provided by laboratories in Japan and the U.S.A., and the remaining three from our own laboratory . The serological relationships between these strains were determined by rotavirus subgroup-specific enzyme-immunoassays (ELISA) and neutralisation of infectivity with serotype-specific antisera . All the isolates clearly belonged to one of two subgroups, but four distinct serotypes were represented . Furthermore, the recently reported fourth serotype (ST4) and two isolates from Japan (Hochi and Hosokawa) appeared to be related to a South American rotavirus strain reported in 1978 as a probable fourth serotype.

Med Microbiol Immunol (Berl), 1984, 172(4), 207 - 13
Hepatitis A-virus in cell culture . IV . Comparison of cell-culture-produced HAV with stool-derived HAV in diagnostic test systems; Flehmig B et al.; Hepatitis A-virus produced in Frhk-4/R cells is used and compared with stool-derived HAV as an antigen in diagnostic test systems . It is shown that the two antigens react identically in the anti-HAV test with anti-HAV-IgG positive human sera . In the anti-HAV-IgM test, too, stool-derived HAV and cell-culture-produced HAV react very similarly . The titres of anti-HAV-IgG and anti-HAV-IgM positive sera, obtained with stool and cell-culture HAV in quantitative antibody determinations, are shown to be identical . It is shown that the problem of HAV antigen production can now be solved by propagation of HAV with Frhk-4/R cells and that this antigen is very useful in diagnostic test systems.

Peptides, 1984 Jan-Feb, 5(1), 47 - 51
Angiotensin II mediates beta endorphin like immunoactivity release in rat pituitary cell culture; Sobel DO; The effect of angiotensin II (Ang II) on pituitary beta endorphin like immunoactivity (beta END-LI) release was studied in monolayer culture of normal rat pituicites . Ang II stimulated beta END-LI release into the culture media . This release of beta END-LI increased with longer incubation time and with higher doses of Ang II . The beta END-LI response was similar to the pattern of Ang II mediated ACTH release . Ang II stimulated beta END-LI release was blocked by cycloheximide and decreased by corticosterone (5 nmol/l) . Successively higher concentrations of {SAR GLY}Ang II, a known Ang II antagonist, induced greater inhibition of Ang II stimulated beta END-LI release . Gel chromatography of pooled media from control and Ang II stimulated cells revealed three peaks of beta END-LI which migrated with the void volume, beta lipotropin (beta LPH) and beta endorphin . The relative amount of beta END-LI in these peaks {(BEND-LI peak + total beta END-LI in column) x 100%} from media of control and stimulated cells were as follows: (1) Void 7% and 19% (2) beta LPH 50% and 52% (3) beta endorphin 43% and 29%.

J Infect, 1984 Jan, 8(1), 22 - 7
A simple immunoperoxidase method for detecting enteric adenovirus and rotavirus in cell culture; Cevenini R et al.; A technique which includes the use of indirect immunoperoxidase antibody (IPA) has been developed for detecting enteric adenovirus and rotavirus antigens in cell cultures and has been compared with immunofluorescence antibody assay (IFA) . The IPA technique was as sensitive as the IFA . The number of positive cells detected by both techniques in tissue cultures was the same; false positive results were not observed . The applicability of IPA in clinical virology is discussed.

Proc Soc Exp Biol Med, 1984 Jan, 175(1), 84 - 7
Viral enhancement and interference induced in cell culture by hepatitis A virus: application to quantitative assays for hepatitis A virus; Hurni WM et al.; Hepatitis A virus (HAV) growing in human diploid lung fibroblast (MRC5) monolayers can either interfere with or enhance the cytopathic effect of Newcastle Disease virus (NDV) challenge . Enhancement of NDV occurred if HAV-infected monolayers were challenged with a low multiplicity of infection of NDV and incubated at 35 degrees C . Interference occurred if HAV-infected monolayers were given a high NDV multiplicity of infection and incubated at 32 degrees C . These phenomena were applied to assays for quantifying HAV and may be useful in providing new insights into viral interference and enhancement.

Endocrinology, 1984 Jan, 114(1), 222 - 6
Insulin-mediated inhibition of tyrosinase activity and protein synthesis in melanoma cell cultures; Fuller BB et al.; Insulin lowers basal levels of tyrosinase activity and inhibits the MSH-induced increase in tyrosinase in Cloudman S-91 mouse melanoma cell cultures . Insulin exerts its inhibitory effects in a typical dose-response manner, with maximal inhibition of enzyme activity occurring at 10-7 M . At maximal inhibition, tyrosinase activity is reduced to approximately 50% of the control levels . This inhibition precedes the observed inhibitory effect on cellular proliferation . Insulin not only lowers cell responsiveness to MSH, but also inhibits the tyrosinase stimulation produced by either theophylline or (Bu)2cAMP . Neither control levels nor MSH-mediated elevated cellular levels of cAMP were altered by insulin (10-7 M) . These findings suggest that insulin exerts its inhibitory effects at a site distal to cAMP production . The inhibitory effect of insulin on tyrosinase activity could not be mimicked by either (Bu)2cGMP or 8-bromo-cGMP, suggesting that insulin does not exert its effects by altering cellular levels of this nucleotide . Insulin reduces the rate of incorporation of {3H}leucine into trichloroacetic acid-precipitable material by 50%, a finding which suggests that insulin may exert its inhibitory effects on tyrosinase activity and perhaps on cellular proliferation by causing a general reduction in protein synthetic rates.

J Clin Endocrinol Metab, 1984 Jan, 58(1), 134 - 42
A pituitary tumor producing high molecular weight adrenocorticotropin-related peptides: clinical and cell culture studies; Fuller PJ et al.; The tissue-specific processing of proopiomelanocortin (POMC), the precursor of ACTH, beta-endorphin, and their related peptides, is currently of considerable interest . We report a patient with a large aggressive pituitary tumor, Cushing's syndrome, and hyperpigmentation managed by transsphenoidal hypophysectomy, bilateral adrenalectomy, and sellar irradiation . Preoperatively, plasma levels of immunoreactive ACTH (ir-ACTH; 280 ng/liter) and beta-endorphin (ir-beta EP; 520 ng/liter) were moderately elevated . Chromatography of the plasma showed two peaks of ACTH immunoreactivity, with the major peak eluting in the void volume, and two major peaks of ir-beta EP, corresponding to the elution positions of beta-lipotropin and beta-endorphin standards . Plasma ir-ACTH and ir-beta EP were not suppressed by high doses of glucocorticoid or bromocriptine, a degree of autonomy more commonly found with POMC production from ectopic sources than that from pituitary tumors . Tissue removed at operation was enzymatically dispersed, and the cells were cultured in suspension, propagated, and passaged sequentially for over 20 passages . Using this cell line, we demonstrated that the biosynthesis of POMC, its pattern of processing, and the release of POMC/ir-beta EP/ir-ACTH in vitro were consistent with the in vivo evidence of autonomous secretion and abnormal processing of POMC by this pituitary tumor.

Immunol Lett, 1984, 8(4), 165 - 8
Depression of direct plaque-forming cell response in mouse spleen cell cultures by aggregated IgG2b-induced factors; La Via MF et al.; Mouse spleen cells were treated with concanavalin A (Con A) or aggregated mouse IgG2b for 48 h in culture . When cells thus treated were added to fresh mouse spleen cell cultures immunized with SRBC they depressed the response of B lymphocytes as measured by enumerating plaque forming cells (PFC) on the fourth day of culture . When supernatant from cells cultured with IgG2b was added to immunized cultures this resulted in depression of PFC generation similar to that observed by addition of treated cells . The depression observed was essentially in the same range as that observed by addition of Con A treated cells or their supernatant . These observations extend previous work suggesting that IgG2b-induced PFC depression may result from activation of suppressor T cells with elaboration of soluble suppressor factors . This mechanism of immunomodulation may be important in the pathogenesis of immune complex disorders.

Exp Cell Res, 1984 Jan, 150(1), 68 - 76
A system for the study of epidermal G1-chalone in cell culture . The rat tongue epithelial cell line RTE2; Richter KH et al.; A pig-skin preparation enriched in epidermal G1-chalone when administered to cells of the rat tongue epithelial line RTE2 at concentrations of 3-300 micrograms/ml (dry mass) caused a 60% reduction in cell number . Three other cell lines showed essentially no growth inhibition during chalone treatment . The kinetics of chalone inhibition were similar to those observed in mouse epidermis in vivo . Five hours after the addition of chalone preparation in fresh medium a decrease in the rate of DNA synthesis was observed . Maximum inhibition at 12 h was followed by a subsequent increase in DNA synthesis, reaching control values again after 30 h . The inhibitory effect was dose-dependent up to 3 micrograms/ml . At higher concentrations the degree of inhibition remained constant at about 50% of the control up to 300 micrograms/ml . Removal of added chalone by changing the medium at the time of maximum inhibition gave rise to a complete recovery within 9 h . These results indicate a cell-line specific, non-toxic and reversible inhibitory effect of the chalone preparation which resembles that observed in the living animal . The RTE2 cell line may thus be considered to provide a highly sensitive experimental system suitable for more detailed studies on the mechanism of action of epidermal G1-chalone.

Clin Exp Immunol, 1984 Jan, 55(1), 99 - 105
Neutral proteinases induce rheumatoid factor production in mouse spleen cell cultures; Vischer TL; Mouse spleen cells were cultured for 4 days in RPMI 1640 medium with 5% fetal calf serum . The neutral proteinases trypsin and plasmin, and bacterial lipopolysaccharide LPS, all polyclonal B lymphocyte activators, stimulated the development of immunoglobulin producing cells as detected by the protein A plaque assay . At the same time, direct plaque forming cells reacting with mouse, human and rabbit IgG and the Fc fragment of human IgG were induced by the stimulants . The plaques could be inhibited by free IgG or Fc fragment . In the culture supernatants, IgM and IgM anti-IgG antibodies were detected by enzyme linked immunosorbent assays . Both general IgM and IgM anti-IgG antibodies increased under the influence of the proteinases and of LPS . The results are discussed in relation to rheumatoid factor production during inflammatory diseases.

Med Microbiol Immunol (Berl), 1984, 173(2), 95 - 103
Metabolic studies on vaccinia-virus-infected oligodendrocytes in brain cell cultures; Beranek CF et al.; Twelve-day-old cultures of dissociated newborn mouse brain were infected with neurotropic vaccinia virus strain WR . Using the indirect immuno-fluorescence staining technique the total destruction of galactocerebroside (GL) or myelin basic protein (MBP)-positive oligodendrocytes could be detected after 72 h of infection . The activity of the oligodendrocyte-specific enzymes, cerebroside sulfotransferase (CST) and 2'3'-cyclic nucleotide 3'-phosphohydrolase (CNP), was 27% and 17% respectively of the activity in noninfected controls . This reduction was not a result of viral-induced inhibition of host protein synthesis . In cultures treated with puromycin GC- and MBP- positive oligodendrocytes were detectable at a time at which no CST or CNP activity could detected.

J Neural Transm, 1984, 59(4), 309 - 17
Herpes simplex virus persistence in mouse neuroblastoma (C 1300) cell cultures: role of interferon; Dawson GJ et al.; The Mp strain of herpes simplex virus type 1 (HSV1) induced a persistent infection in the mouse C 1300 neuronal cell line (clone N 115) . C 1300 cultures infected at an MOI of 0.01 or 0.001 survived the initial infection and continued to produce infectious virus and viral antigens for 185 days and 31 days, respectively . Viral antigens were not detected in cultures no longer producing infectious virus; these "cured" cultures had comparable susceptibility to reinfection with HSV as previously uninfected C 1300 cells . While significant amounts of interferon were produced by C 1300 cells when challenged with Newcastle Disease Virus (NDV) or when treated with poly I:C, HSV-induced interferon could not be detected in either the acutely or persistently infected cell lines . The persistent state was not significantly altered by the addition of 1,000 units/ml of murine interferon alpha plus beta (MuIFN alpha + beta), nor was it affected by the addition of antibody to MuIFN . It appears that IFN does not play an important role in the establishment and/or maintenance of viral persistence in this neuronal system.

Placenta, 1984 Jan-Feb, 5(1), 41 - 53
Identification and estimation of cell types in mixed primary cell cultures of early and term human placenta; Contractor SF et al.; Placental villi of early and term human placentae were dissociated with trypsin and mixed cell cultures were established . The different cell types were identified and estimated over a 14-day culture period using antibodies to keratin and vimentin filaments and their capacity to phagocytose yeast . The three main cell types were found to be epithelial-, macrophage- and fibroblast-like cells . The epithelial-like cells can be further divided into the multinucleated and the small- and medium-sized round cells, and these are most likely to be derived from the trophoblast . The cellular composition of cultures were different for early and term placentae and also varied characteristically over the 14-day cultures period.

Cell Immunol, 1984 Jan, 83(1), 1 - 13
Cell culture of mammalian thymic epithelial cells: growth, structural, and antigenic properties; Sun TT et al.; The thymus plays an important role in the maturation and differentiation of T lymphocytes . Many of its functions have been attributed to its epithelial component . Past in vitro studies of putative thymic epithelial cells have been hampered by the inability to produce well-characterized cultures of these cells . Using lethally irradiated 3T3 cells as a feeder layer, we have succeeded in growing virtually pure cultures of thymic epithelial (TE) cells from rabbits, mice, and humans . Antikeratin staining provides an unambiguous criterion for positive identification of the epithelial cells . These cells were found to lack Ia-like or theta-like antigens . The ability to culture large quantities of mammalian TE cells should allow for their detailed functional characterization.

Am J Clin Pathol, 1984 Jan, 81(1), 43 - 7
Rapid detection of herpes simplex virus using a combination of human fibroblast cell cultures and peroxidase-antiperoxidase staining; Meyer MP et al.; A method for rapid detection of Herpes simplex virus (HSV), using a combination of human fibroblast cell cultures with peroxidase-antiperoxidase (HF-PAP) staining after 18-24 hours of incubation was developed . The sensitivity of the HF-PAP, compared with viral isolation using primary rabbit kidney and human fibroblast cells, was 88.4% with 20.7% of clinical Herpes isolates detected by HF-PAP before any signs of cytopathic effect (CPE) of HSV were present . The specificity of the new procedure was 98.8%, and overall 739 of 761 specimens (97.1%) were identified correctly by HF-PAP . This system can be used to confirm preliminary or questionable CPE . All of the specimens that exhibited possible CPE at 18-24 hours and from which the virus eventually was isolated were also positive by HF-PAP . However, this method was less sensitive for specimens with smaller amounts of virus . Thus, this method provides a rapid and improved, although not foolproof, means of HSV detection.

Teratog Carcinog Mutagen, 1984, 4(4), 349 - 64
Development of a mouse embryo limb bud cell culture system for the estimation of chemical teratogenic potential; Guntakatta M et al.; High-density cultures of mouse embryo limb bud cells differentiate and synthesize an extracellular matrix containing sulfated proteoglycans . Since these in vitro events are related to those that occur during fetal development, we have investigated the use of cultured limb bud cells for the analysis of the activities of chemical teratogens . We have established the conditions for the use of the radiochemicals 35SO=4 and 3H-thymidine for the assessment of chemical effects on sulfated proteoglycan and DNA synthesis, respectively, in mouse limb bud cells . By performing double-labeling experiments in the presence of test chemicals, the preferential inhibition of either proteoglycan synthesis or DNA synthesis could be demonstrated for 19 of 22 known mouse teratogens tested . The five nonteratogens tested did not cause significant differences in the inhibition of either macromolecular class . The overall predictive accuracy of the system was approximately 89%, and the false-negative rate was approximately 14.8% . No false positives were observed . These data showed that this cell culture system differentiated between the activities of a limited set of selected mouse teratogens and nonteratogens, suggesting that cultured mouse embryo limb bud cells may constitute the basis for the development of a powerful tool for analyses of chemical teratogenic potential.

Adv Exp Med Biol, 1984, 165 Pt A, 373 - 9
IMP dehydrogenase mutants: cell culture model for hyperuricemia; Ullman B; These studies with wild-type and mutant cells defective in IMP dehydrogenase and the previous data with the adenylosuccinate synthetase-deficient cell line suggest that among the clinical population with dominantly inherited hyperuricemia, patients with partial deficiencies in these enzymes exist . It is hoped that these pharmacogenetic cell culture models for overproduction hyperuricemia will lead to the initiation of a search for hyperuricemia patients with either of these deficiencies . If such patients are found it may be possible to design chemotherapeutic regimens by which effectors (inhibitors) of purine synthesis might ameliorate the overproduction of purines by the de novo pathway.

Clin Exp Hypertens A, 1984, 6(10-11), 1829 - 32
Angiotensin-converting enzyme, enkephalinase A and aminopeptidases in the breakdown of enkephalin--studies in cell cultures; Lentzen H et al.; Neuroblastoma cells (N1E-115) and glial cells possess aminopeptidases, dipeptidyl carboxypeptidases and dipeptidyl aminopeptidases, which are located in the plasma membranes . Neuronal cells possess angiotensin-converting enzyme (ACE), which splits the Gly-Phe bond and generates Tyr-Gly-Gly from enkephalin . They have no enkephalinase A . In contrast, glial cells possess enkephalinase A but no ACE . Not only the dipeptidyl carboxypeptidases on neuronal and glial cells are different . These cells have aminopeptidases which have to be distinguished as well . This differentiation is made by using bestatin as an aminopeptidase inhibitor and reveals peptidases of high and poor bestatin-sensitivity.

Arch Oral Biol, 1984, 29(10), 827 - 31
Mechanical and hormonal stimulation of cell cultures derived from young rat mandible condyle; Shimshoni Z et al.; Collagenase digestion of young rat condyles released cells which were grown in culture during two weeks . Morphologically, two populations of cells were distinguished, one of which reacted to alkaline phosphatase and resembled chondroblast or osteoblast-like cells . Parathyroid hormone stimulated a 2-fold increase in cellular cyclic AMP, whereas calcitonin had no effect . Physical forces activated cellular cyclic AMP to a 2.5-fold of control levels and increased the incorporation of radioactive thymidine into DNA by 50 per cent . In contrast to cultured bone cells, the response to physical forces was not inhibited by indomethacin in cultured condyle cells . It seems, therefore, that condyle cells are specific in their response to bone-seeking hormones and physical forces.

Arch Virol, 1984, 82(1-2), 119 - 23
Isolation of murine rotavirus in cell cultures . Brief report; Tajima T et al.; Murine rotavirus was isolated in primary monkey kidney cells . Electrophoretic pattern of genome RNA of murine rotavirus was different from that of human rotaviruses . Oral administration of cultured murine rotavirus caused mild diarrhea in newborn BALB/C mice.

Med Microbiol Immunol (Berl), 1984, 173(1), 9 - 17
Hepatitis A-virus in cell culture . V . Neutralizing antibodies against hepatitis A-virus; Zahn J et al.; A test system for the detection of neutralizing antibodies against hepatitis A-virus (anti-HAV-Nt) is presented . The anti-HAV-Nt assay is performed with Frhk-4/R cells and the hepatitis A virus (HAV) strain GBM/Frhk-4/R which has been adapted to these cells . Non-neutralized HAV is demonstrated 14 days after infection of Frhk-4/R cells using a radio-immunoassay for detecting newly grown HAV . The influence of differing amounts of HAV on the anti-HAV-Nt titre and the effect of variations in incubation time of virus-serum mixtures are described . The time course of anti-HAV-Nt is shown in sera from a hepatitis-A patient which were taken at different stages of the disease . Anti-HAV-Nt is compared with anti-HAV and anti-HAV-IgM . It is shown that anti-HAV-Nt correlates closely with anti-HAV and separated anti-HAV-IgG, but only slightly with anti-HAV-IgM . The test system presented makes possible the demonstration of neutralizing antibodies against HAV in gamma-globulin preparations and during vaccination studies.

Arch Immunol Ther Exp (Warsz), 1984, 32(4), 459 - 65
Cell cultures of spotted souslik--preliminary characterization of their growth and sensitivity to viruses; Kandefer-Szerszen M et al.; Cell cultures from the cutaneo-muscular tissue of fetuses (SE), and from lungs (SL), kidneys (SK), testes (STe) of adult spotted sousliks were obtained . Cells from lungs (SL) were viable at 4 degrees C three times longer than fibroblastic cells of human embryos (HEF) . At 37 degrees C the growth rate of SL, HEF and L929 cells was similar . However, at 26 degrees C SL cells grew faster than HEF or L929 cells and their population doubling time was shorter . The cultures of SE, SL and SK cells were sensitive to vesicular stomatitis virus (VSV), Newcastle disease virus Radom (NDV-R) and Hertfordshire (NDV-H) strains, to vaccinia virus and herpes simplex virus type 1 and type 2 . All these viruses were able to reproduce in souslik cell cultures and caused characteristic CPE.

Prog Neuropsychopharmacol Biol Psychiatry, 1984, 8(4-6), 481 - 6
Cell culture as models for studying neural functions; Hamprecht B; Two cell culture systems were used for studies of neural functions in vitro . A neuronal hybrid cell line (neuroblastoma x glioma hybrid cells) and primary glial-rich cultures of newborn murine brain . The level of cyclic AMP in both systems is regulated by two groups of hormones, those that stimulate and those that inhibit formation of cyclic AMP . Among the inhibitory hormones active on the hybrid cells are opioids . Therefore the cells are being used in the elucidation of action of opioids . The list of stimulating and inhibitory hormones regulating the primary glial-rich cultures includes several peptide hormones such as the gastrointestinal peptides secretin and vasoactive intestinal peptide, the calcaemic hormones parathyrin and calcitonin, adrenocorticotropin and melanotropins, and somatostatin . Noradrenaline (via alpha- and beta-adrenergic receptors) and adenosine (via A1 and A2 receptors) inhibit and stimulate cyclic AMP synthesis in the primary glial-rich cultures . Bradykinin slowly hyperpolarizes the hybrid cells and elicits formation of cyclic GMP . Both responses desensitize rapidly . Substance P increases the permeability of hybrid cells for Na+, as measured by using 14C-guanidinium as substitute for Na+ . Hybrid cells actively accumulate taurine, an amino acid that appears to fulfill important functions in the nervous system . The transport of taurine across the plasma membrane is highly specific for and strictly dependent on Na+ . The pumped station hypothesis of taurine action in the nervous system views taurine gradient plus taurine carrier as a transport system for the elimination of sodium from neurons during phases of high neuronal activity.

Brain Res, 1983 Dec 19, 289(1-2), 99 - 107
Action potential mechanism of mammalian cortical neurons in cell culture; Dichter MA et al.; Rat cortical neurons in 4- to 7-week-old dissociated cell cultures have action potentials (APs) which are tetrodotoxin-sensitive and appear to be generated by conventional sodium mechanisms . Approximately 10% of neurons can also generate APs when sodium channels are blocked . These latter APs are smaller, slower and sensitive to cobalt . When voltage sensitive potassium channels are blocked by TEA or when Ba is substituted for calcium, all the neurons are capable of generating large, overshooting, slow and prolonged, calcium-dependent APs . The amplitudes of these APs are proportional to the log of the divalent cation concentration and the APs are blocked by either cobalt or verapamil . We conclude that under physiological conditions, calcium currents are not likely to play a major role in the AP morphology in 'mature' neurons, but are more likely to be involved with other aspects of cell excitability and neurotransmitter function . APs in younger neurons (3-4 weeks in vitro) have a large calcium component even under physiological conditions.

Brain Res, 1983 Dec 12, 288(1-2), 354 - 8
Benzodiazepine receptor binding by membranes from brain cell cultures following chronic treatment with diazepam; Prezioso PJ et al.; Murine brain cells differentiated for 19 days in culture before treatment for 5 days with 10 microM diazepam . Radioligand binding to the benzodiazepine receptors on membranes obtained from these cultures was determined by the filtration assay method . Decreased binding was observed in the membranes from treated cultures relative to untreated cells . However, this decrease appears to be due, at least in part, to competition from residual drug in the assay system despite extensive cell and membrane rinses . These data emphasize the difficulty in ascribing the mechanism of benzodiazepine tolerance observed clinically with chronic treatment to receptor down-regulation as determined by binding assays.

Biochemistry, 1983 Dec 6, 22(25), 5687 - 92
Effects of 3 beta-{2-(diethylamino)ethoxy}androst-5-en-17-one on the synthesis of cholesterol and ubiquinone in rat intestinal epithelial cell cultures; Sexton RC et al.; The relationship between cholesterol and ubiquinone synthesis in rat intestinal epithelial cell cultures was examined by using 3 beta-{2-(diethylamino)ethoxy}androst-5-en-17-one hydrochloride (U18666A) . Addition of U18666A to cells caused a greater than 90% inhibition of incorporation of {3H}acetate into cholesterol and an apparent large increase in the incorporation of {3H}acetate and {3H}mevalonate into ubiquinone . However, the incorporation of 4-hydroxy{U-14C}benzoate, a ring precursor of ubiquinone, was unchanged . The apparent increase of 3H incorporation into ubiquinone was found to be due to the formation of a contaminant that has been identified as squalene 2,3:22,23-dioxide . Following incubation of cells with U18666A, its removal from the medium resulted in a decrease in squalene 2,3:22,23-dioxide labeling and a corresponding increase in the polar sterol fraction . These results demonstrate that U18666A inhibits the reaction catalyzed by 2,3-oxidosqualene cyclase (EC 5.4.99.7) . As a result, the isoprenoid precursors are diverted not to ubiquinone as has been suggested but to squalene 2,3:22,23-dioxide, a metabolite not on the cholesterol biosynthetic pathway . Removal of the drug allows cyclization of squalene 2,3:22,23-dioxide, leading to formation of compounds with chromatographic properties of polar sterols.

Environ Res, 1983 Dec, 32(2), 466 - 73
Cellular toxicity in Chinese hamster ovary cell cultures . II . A statistical appraisal of sensitivity with the rabbit alveolar macrophage, Syrian hamster embryo, BALB 3T3 mouse, and human neonatal fibroblast cell systems; Garrett NE et al.; Chinese hamster ovary, rabbit alveolar macrophage, Syrian hamster embryo, BALB 3T3 mouse, and human neonatal fibroblast cells were employed in a statistical evaluation of the relative sensitivity of the cells to toxic substances . The cells were exposed to 1,2,4-trichlorobenzene, 2,4-dimethylphenol, Aroclor 1248, cadmium chloride, lead sulfate, nickel nitrate, lead oxide-coated fly ash, and a fine particulate from coal combustion . A filter-disk technique was used to measure the inhibition of protein and DNA synthesis . A quantitative ranking of cell-system sensitivity was determined from comparisons of statistically significant differences (P less than or equal to 0.01) in protein and DNA synthesis expressed as a percentage of control . An overall ranking of sensitivity showed that rabbit alveolar macrophages, Syrian hamster embryo cells, and Chinese hamster ovary cells were more sensitive than another of the five cell systems in 75, 68, and 62% of the experiments, respectively . The corresponding values for BALB 3T3 mouse and human neonatal fibroblast cells were 38 and 28%, respectively, under our experimental conditions . Detailed data on the control cell cultures are also presented.

Appl Biochem Biotechnol, 1983 Dec, 8(6), 505 - 13
Cell culture on polymers prepared by radiation-induced grafting of various monomers; Yoshii F et al.; The adhesion and growth of tissue cells on polymers prepared by radiation grafting was investigated . The apparent rates of initial attachment and growth of Chang liver and C6 cells were promoted on surfaces with increased wettability and with a heterogeneous structure for grafted polyvinyl fluoride film . The degree of cell attachment and growth on surfaces having a dense microblock structure, formed by grafting of methyl methacrylate in acetone solvent, was greater than that caused by other factors, such as wettability.

Xenobiotica, 1983 Dec, 13(12), 725 - 30
Induction of hepatic cytochrome P-450 and drug metabolism by metyrapone in the rat: relevance to its effects in rat-liver cell culture; Lake BG et al.; The ability of metyrapone to induce hepatic cytochrome P-450 and xenobiotic metabolism in rats and rat hepatocyte culture has been studied . Metyrapone is a phenobarbitone-type inducer in rats . When rat hepatocytes were cultured with beta-naphthoflavone, a polycyclic hydrocarbon-type induction pattern was observed . In contrast, the observed induction pattern of both phenobarbitone and metyrapone in cell culture differed from that obtained with these compounds in vivo . The relationship between the nature of the induction of hepatic xenobiotic metabolism by metyrapone in vivo and its effects in liver-cell culture are discussed.

Dev Biol, 1983 Dec, 100(2), 478 - 88
Regenerating adult chicken skeletal muscle and satellite cell cultures express embryonic patterns of myosin and tropomyosin isoforms; Matsuda R et al.; Regenerating areas of adult chicken fast muscle (pectoralis major) and slow muscle (anterior latissimus dorsi) were examined in order to determine synthesis patterns of myosin light chains, heavy chains and tropomyosin . In addition, these patterns were also examined in muscle cultures derived from satellite cells of adult fast and slow muscle . One week after cold-injury the regenerating fast muscle showed a pattern of synthesis that was predominately embryonic . These muscles synthesized the embryonic myosin heavy chain, beta-tropomyosin and reduced amounts of myosin fast light chain-3 which are characteristic of embryonic fast muscle but synthesized very little myosin slow light chains . The regenerating slow muscle, however, showed a nearly complete array of embryonic peptides including embryonic myosin heavy chain, fast and slow myosin light chains and both alpha-fast and slow tropomyosins . Peptide map analysis of the embryonic myosin heavy chains synthesized by regenerating fast and slow muscles showed them to be identical . Thus, in both muscles there is a return to embryonic patterns during regeneration but this return appears to be incomplete in the pectoralis major . By 4 weeks postinjury both regenerating fast and slow muscles had stopped synthesizing embryonic isoforms of myosin and tropomyosin and had returned to a normal adult pattern of synthesis . Adult fast and slow muscles yielded a satellite cell population that formed muscle fibers in culture . Fibers derived from either population synthesized the embryonic myosin heavy chain in addition to alpha-fast and beta-tropomyosin . Thus, muscle fibers derived in culture from satellite cells of fast and slow muscles synthesized a predominately embryonic pattern of myosin heavy chains and tropomyosin . In addition, however, the satellite cell-derived myotubes from fast muscle synthesized only fast myosin light chains while the myotubes derived from slow muscle satellite cells synthesized both fast and slow myosin light chains . Thus, while both kinds of satellite cells produced embryonic type myotubes in culture the overall patterns were not identical . Satellite cells of fast and slow muscle appear therefore to have diverged from each other in their commitment during maturation in vivo.

Infect Immun, 1983 Dec, 42(3), 1168 - 75
Characterization of Spiroplasma mirum (suckling mouse cataract agent) in a rabbit lens cell culture; Megraud F et al.; Spiroplasma mirum (suckling mouse cataract agent) was studied in an epithelial cell line AG-4676, derived from rabbit eye lens . Rabbit eye lens is a natural target tissue of S . mirum infection . The organism grew rapidly in this cell line, reaching titers of 10(7) to 10(9) color change units per ml at 7 days after infection . This is the same level as that achieved in SP-4 medium designed specifically for S . mirum . No lag period was apparent in growth in AG-4676 . S . mirum did not grow in Dulbecco minimal essential medium-10% fetal bovine serum, the medium for AG-4676, indicating the need for cells or a cellular product . S . mirum-infected AG-4676 cells exhibited vacuolization and granulation and an increase in polynucleation compared with uninfected controls (36/100 versus 14/100, P less than 0.001) . Infection significantly decreased the growth rate of AG-4676, especially late in the growth cycle . In a representative experiment, growth of AG-4676 at 11 days was reduced from 9 X 10(5) to 2 X 10(4) cells by S . mirum infection . S . mirum grew to high titers in conditioned medium of AG-4676, obtained from cell-free supernatants of 1- to 5-day-old AG-4676 cultures . This growth promotion was not due to osmotic conditioning of the medium . Preliminary characterization of this growth promotion substance showed it to be active after 0.22-micron filtration, heating at 56 degrees C for 30 min, freezing and thawing, and dilution at 10(-1) but not 10(-2) . AG-4676-propagated S . mirum produced death or cataracts in suckling Wistar rats at the same frequency (55/60, 91.7%) as SP-4-propagated organisms (60/65, 92.3%).

Environ Res, 1983 Dec, 32(2), 455 - 65
Cellular toxicity in Chinese hamster ovary cell cultures . I . Analysis of cytotoxicity endpoints for twenty-nine priority pollutants; Garrett NE et al.; Chinese hamster ovary cells were exposed to 29 toxic chemical substances which were representative of several classes of compounds listed by the Natural Resources Defense Council Consent Decree as priority toxic pollutants . After cell cultures were exposed to the test substance, cell samples were assayed for protein and DNA synthesis, ATP, cell number, and viability . A filter-disk technique employing a batch-washing procedure was used for the determination of protein and DNA synthesis . Dose-response data were obtained for 15 of the more toxic agents including chlorinated aromatics, metallic compounds, phenols, and polychlorinated biphenyls . Estimates of the sample concentrations necessary to produce a 50% reduction in response were used to compare cytotoxicity endpoints . ATP and protein synthesis were approximately equally effective as indicators of cellular toxicity . Cadmium chloride, nickel nitrate, arsenic trioxide, and potassium chromate produced a more pronounced effect on DNA synthesis than on ATP or protein synthesis . The dose-response relationship for protein and DNA synthesis was a smooth, continuous function for responses as low as 1 to 2% of the control . On a log-log scale, the dose-response relation yielded a unique pattern for several chemical classes . A ranking of the compounds based on their inhibition of DNA synthesis was compared to a ranking obtained from the literature for whole-animal toxicity . With the exception of the polychlorinated biphenyls, the in vitro results correlated well with animal test data.

Hum Immunol, 1983 Dec, 8(4), 249 - 54
Strong HLA-DR expression in T cell cultures after activation is necessary for IL-2-dependent proliferation; Effros RB et al.; We have examined the appearance of DR antigens on human T cells activated by PHA, using a monoclonal anti-DR framework antibody and a large panel of human HLA typing sera . Strong DR expression within the culture by day 8 was associated with the ability of cells to grow for long periods in IL-2 containing medium, whereas weak or absent DR expression was predictive of poor in vitro growth . All the cells responded equivalently to initial stimulation with PHA . These data support the hypothesis proposed by Moretta et al . to explain blocking of IL-2-dependent proliferation by an anti-DR antibody--that DR molecules may be involved in the transmission of signals by IL-2.

Tropenmed Parasitol, 1983 Dec, 34(4), 225 - 8
Pleomorphism in trypomastigotes of Trypanosoma cruzi from blood and cell culture; Urdaneta-Morales S; The sequential variation in the body type of trypomastigotes of the 'Brazil' strain of Trypanosoma cruzi was followed in 4 experimental models: Cultures of Vero fibroblasts incubated at 37 degrees C . Cultures of fish epithelial cells (Pimephales promelas) incubated at 37 degrees C . Blood of C3H mice inoculated with trypomastigotes from 37 degrees C Vero cell culture . Blood of C3H mice inoculated with trypomastigotes from blood of CF1 mice . In both animal models, the picture was similar: a great preponderance of the slender forms in the first days post-infection, followed by a rapid decline while the broad forms reached a peak level; the broad forms in turn declined in the final days, while the stout forms rose sharply in relative number . In the cell culture models, the same tendencies were visible, though less strongly marked . The possible biological and epidemiological significance of this constant pattern of pleomorphism is discussed.

Am J Vet Res, 1983 Dec, 44(12), 2376 - 8
Variation of virus replication in primary bovine kidney cell cultures derived from 68 three-day-old calves; Stauber EH et al.; Primary kidney cell cultures were prepared from 68 three-day-old calves . Complete monolayers of these cultures were infected separately with viral diarrhea, infectious bovine rhinotracheitis, parainfluenza, and adenovirus 7 viruses . The yield of virus from all infected cultures was calculated by plaque titer assay after 2 to 4 days' incubation . The variation of virus yield was substantial between individual cultures.

J Pharmacol Exp Ther, 1983 Dec, 227(3), 779 - 89
Multiple actions of phenytoin on mouse spinal cord neurons in cell culture; McLean MJ et al.; Concentration-dependence of multiple actions of phenytoin (PT) on mouse spinal cord neurons in primary dissociated cell culture was studied using intracellular microelectrode recording techniques . At concentrations of 2 to 50 micrograms/ml, PT did not alter resting membrane potential or input resistance . At 1 to 2 micrograms/ml, equivalent to therapeutic cerebrospinal fluid concentrations, PT limited the ability to sustain high-frequency repetitive firing of action potentials during long (500-2000 msec) depolarizing current pulses . There was a progressive reduction of maximal rate of rise (Vmax) of action potentials during the train until firing failed . Recovery of Vmax of single action potentials after repetitive firing was also prolonged . PT did not reduce Vmax of a single action potential at 1 to 2 micrograms/ml, but did so at 3 to 40 micrograms/ml in a voltage-dependent manner . Hyperpolarization partially reversed this reduction of Vmax . Thus, PT may slow recovery of sodium channels from inactivation . At concentrations above 3 micrograms/ml, PT reduced spontaneous neuronal firing with progressive increase in the number of quiescent neurons, reduced calcium-dependent action potential duration and amplitude, eradicated convulsant-induced paroxysmal bursting and augmented postsynaptic responses to iontophoretically applied gamma-aminobutyric acid . Glutamic acid responses were unaffected at PT concentrations of 10 micrograms/ml or less . These actions occurred at concentrations equivalent to toxic cerebrospinal fluid levels in patients and may be related to PT-induced toxicity . We suggest that limitation of sustained high-frequency repetitive firing may account, at least in part, for the anticonvulsant efficacy of PT.

Brain Res, 1983 Nov 21, 279(1-2), 207 - 16
Development of cholinergic properties in nerve cell cultures in the presence of brain extract; Barakat I et al.; Cells dissociated from cerebral hemispheres of 6-day-old chick embryos were cultured either in standard nutrient medium or in the presence of a brain extract from 8-day-old chick embryo . Morphological observations showed the development of bipolar and multipolar neurons in both culture conditions and acetylcholinesterase activity was found in all neuronal cells . Brain extract stimulated the morphological maturation of neurons, expressed by the formation of fiber bundles, fine structural maturation and development of synapses rich in clear vesicles . Furthermore, acetylcholinesterase and choline acetyltransferase activities were higher in the cultures treated with brain extract . In these cultures, the values of choline acetyltransferase activity reached a peak at 10 days and then decreased . These observations are discussed with particular reference to proliferation, maturation and degeneration of cholinergic neurons.

In Vitro, 1983 Nov, 19(11), 807 - 10
Success rate of primary human endothelial cell culture from umbilical cords is influenced by maternal and fetal factors and interval from delivery; Balconi G et al.; There is increasing interest in human umbilical cord vein as a source of endothelial cells . This paper shows that success in setting up cultures of human endothelial cells from umbilical cords depends not only on culture conditions, as so far proposed, but also on factors preceding the harvesting of the cells . In particular, the mother's smoking habit and the use of umbilical cord within 1 h of delivery have been shown to impair success of the culture . Age, parity, diabetes, and hypertension of the mother, type of delivery, and sex and weight of the newborn did not significantly influence the possibility of establishing successful endothelial cell culture.

J Steroid Biochem, 1983 Nov, 19(5), 1557 - 62
Melanocyte-stimulating hormone and prolactin secretion in cell culture of estrogen-induced pituitary tumors in the hamster; Peebles DC et al.; Pituitary cells from hamsters bearing diethylstilbestrol induced renal adenocarcinomas were cultured in vitro . Dispersed cells in plastic dishes were viable for up to two weeks in Dulbecco's modified Eagle's medium supplemented with 17.5% of 6:1 horse serum to fetal calf serum . The secretion of alpha-melanocyte stimulating hormone and prolactin into the medium were measured by radioimmunoassay . The concentrations of both were elevated by day 3 in the medium from the hyperplastic pituitaries obtained from the estrogen treated, tumor bearing hamsters . Neither DES (10(-8)M) nor tamoxifen (10(-7)M) influenced the secretion of either hormone and neither altered either cell number or DNA synthetic activity as measured by thymidine incorporation . The secretion of hormones and the growth of the pituitary cells were, however, decreased by charcoal treatment of the serum . The results suggest that the elevation of serum alpha-MSH and prolactin observed in DES implanted hamsters is due to pituitary secretion of the hormones but that DES probably does not act directly on the pituitary to control the secretion.

J Neurosci, 1983 Nov, 3(11), 2395 - 402
Differential regulation of acetylcholine sensitivity and alpha-bungarotoxin-binding sites on ciliary ganglion neurons in cell culture; Smith MA et al.; Levels of acetylcholine (ACh) sensitivity and numbers of alpha-bungarotoxin (alpha-Bgt)-binding sites have been measured for chick ciliary ganglion neurons grown in cell culture under various conditions . The two properties were found not to change in parallel . Neurons maintained in culture medium supplemented with embryonic eye extract developed high levels of ACh sensitivity and low numbers of alpha-Bgt-binding sites, whereas neurons grown in medium containing elevated K+ concentrations displayed the reverse . Neurons from media containing both eye extract and elevated K+ concentrations had both low levels of sensitivity and low numbers of toxin sites . The growth conditions do not alter the basic binding properties of the ACh receptors and alpha-Bgt-binding sites . Both the ACh receptor dose-response characteristics and the pharmacological properties of the toxin-binding sites were similar for neurons grown in media containing eye extract or elevated K+ concentrations . The inhibitory effects of eye extract on development of alpha-Bgt-binding sites appeared to be specific: eye extract had previously been shown to stimulate neuronal growth and cholinergic development, and in the present study eye extract enhanced development of ACh sensitivity and had no effect on mechanisms responsible for binding and accumulation of tetanus toxin . Eye extract did not block alpha-Bgt binding in competition binding experiments and did not cause redistribution of toxin sites away from the neuronal soma . These results demonstrate that ACh sensitivity and alpha-Bgt-binding sites can be independently regulated on the neurons and suggest that the two membrane properties are associated with separate membrane components.

Antimicrob Agents Chemother, 1983 Nov, 24(5), 674 - 8
Biological profile and response to anti-pneumocystis agents of Pneumocystis carinii in cell culture; Pifer LL et al.; Although the growth characteristics of Pneumocystis carinii have been described in several cell culture systems, the response of this organism to the drugs of choice, trimethoprim-sulfamethoxazole and pentamidine isethionate, have not been described in vitro . The effect of various concentrations of drugs against P . carinii on the growth of this potentially hazardous opportunistic organism as well as the methodology for in vitro assay of these agents have been detailed . Fluorescence profiles illustrating size ranges of trophozoites and cysts derived from cell culture are described.

J Immunol, 1983 Nov, 131(5), 2508 - 14
In vitro induction of cytotoxic polymorphonuclear leukocytes by supernatant from a concanavalin A-stimulated spleen cell culture; Inoue T et al.; Rat polymorphonuclear leukocytes (PMN) pretreated with supernatant of a spleen cell culture stimulated with concanavalin A (Con A sup) inhibited 3H-thymidine uptake of various syngeneic, allogeneic, and xenogeneic tumor cells . Con A sup-treated PMN were cytostatic not only to tumor cells but also to embryonal fibroblasts and normal Con A blasts . Stimulated PMN also killed some tumor cells, as shown by 3H-uridine release assay . Cytostasis by Con A sup-treated PMN began at a very early phase of incubation, but in the cytolysis assay, incubation for more than 12 hr was required to obtain significant killing . The time required for activation of cytostatic PMN by Con A sup was short (5 min was enough) if high titers of Con A sup were used for activation . PMN-stimulating activity of Con A sup was not abrogated by treatment at 60 degrees C for 30 min or by change of pH from 1 to 11 . The mechanisms of cytotoxicity by Con A sup-treated PMN are discussed.

Vopr Virusol, 1983 Nov-Dec, 28(6), 673 - 5
{Effect of ribamidil on the reproduction of the herpes simplex virus in a CV1 cell culture}; Linitskaia GL et al.; Cytotoxic properties and antiviral activity of abnormal nucleoside ribamidile were studied on the model of herpes simplex virus type 1 in CV1, continuous cell culture . The drug produced no cytotoxic effect even in concentrations of 500 and 250 micrograms/ml but had a marked capacity to inhibit herpes simplex virus reproduction in this system in concentrations of 125 and 62.5 micrograms/ml . Antiviral effect of ribamidile was enhanced by its combined use with 5'-deoxy-5'-fluorothymidine.

Antimicrob Agents Chemother, 1983 Nov, 24(5), 819 - 22
Kinetic analysis in cell culture of the reversal of antiherpes activity of nucleoside analogs by thymidine; Larsson A et al.; The inhibition of herpes simplex virus type 1 plaque formation by acyclovir, bromovinyldeoxyuridine, 9-(3,4-dihydroxybutyl)guanine, and 9-(4-hydroxybutyl)guanine at different thymidine concentrations was analyzed in Lineweaver-Burk plots . Linear competitive patterns between thymidine and the nucleoside analogs were observed for the inhibition of herpes simplex virus type 1 plaque formation . A new constant, the reversal constant Kr, was introduced to describe the sensitivity in cell culture of an antiviral drug to the reversal of its viral activity by a metabolite (e.g., thymidine).

Tsitologiia, 1983 Nov, 25(11), 1296 - 301
{Effect of the tumor promotor 12-o-tetradecanoyl phorbol-13--acetate on cyclic adenosine monophosphate levels in cell cultures . Relation to cell proliferation}; Ganelina LSh et al.; A considerable increase in the level of cyclic adenosine monophosphate (cAMP) was found in hepatoma cultures (clones G-10, G-1c) and L-cells (clones Lebr 625, Lebr f . s.) in 30, 60 and 120 minutes after their treatment with the tumor promoter 12-o-tetradecanoyl-phorbol-13-acetate (TPA) . In the mutant cells with changed membranes (clones G-1c, Lebr 625, Lebr 625 f . s.) the rising of cAMP was less expressed under the influence of TPA . The contents of cAMP decreases to the control level by 22 hours after their TPA treatment . A consequence of biochemical changes, leading to the tumor growth after TPA treatment of the cells, has been proposed . A considerable increment in cAMP amount is supposed to be a trigger in this chain.

Radiobiologiia, 1983 Nov-Dec, 23(6), 819 - 22
{Beta-adrenergic mechanism of the radio-protective effect of isoproterenol in a mammalian cell culture}; Chirkov IuIu et al.; A specific beta-agonist, isoproterenol, increased the intracellular content of cyclic AMP and decreased the radiosensitivity of Chinese hamster fibroblasts . Beta-antagonist, propranolol, blocked the manifestation of the effect of isoproterenol . Isoproterenol did not affect either the intracellular level of cyclic AMP or the radiosensitivity of B-82 cells which had no beta-adrenoreceptors.

Dev Biol, 1983 Nov, 100(1), 166 - 71
Tracing the development of oligodendrocytes from precursor cells using monoclonal antibodies, fluorescence-activated cell sorting, and cell culture; Abney ER et al.; We have used antibody and complement-mediated cell killing, fluorescence-activated cell sorting and tissue culture to study the development of rat oligodendrocytes . We show that (1) three ligands that bind to the majority of CNS neurons (the monoclonal antibodies A4 and A2B5 and tetanus toxin) also bind to immature oligodendrocytes and to precursor cells in 14-day embryonic rat brain that develop into oligodendrocytes in vitro; and (2) precursor cells in 17- to 18-day embryonic rat optic nerve can develop into oligodendrocytes in vitro in the absence of living neurons.

Biosci Rep, 1983 Nov, 3(11), 1027 - 34
Tyrosinase activity in primary cell culture of amelanotic melanoma cells; Slominski A et al.; After transfer of the Ab amelanotic melanoma cells from in vivo to in vitro growth conditions tyrosinase activity in their soluble fraction rapidly increased . This increase lasted to the middle of the logarithmic phase of growth and was followed by a decrease of tyrosinase activity, which was accompanied by accumulation of melanin in the cells . Calf serum stimulated simultaneously tyrosinase activity, melanin synthesis, and proliferation of the melanoma cells . Acrylamide-gel electrophoresis patterns of soluble tyrosinase from the Ab melanoma cells cultured in vitro consisted of two bands, similarly as soluble tyrosinase from the Ma melanotic melanoma cells freshly isolated from solid tumors.

Aktuelle Gerontol, 1983 Nov, 13(6), 223 - 5
{Cell culture as an experimental system for studying aging processes}; Steinhardt M; The investigation of cellular aging has mainly been limited by experimental and descriptive work on two separate cell culture systems . On one hand studies were performed on in vitro aging, comparing fibroblasts in early and late passage . On the other hand, fibroblasts derived from donors of different age were compared (in vivo aging) . Recently, results have been achieved, stating that some of the investigated parameters change similarly in both systems . Therefore it is of great importance to find criteria for cellular aging, not only to precise the results in both cell culture systems, but also to be able to draw conclusions concerning aging of the organism in vivo.

Vet Rec, 1983 Oct 29, 113(18), 413 - 4
Direct isolation of the agent of enzootic abortion of ewes (Chlamydia psittaci) in cell cultures; Johnson FW et al.; Isolation of Chlamydia psittaci in McCoy cell coverslip cultures from clinical material from field cases of enzootic abortion of ewes proved more sensitive than diagnosis by examination of smears stained by Ziehl-Neelsen . Enzootic abortion could be diagnosed in the absence of fetal membranes by culture of fetal lung or liver tissue.

Presse Med, 1983 Oct 29, 12(38), 2383 - 7
{Study of thyroid autoimmunity in man . Contribution of cell cultures to the study of antibodies stimulating the thyroid gland in Basedow's disease}; Yeni P et al.; The value of thyroid cell cultures in vitro has been demonstrated in auto-immune thyroiditis in animals . We have used the same method in man to investigate for serum thyroid-stimulating antibodies (TSAb) by measuring cyclic AMP production in thyroid cell cultures incubated with immunoglobulins from patients with Graves' disease . The specificity of the reaction is strictly directed either against TSH or against immunoglobulins from patients with Graves' disease; 92% of the sera of our untreated Graves' disease patients were positive . The value of this technique compared to the other methods used for detecting TSAb is discussed.

Nucleic Acids Res, 1983 Oct 11, 11(19), 6611 - 29
Sequential stimulation of cellular RNA synthesis in polyoma-infected mouse kidney cell cultures; Matter JM et al.; Lytic infection with polyoma virus leads in Go-arrested primary mouse kidney cell cultures to a mitotic host response . In the present work we focused our attention on cellular RNA synthesis shortly after onset of polyoma T-antigen synthesis . Onset of polyoma-induced stimulation of 45S pre-rRNA synthesis was determined by hybridization of total cellular RNA with a plasmid (pMrSalB) containing the 5'-end of the mouse ribosomal gene and of the other cellular RNA species by standard biochemical analysis of cellular fractions . The results showed that polyoma-induced stimulation of cellular hnRNA (hnRNP) synthesis, the earliest presently known host cell reaction, preceded onset of stimulated 45S pre-rRNA synthesis and that the latter was paralleled by polyoma-induced stimulation of 5S RNA, tRNA and overall protein synthesis . The polyoma-induced mitotic response is similar to that triggered by simian virus 40 and by certain nonviral mitogens.

Biochim Biophys Acta, 1983 Oct 4, 760(1), 1 - 12
Antibodies to the heparan sulfate proteoglycans synthesized by endothelial cell cultures; Buonassisi V et al.; We have determined, using polyacrylamide gel electrophoresis, that endothelial cell cultures derived from rabbit aorta synthesize a wide spectrum of heparan sulfate proteoglycans . The electrophoretically slower-moving heparan sulfate proteoglycans have been isolated from the endothelial cell culture medium . Antibodies to these proteoglycan species have been raised in the goat . The goat antiserum binds selectively the heparan sulfate proteoglycans that were used for the immunization and does not cross-react with the other (faster moving) species . Only a moderate level of cross-reactivity was observed with the heparan sulfate proteoglycans synthesized by another cell line (presumably smooth muscle cells) of vascular derivation . These results support the suggestion that structural differences in the heparan sulfate proteoglycans are responsible for certain differences in function between the various cell types of the vessel wall.

Tsitologiia, 1983 Oct, 25(10), 1216 - 8
{Relationship between bovine serum composition and its capacity to stimulate cell culture growth}; Igudin LI et al.; A relationship has been studied between the growth-stimulating activity and chemical composition of bovine serum (BS) that is widely used for cell culture growth and virus replication . A direct correlation was shown between the growth-promoting activity of bovine serum, manifested on the model of chick embryo fibroblasts, and the contents of phospholipids, total lipids, proteins, and calcium in this serum . A significant inverse association has also been revealed between the growth-promoting activity of the serum and the amounts of cholesterol and glucose in this.

Tsitologiia, 1983 Oct, 25(10), 1204 - 7
{Simultaneous identification of nucleolus organizer regions and differential chromosome staining in continuous human cell cultures}; Narovlianskii AN et al.; The application of NORs and R-bands for simultaneous staining of nucleolar organizer regions and banding chromosomes of stable human line is proposed . This technique was applied to studies on the human stable cell line J-96.

J Lipid Res, 1983 Oct, 24(10), 1304 - 9
26-hydroxycholesterol: regulation of hydroxymethylglutaryl-CoA reductase activity in Chinese hamster ovary cell culture; Esterman AL et al.; The effect of 26-hydroxycholesterol and other intermediates in bile acid synthesis on HMG-CoA reductase activity was studied in Chinese hamster ovary (CHO) cell culture . Incubation of CHO cells for 5 hr in 0.25 microM 26-hydroxycholesterol caused a 40% inhibition of HMG-CoA reductase activity . All other intermediates tested including 3 beta-hydroxy-5-cholenoic acid and cholest-5-ene-3 beta,7 alpha,26-triol, oxidation products of 26-hydroxycholesterol, had little or no inhibitory effect . It is proposed that 26-hydroxycholesterol has a selective biological role in the regulation of cholesterol synthesis.

J Clin Invest, 1983 Oct, 72(4), 1420 - 6
Metabolism of estradiol in liver cell culture . Differential responses of C-2 and C-16 oxidations to drugs and other chemicals that induce selective species of cytochrome P-450; Schneider J et al.; The oxidative metabolism of estradiol (the natural estrogen 2,3,5(10)-estratriene-3,17 beta-diol) at positions C-2 and C-16 was examined in primary cultures of chick embryo liver cells using estradiol which was labeled with 3H specifically at either the C-2 or C-16 position as the substrate . Oxidation of estradiol by the cultured liver cells was assessed by the release of 3H which accumulated as 3H2O in the culture medium; both C-2 and C-16 oxidative reactions were detectable in the liver cell cultures by this technique . When incubated with a concentration of estradiol substrate close to the Michaelis constant (Km), approximately 45.8 pmol {2-3H}estradiol and 5.0 pmol {16-3H}estradiol/mg protein per minute underwent oxidative metabolism in untreated cells . Total amounts of oxidized product formation after 2 h of incubation were 28 and 5 pmol/mg protein for C-2 and C-16 oxidation, respectively . Treatment of cultures with phenobarbital or 2-propyl-2-isopropylacetamide significantly increased oxidation at C-16 (1.9-fold and 2.6-fold greater than control values, respectively), whereas no significant change in C-16 oxidation was observed after treatment of the cultures with 3-methylcholanthrene, benzo{a}pyrene, or benz{a}anthracene . The latter chemicals, however, were found to increase the extent of oxidation at C-2 significantly (i.e., 1.5-2.2-fold increases over control values) . The increase in C-2 oxidation after treatment of cultures with phenobarbital or 2-propyl-2-isopropylacetamide was significantly less than that observed for oxidation at C-16 . The apparent Km values for these oxidations in control cultures were 23.5 and 30.3 microM for C-2 and C-16 oxidation, respectively; corresponding maximum velocity (Vmax) values were 119 and 11.7 pmol/mg protein per minute, respectively . These data indicate that the C-2 and C-16 oxidations of estradiol take place in cultured avian hepatocytes and that the extent of metabolism at these positions on the hormone molecule can be altered by chemicals, such as drugs and polycyclic aromatic hydrocarbons, which induce distinctive species of cytochrome P-450 in the liver.

J Cell Physiol, 1983 Oct, 117(1), 123 - 7
Increase of latent HMG-CoA reductase activity with increasing density of cell cultures; Chen HW; The total (active latent) activity of HMG-CoA reductase declined linearly with increasing cell density in cultures of three lines of mammalian cells . The active form disappeared almost entirely under this condition, while the latent (presumably phosphorylated) form increased to some extent . The disappearance of active HMG-CoA reductase with concomitant increase in the proportion of latent HMG-CoA reductase was correlated with the decline in cellular multiplication and sterol synthesis . These results suggest that interconversion of HMG-CoA reductase between active and inactive forms through phosphorylation-dephosphorylation can be associated with changes in the rate of cellular proliferation in cell cultures . However, the decreased rate of sterol synthesis followed more closely the slower disappearance of the total HMG-CoA reductase activity than the rapid decrease of the active form of the reductase alone . Therefore, changes in the rate of cellular proliferation can affect the interconversion of HMG-CoA reductase between active and inactive forms through reversible phosphorylation . However, phosphorylation of the enzyme to the inactive form appears not to be the mechanism by which the sterol synthetic rate is regulated in confluent cell cultures . Rather, the amount of total HMG-CoA reductase determines the rate of sterol synthesis.

J Clin Oncol, 1983 Oct, 1(10), 627 - 34
Marrow cytogenetic and cell-culture analyses of the myelodysplastic syndromes: insights to pathophysiology and prognosis; Gold EJ et al.; Marrow cytogenetic and granulocyte-macrophage colony formation (CFU-GM) studies were performed on 34 previously untreated patients with documented myelodysplastic syndromes seen between January 1978 and June 1982 . All patients were managed without chemotherapy until progression to acute leukemia was observed . All 10 patients with exclusively abnormal marrow metaphases developed acute leukemia (100%) while only one (7%) of 14 patients with solely normal marrow metaphases subsequently developed leukemia (p less than 0.001) . Three (42%) of the seven patients with both normal and abnormal marrow metaphases developed acute leukemia . Fifteen (86%) of the 19 patients with either large cluster or no growth patterns developed acute leukemia while only two (13%) of 15 patients with either small cluster or colony forming growth patterns developed acute leukemia (p less than 0.001) . Abnormal marrow cytogenetic status correlated with abnormal marrow CFU-GM growth pattern (p less than 0.05) . Analysis of CFU-GM sensitivity to inhibition by prostaglandin E was performed in 12 patients . Nine patients showed CFU-GM refractoriness to inhibition by prostaglandin E . Seven of these patients eventually developed leukemia . Three patients had CFU-GMs which were initially sensitive to prostaglandin E inhibition . In these three patients, a loss of CFU-GM sensitivity to prostaglandin E was observed prior to their progression to morphologically identifiable acute leukemia.

J Endocrinol Invest, 1983 Oct, 6(5), 369 - 74
Effect of prostaglandin E2 on adenylate cyclase system in human kidney cell cultures: interactions with parathormone and arginine-vasopressin; Rotella CM et al.; We have used human kidney cortical and medullary cells in primary culture as an experimental model, in order to study the effect of prostaglandin E2 on the adenylate cyclase-cyclic AMP system at renal level, in comparison with that of parathormone and desamino 1-D-arginine 8-vasopressin . Prostaglandin E2 is a more potent stimulator of cyclic AMP accumulation in cortical cells than in medullary ones, the minimal effective dose being 3 nM in the cortex and 20 microM in the medulla . The maximal response was obtained with 30 microM in cortical cells and with 0.3 mM in medullary ones . The effects of combining submaximal and maximal active doses of the three hormones were always additive when tested on cortical cells . On the contrary, prostaglandin E2, at doses effective on the medullary cells adenylate cyclase system, produced an increase of cyclic AMP accumulation significantly lower than expected when incubated with submaximal and maximal amounts of desamino 1-D-arginine 8-vasopressin . Present data provide evidence for the existence of distinct receptors for parathormone, arginine-vasopressin and prostaglandin E2 at cortical level; the possible role of prostaglandin E2 in the counter-regulation of arginine-vasopressin action on medullary cells in unlikely, in view of the high prostaglandin E2 concentration which is necessary to exert this effect.

Brain Res, 1983 Sep 26, 275(2), 369 - 72
Normal proliferation rate of galactocerebroside positive oligodendrocytes in brain cell cultures of the hypomyelinated mouse mutant jimpy; Bologa L et al.; Proliferation of oligodendrocytes from the jimpy (jp) hypomyelinated mouse mutant was studied in dissociated brain cell cultures . This was done by combining anti-galactocerebroside (GC) immunostaining (for identifying oligodendrocytes) with {3H}thymidine autoradiography (for identifying proliferating cells) . Previously we showed that the expression of GC in culture by jp oligodendrocytes is not altered by the jp mutation . Present results show that in 7-, 14- and 21-day-old jp cultures oligodendrocytes proliferate at a rate similar to that of normal GC+ oligodendrocytes . This indicates that, in jp brain cell cultures, oligodendrocytes which are not affected by mutation in their capability to express GC are also unaffected with regard to their proliferation rate.

Toxicol Appl Pharmacol, 1983 Sep 15, 70(2), 181 - 7
Use of heart cell cultures as a tool for the evaluation of halothane arrhythmia; Miletich DJ et al.; The effects of various combinations of epinephrine-halothane and epinephrine-enflurane were tested on beating myocardial muscle cells cultured from 2- to 3-day-old rats . A series of culture plates containing myocytes were exposed to 0.5, 1.0, 1.5, and 2% halothane . At each halothane concentration, 9 ng of epinephrine was added, and the rate of contraction and rhythm of myocytes were observed . With increasing halothane concentrations, a significant and progressive increase in the percentage of plates demonstrating arrhythmia was observed . In a separate series of experiments, doses of epinephrine were added following exposure to 1.5% halothane . As the dose of epinephrine was increased progressively more plates displayed arrhythmia . In addition, culture plates were exposed to enflurane (3 and 6%), and epinephrine was added to each plate . No arrhythmia was observed in any of the 3% enflurane exposed plates . However at 6%, 100% of the plates displayed arrhythmia . In another series of experiments the efficaciousness of quinidine, procaine amide, lidocaine, propranolol, and verapamil in converting cell culture arrhythmia to normal rhythm following epinephrine and halothane was tested . Quinidine converted 96% of all arrhythmic plates to normal rhythm, procaine amide 80%, lidocaine 50%, and propranolol 10% . Verapamil failed to convert any arrhythmic plates to normal rhythm . It was concluded from this study that halothane directly "sensitizes" heart cells in tissue culture, and that the "sensitization" process is a linear, dose-dependent phenomenon.

Neurochem Res, 1983 Sep, 8(9), 1197 - 202
Effects of N-LAAM on {3H}etorphine binding in neuronal-enriched cell cultures; Gibson DA et al.; Stereospecific {3H}etorphine binding sites are present in neuronal-enriched cell cultures dissociated from 7-day-old chick embryonic brain . Moreover, binding was regulated by both ions and GTP in a manner similar to that of in vivo brain tissue . When cultures were exposed to N-LAAM (10(-6) M) from day 6 to day 7 or 8 and assayed for binding at day 8, Bmax was decreased and KD was increased . These findings support our view that primary neuronal cultures are a suitable model with which to study interactions of drugs with opiate receptors.

Mol Cell Biol, 1983 Sep, 3(9), 1648 - 55
Formation of cytoplasmic heat shock granules in tomato cell cultures and leaves; Nover L et al.; Biochemical and electron microscopic analyses of heat-shocked suspension cultures of Peruvian tomato (Lycopersicon peruvianum) revealed that a considerable part of the dominant small heat shock proteins (hsps) with an Mr of approximately 17,000 are structural proteins of newly forming granular aggregates in the cytoplasm (heat shock granules), whose formation strictly depends on heat shock conditions (37 to 40 degrees C) and the presence or simultaneous synthesis of hsps . However, under certain conditions, e.g., in preinduced cultures maintained at 25 degrees C, hsps also accumulate as soluble proteins without concomitant assembly of heat shock granules . Similar heat shock-induced cytoplasmic aggregates were also observed in other cell cultures and heat-shocked tomato leaves and corn coleoptiles.

Brain Res, 1983 Sep, 285(3), 390 - 5
Survival of HRP-labeled spinal motoneurons of chick embryo in tissue and cell cultures; Tanaka H et al.; Motoneurons in the lumbar spinal cord of chick embryos 6 days old, the age when their cell death starts, were labeled in vivo by retrograde axonal transport of horseradish peroxidase (HRP) and then cultured as cord fragments or isolated cells . In Eagle's minimum essential medium with no additives, spinal cord cells survived well, but among them, the survival of HRP-labeled motoneurons was poor . Muscle-conditioned medium and a mixture of horse serum (HS) and whole chick embryo extract (CEE) promoted motoneuron survival in both suspension culture of spinal cord fragments and monolayer culture of spinal cord cells . It appeared that multiple factors may be involved in the increased extent of motoneuron survival, because HS or CEE alone showed only weak activity in contrast with the combination of HS and CEE.

In Vitro, 1983 Sep, 19(9), 693 - 8
Variation in composition of media and reagents used in the preparation of cell cultures from human and other animal tissues: Dulbecco's, Earle's, and Hanks' balanced salt solutions; Burke CN et al.; When comparison was made of directions given for three salt solutions commonly used in cell culture preparations and identified as Dulbecco's, Earle's, and Hanks', variations in composition were found . Some significantly alter the suitability of the materials for the intended use . Other differences may have less effect . This brief review reveals a tendency among researchers to follow procedures obtained from colleagues for the preparation of laboratory reagents and media, to cite the original publication rather than their colleagues' work as the source of the information, and to fail to compare the two for differences . Some failures in cell culture propagation may be attributed to similar instances with other published but incorrectly cited work . Tables are provided that facilitate comparison of the correct original formulations with variants from selected published sources.

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1983 Sep, 16(3), 183 - 9
Preparation of a Toxoplasma gondii agar gel immunodiffusion test antigen from ovine fetal kidney cell cultures; Chang GN et al.; A toxoplasma gondii antigen prepared from the cell-free supernatant of ovine fetal kidney (OFK) cell cultures produced a sharp precipitation line against anti-Toxoplasma serum in an agar-gel immunodiffusion (AGID) test . Results obtained from the AGID and indirect hemagglutination (IHA) tests on 46 human and nine rabbit sera were compared . Thirty-three percent of the serum samples, which were negative for the Toxoplasma IHA test, were positive when assayed by the AGID method . The AGID test appears to be superior to the IHA test for detecting low titers of antibodies against T . gondii . After 70 generations of serial passages through the OFK cell cultures, T . gondii still produced the AGID test antigen.

Yale J Biol Med, 1983 Sep-Dec, 56(5-6), 679 - 83
Mycoplasma pneumoniae attachment to WiDr cell cultures: competitive inhibition assays; Chandler DK et al.; Attachment of radiolabeled M . pneumoniae to human WiDr cell culture monolayers was dependent on the WiDr cell density and the concentration of M . pneumoniae . Saturation of confluent monolayers grown on 5 mm coverslips was attained with only 40 micrograms of M . pneumoniae protein . Preincubating the WiDr monolayers with unlabeled M . pneumoniae or with a protein-rich extract prepared from M . pneumoniae inhibited subsequent attachment of radiolabeled organisms . Attachment inhibition by the M . pneumoniae extract provided a quantitative assay for mycoplasmal binding components . Treatment of radiolabeled M . pneumoniae with orosomucoid, ceruloplasmin, and gangliosides inhibited attachment to WiDr cells . These sialoglycoconjugates may be structural analogues of the target cell receptor.

Cancer Lett, 1983 Sep, 20(2), 165 - 71
Effects of butylated hydroxyanisole and butylated hydroxytoluene on 7,12-dimethylbenz{a}anthracene-DNA adduct formation in mouse embryo cell cultures; Sawicki JT et al.; Neither butylated hydroxyanisole (BHA) nor butylated hydroxytoluene (BHT) significantly reduced overall 7,12-dimethylbenz{a}anthracene (DMBA)--DNA adduct formation in mouse embryo cell cultures . However, analysis of DMBA--DNA adducts by Servacel DHB chromatography and high-pressure liquid chromatography showed that treatment of cells with BHA, but not with BHT, resulted in a decreased contribution from the syn bay region dihydrodiol epoxide to overall binding.

J Cell Sci, 1983 Sep, 63, 155 - 71
Role of the cellular matrix in haemopoiesis . I . Synthesis of glycosaminoglycans by mouse bone marrow cell cultures; Gallagher JT et al.; Haemopoietically active mouse bone marrow cultures, incubated for 48 h with {3H}glucosamine and Na2(35)SO4, synthesized radiolabelled hyaluronic acid, heparan sulphate and chondroitin sulphate . Heparan sulphate was enriched in a trypsin extract of the adherent cells whereas hyaluronic acid and chondroitin sulphate were distributed mainly to the culture medium . Analysis of nitrous acid scission products of heparan sulphate by gel chromatography demonstrated the close association of N- and O-sulphate groups along the polysaccharide chain . Chondroitinase AC degradation established the copolymeric nature of chondroitin sulphate in which about 38% of the hexuronic acid residues were in the form of GlcUA . Studies on non-haemopoietic cultures, derived from W/Wv mice or from normal marrow cells maintained in foetal calf serum instead of horse serum, indicated that adherent stromal cells were the major source of glycosaminoglycans.

In Vitro, 1983 Sep, 19(9), 672 - 82
Mycoplasmal infection of insect cell cultures; Steiner T et al.; Twenty-five cell cultures of three insect orders from eight laboratories were tested for mycoplasmal infection . Acholeplasma laidlawii was detected in one culture, an incidence of 4.0% . A . laidlawii, Mycoplasma orale, M . arginini, but not M . hyorhinis, could establish infections of drosophila Dm-1 cell cultures at 25 degrees C . In prospective studies, drosophila Dm-1 cultures were intentionally infected with broth-propagated A . laidlawii and M . hyorhinis . M . hyorhinis did not grow and was eliminated from the Dm-1 cultures during consecutive passages . A . laidlawii grew without obvious cytopathic effects during six weekly passages; titers of over 10(7) CFU/ml were recorded at Passages 2 and 5 (p2 and p5) . Minimal cell culture infectious doses were also determined during these studies . 0.1 milliliter cell samples were inoculated into Leighton tubes containing either fresh M1A culture medium or 3T6 indicator cells in McCoy's 5a medium . After 4 d of incubation at 25 and 37 degrees C, respectively, the cover slips were stained by DNA fluorochrome Hoechst 33258 (A . laidlawii) or by specific fluorescein-conjugated antiserum (M . hyorhinis) . At p2 with both mycoplasma species, the procedure using M1A medium and incubation at 25 degrees C without 3T6 cells was inferior to indicator cells . In five of six experiments at least a two-log higher titer of mycoplasmas was needed to be detected with M1A and 25 degrees C . At p5 no difference could be found . Uridine phosphorylase assays of Dm-1 cultures infected with A . laidlawii, M . hyorhinis, M . orale, and M . arginini gave clearly positive results only with A . laidlawii . The ratio of incorporated uridine to incorporated uracil method yielded false positives with two drosophila cell lines . Suggestions for assay of mycoplasmas in invertebrate cell cultures are given.

Cancer Res, 1983 Sep, 43(9), 4132 - 5
Evidence that binding of 7,12-dimethylbenz(a)anthracene to DNA in mouse embryo cell cultures results in extensive substitution of both adenine and guanine residues; Dipple A et al.; Primary mouse embryo cell cultures were grown in the presence of {14C}guanine, labeling primarily deoxyguanosine residues in the cellular DNA, or in the presence of {14C}adenine, labeling both deoxyguanosine and deoxyadenosine residues in the cellular DNA . These cultures were subsequently exposed to 7,12-{3H}dimethylbenz(a)anthracene for 24 hr . The DNA was isolated and hydrolyzed to deoxyribonucleosides, and the 7,12-dimethylbenz(a)anthracene:deoxyribonucleoside adducts were separated chromatographically allowing the three major adducts found to be identified as bay-region anti-dihydrodiol-epoxide:deoxyguanosine and :deoxyadenosine adducts and a bay-region syn-dihydrodiol-epoxide:deoxyadenosine adduct . Therefore, in contrast to what is known for benzo(a)pyrene, substantial amounts of deoxyadenosine adducts are formed with the more potent carcinogen, 7,12-dimethylbenz(a)anthracene.

Cell Mol Neurobiol, 1983 Sep, 3(3), 255 - 62
Simultaneous determination of leu-enkephalin localization and {3H} gamma-aminobutyric acid uptake in rat striatal cell cultures; Mazurkiewicz JE et al.; The purpose of this study was to investigate the coexistence of gamma-aminobutyric acid (GABA) and leu-enkephalin in single neurons from the corpus striatum . Monolayer cell cultures, started from newborn rat corpus striatum, were grown in serum-free medium and examined using GABA autoradiography and leu-enkephalin immunocytochemistry in a double-label protocol . Examples of cells were found which were positive for one or the other neurotransmitter or for neither transmitter, but not for both . Furthermore, cells which appear similar by morphological criteria alone differed in transmitter specificity . We conclude that the two transmitters tested are not localized within single cells and that morphology alone is inadequate to identify functional cell classes in this area.

J Nat Prod, 1983 Sep-Oct, 46(5), 626 - 32
{Inhibitory effects of some crude and semi-purified extracts of indigenous French plants on the multiplication of human herpesvirus 1 and poliovirus 2 in cell culture}; Suganda AG et al.; Within a valorization program of natural regional resources, 50 ethanolic extracts of 41 indigenous plants have been subjected to chemical tests and antiviral screening . Four plants: Bryonia dioica, Anthyllis vulneraria, Matricaria chamomilla, and M . inodora inhibit the growth of poliovirus . Furthermore, three (A . vulneraria, M . Chamomilla, and M . inodora) have an antiherpetic effect.

Environ Health Perspect, 1983 Sep, 51, 257 - 65
Interactions of chrysotile and benzopyrene in a human cell culture systems; Stephens RE et al.; The risk of lung cancer related to asbestos exposure has been shown to increase disproportionately by cigarette smoking, suggesting a synergistic effect . Differing lengths of NIEHS chrysotile with benzopyrene {B(a)P, B(e)P} (organic by-products of combustion) were applied on normal human fibroblasts (cell line CI) to test for cytotoxicity (survival determined by colony-forming efficiency), binding of benzopyrene to DNA, and the production of benzopyrene metabolites . At concentrations of 100 micrograms/mL, NIEHS short chrysotile was more cytotoxic than NIEHS intermediate chrysotile (3% and 17% survival, respectively); B(a)P and B(e)P concentrations up to and including 10 microM were not cytotoxic . Simultaneous application of NIEHS short chrysotile with B(a)P or B(e)P did not decrease survival synergistically . On the contrary, application of B(a)P simultaneously with NIEHS intermediate chrysotile resulted in increased survival over that of intermediate chrysotile alone (25% and 17% survival, respectively) . There were low levels of B(a)P bound to DNA in the presence of NIEHS short chrysotile or NIEHS intermediate chrysotile . Measurable levels of B(a)P-DNA adducts were formed both in the absence and in the presence of each size of NIEHS chrysotile . However, there was no strong indication of a perturbation of the level of DNA-B(a)P binding following simultaneous administration of increasing levels of asbestos in addition to 1 microM hydrocarbon . The asbestos had no demonstrable influence on the level of B(a)P metabolism during the 24-hr period following simultaneous exposure of asbestos and hyrdocarbons.(ABSTRACT TRUNCATED AT 250 WORDS)

Ann Pathol, 1983 Sep, 3(3), 245 - 9
{Labial embryonal rhabdomyosarcoma . Value of cell culture and electron microscopy, histogenesis}; Payen L et al.; The authors report the case of a labial embryonal rhabdomyosarcoma of a baby . This appears to be an exceptional localization . The study of the first biopsy which was too superficial leads to the thought of a capillary angioma . At the age of 6 months the lesion is 35 mm in diameter, a lobulated formation, typically botryoid . The excision after a frozen section control was in healthy tissue . At the age of 10 months, there is a local relapse with a nodule of 18 mm with left submandibular lymph nodes . A second surgical operation allows the culture of the tumoral tissue . The conventional histopathological examinations shows the aspect of an embryonal rhabdomyosarcoma . On the ultrastructural examination one can visualize in some cells an intracytoplasmic filamentous material . The cellular proliferation in culture, after May Grunwald Giemsa coloration is monomorphous, spindle shaped and of fibroblastic aspect . The ultrastructural study of this cellular proliferation after trypsinization on the 7th day shows some cells including an intracytoplasmic filamentous material . On the data of the cellular culture, the ultrastructural studies and the review of the literature, the authors discuss the possibility of the relationship between the cells of embryonal rhabdomyosarcomas and the fibroblasts and myofibroblasts . Embryonal sarcoma seems to be a better denomination than embryonal rhabdomyosarcoma, as regards the histogenesis.

J Neurosci, 1983 Sep, 3(9), 1888 - 99
Dissociated neurons from normal and mutant Drosophila larval central nervous system in cell culture; Wu CF et al.; A primary dissociated cell culture of Drosophila larval central nervous system is reported . Divisions of neuroblasts and vigorous outgrowth of neurites could be observed in culture . Within 24 hr cultured cells exhibited characteristic neuronal morphology and unimpaired ability to synthesize and accumulate acetylcholine . This cell culture system renders easy access to experimental analysis of normal neuronal properties and the altered mechanisms in neurological mutants . Single-channel currents induced by acetylcholine and regenerative action potentials were studied in the somata of the dissociated neurons . The appearance of Na channels in these cultured neurons was demonstrated by the cell lethality induced by veratridine and inhibition of the effect by tetrodotoxin . Dissociated neurons from a temperature-sensitive paralytic mutant napts, in which nerve conduction fails at high temperature, were studied in culture . Neuronal growth was not affected by this mutation, nor by tetrodotoxin . However, napts neurons showed greatly reduced sensitivity to veratridine even at 21 degrees C, a temperature at which napts individuals behave normally . This finding indicates expression of the napts phenotype at a level of isolated single cells and provides independent evidence that napts affects Na channel function.

Pharmacol Res Commun, 1983 Sep, 15(8), 709 - 17
Effects of glycosaminoglycan-polysulfate and two non-steroidal anti-inflammatory drugs on prostaglandin E2 synthesis in Chinese hamster ovary cell cultures; Egg D; We have studied the influence of a polysulfated glycosaminoglycan (Arteparon) in comparison with indomethacin and piroxicam on the synthesis of prostaglandin E2 in Chinese hamster ovary cell cultures (CHO) . The two non-steroidal anti-inflammatory drugs (NSAD) were found to be strong inhibitors of prostaglandin E2 (PGE2) synthesis . The inhibition was found to be dose-dependent . Arteparon, an antidegenerative drug for the treatment of osteoarthritis, was also found to inhibit PGE2 synthesis in the CHO cell cultures . The inhibition was not as strong as that of the NSAD and no correlation between the concentration of Arteparon and the inhibition of PGE2 synthesis could be demonstrated.

J Clin Microbiol, 1983 Sep, 18(3), 550 - 3
Rapid detection and identification of herpes simplex virus in cell culture by a direct immunoperoxidase staining procedure; Miller MJ et al.; Cell monolayers were inoculated with 169 fresh and 76 previously frozen clinical specimens and examined for the presence of herpes simplex virus (HSV) by noting the appearance of characteristic cytopathic effect (CPE) and by using direct immunoperoxidase (IP) stain for viral antigen . HSV was detected by IP staining in 40 of 169 (23.7%) monolayers and by CPE in 39 of 169 (23.1%) monolayers inoculated with fresh specimens . All 40 isolates were detected and confirmed by IP staining within 24 h . Although 39 of 40 isolates were detected by CPE, only 9 of 39 (23%) were positive within 24 h . CPE was observed at 2.7 days on the average, but 4 days were required before 90% of the cultures were positive and more than 5 days were required before all HSV isolates were recognized . Similar results were observed for frozen specimens . HSV was detected earlier with IP staining, which demonstrated more extensive infection of cell monolayers inoculated with titrated fresh culture isolates and clinical specimens than did CPE . IP staining reduces the amount of time required for detection and identification of HSV in culture, is readily adaptable for use in the clinical laboratory, and permanent stained preparations can be made.

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