Neurotoxicology, 1992 Summer, 13(2), 401 - 12 Mitochondrial glutathione and methyl iodide-induced neurotoxicity in primary neural cell cultures; Bonnefoi MS; The status of both cytosolic and mitochondrial glutathione was studied in primary cultured cerebrocortical cells from fetal mice using the selective membrane-solubilizing properties of digitonin and after exposure to the monohalomethane methyl iodide . A correlation was found between cell injury (assessed by lactate dehydrogenase leakage 24 hr after exposure) and early loss of mitochondrial glutathione (2 hr after exposure), while cell death did not appear directly dependent on cytosolic glutathione depletion . The antioxidants BW 755C (3-amino-1-{m-(trifluoromethyl)phenyl}-2-pyrazoline) and DPPD (N,N'-diphenyl-p-phenylenediamine), and the glutathione precursor N-acetyl-L-cysteine were used to modify cellular responses to methyl iodide . Prevention of cell injury by these reagents was obtained only under conditions where at least 50% of the normal level of mitochondrial glutathione was preserved after methyl iodide exposure . Mitochondrial metabolic activity (reduction of 3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide, MTT) was affected by exposure to methyl iodide and correlated with mitochondrial glutathione depletion and cytotoxicity . These findings indicate that the mitochondrial glutathione pool and mitochondrial functions may be the most significant intracellular targets of methyl iodide in neural cultures . Moreover, the present work exemplifies the dependence of neural cell viability on the status of mitochondrial functions and suggests that, as in the liver, mitochondrial glutathione is an important component of cellular homeostasis in nervous tissue. Horm Res, 1992, 37 Suppl 1, 25 - 31 Modulation of progesterone secretion by androgens in porcine granulosa cell cultures; Tanabe K et al.; The influence of testosterone, androstenedione and dihydrotestosterone (DHT) on the progesterone (P4) production by cultured porcine granulosa cells was studied in the presence or absence of gonadotropins . Porcine granulosa cells from large follicles (6-12 mm in diameter) were incubated for 2 days with 5% CO2 in air with testosterone, androstenedione and DHT (10(-12), 10(-10), 10(-8) and 10(-6) M) in the presence or absence of luteinizing hormone (LH, 10 ng/ml) or follicle-stimulating hormone (FSH, 30 ng/ml) . P4 and pregnenolone (P5) in conditioned culture media were quantified by their specific RIAs . In the absence of gonadotropins, P4 in media with androgens were not significantly different from controls . In the presence of LH, the addition of testosterone (10(-10), 10(-8) M), androstenedione (10(-8) M) and DHT (10(-8) M) caused a significant 1.3- to 2.3-fold increase in P4 over that caused by LH alone . In contrast, in the presence of FSH, testosterone (10(-12), 10(-10) M), androstenedione (10(-12)-10(-6) M) and DHT (10(-6) M) reduced the levels of P4 by 22% to 41% . The addition of androgens with LH caused a significant increase in P5, while P5 decreased in the presence of FSH . P4/P5 ratios remained unchanged in the presence of both LH and FSH . These data suggest that the P4 production by cultured porcine granulosa cells is modulated in a paracrine or an autocrine fashion by androgens in the presence of gonadotropins, and that androgens may exert their actions partly by altering the activity of cholesterol side chain cleavage enzymes. Calcif Tissue Int, 1992, 51 Suppl 1, S16 - 20 Stimulatory effect of ipriflavone on formation of bone-like tissue in rat bone marrow stromal cell culture; Notoya K et al.; The effects of ipriflavone (IP) (10(-5) M) on bone formation were studied in stromal cells from the femoral bone marrow of young adult rats cultured for 21 days in the presence of beta-glycerophosphate and dexamethasone . Stereoscopic microscopy showed nodule formation after 14 days of culturing, and both the number and the size of the nodules increased with time . The alizarin-red-stained calcified area in the nodules in the IP group was nearly 4 times as large as that in the control after 21 days . Light and electron microscopy revealed the presence of many osteoblast-like cells with developed rough endoplasmic reticulum and Golgi apparatus in the nodules in the control group after 14 days, and a collagenous fibril network was seen among the cells . After 21 days, calcification of the dense collagenous fibril network and bone matrix-like tissue were observed in many nodules, resulting in the formation of bone-like tissue containing osteocyte-like cells . In the IP group, the collagenous fibril network area in the nodules was greater than that in the control after 14 days, and a further increase in both the dense collagenous fibril network area and calcified bone-like tissue area was observed after 21 days . These findings indicate that IP stimulates bone-like tissue formation in the rat bone marrow stromal cell culture, suggesting that the promotion of collagen production by osteoblasts is involved in the stimulation of bone-like tissue formation by IP. Zh Mikrobiol Epidemiol Immunobiol, 1992 Jan, (1), 25 - 8 {The protective activity of preparations made from the tick-borne encephalitis virus grown using different cell cultures}; El'bert LB et al.; The preparations of tick-borne encephalitis (TBE) virus grown in swine embryo kidney cell culture have been shown to possess pronounced protective activity per unit of virion protein E in comparison with TBE virus preparations derived from cell culture 4647 and chick embryo cell culture . The antigenic activity of all virus preparations under study has proved to be practically the same . The role of post-translation modifications of TBE virus protein E in the manifestation of some of its biological properties is discussed. Rev Med Chir Soc Med Nat Iasi, 1992 Jan-Jun, 96(1-2), 111 - 3 {The use of cell cultures in the cytotoxicity testing of dental materials}; Barlean L et al.; On lines of human (HeLa) and monkey (BS-C-1) cell cultures, the cytotoxicity of 15 products of dental use for radicular and coronary fillings, alloys and endodontic antiseptics was analysed . It was found that the simplified method can be used, together with the in vivo and in vitro tests recommended by I.S.O., for determining the bioavailability of dental products. Pharmacol Ther, 1992, 53(3), 355 - 74 Serum-free cell culture; Bjare U; Cell culture is one important tool when studying cellular functions and molecular biology . It is also a basic method in most virological investigations . Serum has been an obligatory component in most cell culture media . During the last decades serum-free, chemically defined media have been developed, that are supplemented with a number of substances with specific cellular activities . The main developments of defined media are presented . Examples are given of investigations with different cell types. Microbiol Immunol, 1992, 36(7), 707 - 19 Focus formation by the human immunodeficiency virus (HIV) in the immobilized MT-4 cell culture and its application to the evaluation of anti-HIV agents; Nakane M et al.; Immunofluorescence studies were performed on the infection of monolayer cultures of immobilized MT-4 cells with human immunodeficiency virus type 1 (HIV-1) . By using the anti-viral p24 monoclonal antibody, we could observe formation of foci of p24 antigen-positive cells within 3 to 4 days when the infection was initiated with a relatively small amount of the virus . Frequency of the focus formation was in proportion to the dose of input virus (ranging from 0.001 to 0.1 PFU/cell), which allowed us to apply this phenomenon to the assay of anti-HIV agents as well as to the estimation of relative infectivity of the virus stocks . When antiviral agents were added to the infected cultures, number of foci as well as the size of each focus was reduced in a concentration-dependent manner . The dose required for reducing the number of foci by 50% was calculated to be 6 ng/ml and 8 ng/ml for tunicamycin (TM) and azidothymidine (AZT), respectively . These values are comparable to those obtained by other current assay methods . In addition, focus reduction assay is also useful in searching for such antiviral agents that would inhibit or block the early step of viral replication cycle. J Physiol, 1992, 451, 553 - 83 Unitary A-currents of rat locus coeruleus neurones grown in cell culture: rectification caused by internal Mg2+ and Na+; Forsythe ID et al.; 1 . We have used whole-cell and single-channel recording to study the transient outward potassium current (A-current) of rat locus coeruleus neurones grown in tissue culture . The A-current was largely inactivated at the resting potential, but could be activated from sufficiently negative holding potentials during steps positive to -50 mV . The current was sensitive to 4-aminopyridine . Another slowly activating, sustained current was similar to a delayed rectifier . 2 . In the on-cell configuration the unitary conductance of channels carrying A-current was 40.9 +/- 2.2 pS (n = 6) with high external potassium (140 mM) and 14.8 +/- 1.4 pS (n = 11) with 3 mM {K+}o . The unitary current-voltage relation was not linear, but had a negative slope at very positive voltages in 3 mM {K+}o . The reversal potential changed with {K}o as expected for a K+ channel . 3 . The open state probability of A-current channels was voltage dependent, reaching a peak of 0.78 +/- 0.17 (seven patches) . The relationships between both activation and inactivation and membrane potential were well fitted by Boltzmann expressions . Activation was half-maximum at a potential 71.9 +/- 11.8 mV (n = 4) positive to the resting potential (approximately -61 mV) . Inactivation was half-complete 29.4 +/- 3.8 mV (n = 4) negative to the resting potential . There was evidence from runs analysis for slow inactivation of channels . 4 . Channels showed frequent visits to substates, the most readily identifiable of which had an amplitude 0.55 +/- 0.04 (n = 5) of the fully open state . Other substates had amplitudes of around 0.25 and 0.75 . Occupancy of substates was greater at negative membrane potentials . 5 . A preliminary analysis of kinetic behaviour, treating visits to substates as openings, shows that open times are distributed as a single exponential . The open time was 16.2 ms (n = 4) at a voltage 100 mV positive to the resting potential, increasing with further depolarization . Closed times are distributed as the sum of three or four exponentials . First latency distributions are strongly voltage dependent and show a delay, giving a sigmoidal rise to the distribution . Increasing temperature increased unitary current and reduced mean open time . 6 . The mechanism of the rectification seen in the unitary current-voltage relationship was examined using excised, inside-out patches.(ABSTRACT TRUNCATED AT 400 WORDS) J Clin Lab Anal, 1992, 6(5), 311 - 4 Impaired cytokine production in whole blood cell cultures from patients with colorectal carcinomas as compared to benign colorectal tumors and controls; Elsasser-Beile U et al.; Cytokine production was investigated in whole blood cell cultures from 74 patients with colorectal carcinomas, 20 patients with benign colorectal tumors, and 314 healthy controls . In the 4 day post induction supernatants the levels of IFN-gamma, IL-1-alpha, IL-2, and TNF-alpha were measured by a sensitive immunoassay . In the blood cell cultures of the patients with colorectal carcinomas significantly lower values of IFN-gamma (P less than or equal to .001), IL-1-alpha (P less than or equal to .001), and IL-2 (P less than or equal to .01) were found as compared to the patients with benign tumors and the controls, although total and differential leukocyte counts were similar in all three groups . A linear correlation between the levels of IFN-gamma and IL-1-alpha and the tumor stages could be shown . Our results suggest that a growing tumor burden may induce increasing immunological deficiencies as reflected by a decreasing cytokine production of the immune cells. Antisense Res Dev, 1992 Spring, 2(1), 41 - 9 Incorporation of radiophosphorus from labeled oligodeoxynucleotides into RNA of mycoplasma in cell cultures; Geselowitz DA et al.; We have found that various mycoplasma species quickly and efficiently incorporate radiophosphorus into their RNA from labeled oligonucleotides added to the medium . The label can be in any of several positions in an oligodeoxynucleotide, and incorporation also occurs efficiently from labeled RNA . Mycoplasmas also incorporate the radiolabel when they infect a mammalian cell culture; the host cells do not . This incorporation presumably involves uptake of the oligodeoxynucleotide followed by digestion to mononucleotides, conversion to ribonucleotides, and incorporation in new RNA . We believe that the processing of oligodeoxynucleotides by mycoplasma could be a source of artifacts in antisense work in cell culture and could have implications for the development of antisense therapeutics . We also suggest ways to exploit the incorporation phenomenon in mycoplasma testing. Acta Otolaryngol, 1992, 112(2), 254 - 8 Contribution of cell culture to the study of the physiology of the stria vascularis and Reissner's membrane; Yeh TH et al.; Cell culture is a well-established tool in cell biology which may overcome the limitations of the experimental methods currently used to investigate the physiology of labyrinthine fluids . Using explant and dissociated cell culture technique, sheets of oriented cells derived from the stria vascularis and Reissner's membrane may be obtained, which allows new experimental approaches such as the patch-clamp technique . Other experimental approaches are considered which might improve our understanding of inner ear physiology. Cytometry, 1992, 13(1), 90 - 102 Multiparametric flow cytometric analysis of radiation-induced micronuclei in mammalian cell cultures; Schreiber GA et al.; A new flow cytometric method is presented that quantifies the frequency of radiation-induced micronuclei in mammalian cell cultures with high precision . After preparing a suspension of main nuclei and micronuclei stained with ethidium bromide and Hoechst 33258, both types of particles are measured simultaneously in a flow cytometer using forward light scatter and three fluorescence emission intensities excited by UV, 488 nm, and by energy transfer from Hoechst 33258 to ethidium bromide . Nonspecific debris overlapping the micronucleus distribution especially in the low fluorescence intensity region was discriminated from micronuclei by calculating ratios of the different fluorescences . The frequencies of radiation-induced micronuclei measured with this new technique agreed well with results obtained by conventional microscopy . The lower limit of the DNA content of micronuclei identified by this technique was found to be about 0.5%-0.75% of the DNA content of G1-phase nuclei . Dose effect curves and the time-dependent induction of micronuclei were measured for two different mouse cell lines. Brain Res Mol Brain Res, 1992 Jan, 12(1-3), 23 - 30 Expression of the proto-oncogene c-fos in three-dimensional fetal brain cell cultures and the lack of correlation with maturation-inducing stimuli; Bardoscia MT et al.; Previous work has shown that aggregating fetal brain cell cultures are able to attain a highly differentiated state, and that their development is greatly enhanced by growth and/or differentiation factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and the protein kinase C-activating tumor promoter mezerein . The present study shows that in these 3-dimensional cultures the peptide growth factors EGF and bFGF as well as mezerein are able to induce the expression of the proto-oncogene c-fos . This induction was rapid and transient, in good agreement with observations reported from a wide variety of cell types in vitro . The maximal levels of c-fos mRNA found after stimulation were low in immature cultures and increased greatly as maturation progressed . Of the three factors tested, mezerein was the most potent inducer of c-fos . In contrast to the peptide growth factors EGF and bFGF which were found to induce c-fos only in glial cells, mezerein was stimulatory in glial cells as well as in neurons . A similar cell type specificity has been observed previously for the maturation-enhancing response in immature aggregate cultures . However, in the present study no correlation was found between the degree of c-fos induction and the extent of the maturation-enhancing stimulation . Immature cultures known to be most sensitive and responsive to these maturation-enhancing agents required relatively high doses of peptide growth factors for the induction of c-fos, and the maximal levels of c-fos mRNA elicited were much lower than those in differentiated cultures which did not show any long-term response to these stimuli.(ABSTRACT TRUNCATED AT 250 WORDS) J Clin Microbiol, 1992 Jan, 30(1), 25 - 35 Detection of enteroviruses in cell cultures by using in situ transcription; Carstens JM et al.; In situ transcription (IST) was shown to be useful for the detection of human enteroviral RNA in cultured cells . A primer to detect a wide variety of enteroviral genomes and a coxsackievirus type B3 genome-specific primer were demonstrated to be efficient in IST assays . Transcription times greater than 10 to 30 min did not significantly improve the acquisition of a specific signal, whereas the signal-to-noise ratio decreased with time . Inclusion of actinomycin D to suppress DNA-dependent DNA polymerase activity in reverse transcriptase decreased the signal that was obtained without improving the signal-to-noise ratio . Use of RNase H-free murine leukemia virus reverse transcriptase in the IST reaction increased the signal versus that obtained by use of the avian myeloblastosis virus enzyme, which contains endogenous RNase H activity . Exogenous RNase H added to the transcription reaction ablated the signal . Background transcription because of poorly hybridized (mismatched) primers was reduced after primer hybridization and prior to the transcription reaction by rinsing fixed cells with 3 M tetramethylammonium chloride at temperatures which dissociate mismatched primer-template duplexes . The rapid detection time and the simplicity of application suggest that IST can be performed with a high specificity for the detection of enteroviral genomic sequences in cultured cells and may be more useful than in situ hybridization for the detection of enteroviral genomes. Cytotechnology, 1992, 9(1-3), 247 - 53 Flow cytometry and two-dimensional electrophoresis (2-DE) for system evaluation of long term continuous perfused animal cell cultures in macroporous beads; Reiter M et al.; Immobilization of r-CHO cells at high density using macroporous polyethylene carriers in a modular fluidized bed reactor is demonstrated . Specific growth rates of the cells are measured by incorporation of BrdU . At a cell density of about 10(8) cells/ml a stable growth rate of 0.004 h-1 was established . Total release of proteins into the culture supernatant during protein-free perfusion was analyzed by 2-DE in various phases of the long-term culture showing very similar patterns indicating a constant pattern of gene expression. Cytotechnology, 1992, 10(1), 53 - 62 Simulation of an iterative learning control system for fed-batch cell culture processes; Fu P et al.; This paper describes an iterative learning control scheme for fed-batch operation where repetitive trajectory tracking tasks are required . The proposed learning strategy is model-independent, and it takes advantage of the repetitive feature of system operations with a certain degree of intelligence and requires only small size of dynamic database for the learning process . The convergence of the learning process is proven . An example of simultaneously tracking two predefined trajectories by iterative learning control with two control inputs is given to illustrate the methodology . Satisfactory performance of the learning system can be observed from the simulation results. Cytotechnology, 1992, 8(1), 5 - 11 Use of fluorescence anisotropy determinations for indicating the physiological status of hybridoma cell cultures; Eyl V et al.; The evolution of lipid compartment fluidity during culture of hybridoma cells was studied by fluorescence polarization measurements . The probe partition between the plasma membrane and intracytoplasmic compartments was determined by a quenching fluorescence method . A progressive decrease of the plasma membrane fluidity was observed during the growth phase with an increase during stationary and degeneration phases of the culture . These data suggest that fluidity parameters could be used to follow the behaviour of hybridoma cell cultures. Biotechnol Prog, 1992 Jan-Feb, 8(1), 19 - 24 Optimal design of the tubular microporous membrane aerator for shear-sensitive cell cultures; Su WW et al.; In this paper, a theoretical analysis of oxygen transport across the tubular microporous membrane is described . This analysis has provided some insight into the optimal design of the membrane aerator . It was found in this study, at fixed inlet pressure, that the overall membrane oxygen transfer rate increases with increased tubing length only up to a certain length, i.e., the "critical length" . When a large membrane surface area is required, the fiber should be divided into parallel segments to increase the overall oxygen transfer rate . A manifold or a gas distributor can then be used to distribute gas into segments of tubing . The length of each segment cannot exceed the critical length . In addition, shorter tube segments should give a higher oxygen transfer rate per unit tube length; however, this advantage is counterbalanced by the fact that gas distribution into huge numbers of parallel tubings may not be uniform. Can J Physiol Pharmacol, 1992, 70 Suppl, S344 - 9 Use of cell cultures to differentiate among effects of various ischemia factors on astrocytic cell volume; Juurlink BH et al.; Mouse astrocytes were subjected to in vitro models of ischemia (hypoxia with or without substrate deprivation, excess potassium, or elevated glutamate) . Three hours of hypoxia alone or with substrate deprivation had little effect upon the morphology of astrocytes but did cause disaggregation of polyribosomes . Excess (12-50 mM) potassium added (as KCl) to a normal isotonic medium also caused no swelling; it did, however, cause a shrinkage of cell volume . When 50 mM potassium was substituted for a similar amount of sodium, marked swelling occurred . Swelling of astrocytes was also seen after addition of glutamate (50 microM to 1 mM) to the culture medium . These results show that ischemia per se does not result in astrocytic swelling; rather, microenvironmental alterations such as rising glutamate levels and changes in the sodium/potassium ratios result in astrocytic swelling . We conclude that one can use astrocytes in culture to dissect out the mechanisms that cause postischemic alterations in astrocytes in vivo. Biosens Bioelectron, 1992, 7(8), 569 - 74 A chemiluminescence fiber-optic biosensor system for the determination of glutamine in mammalian cell cultures; Cattaneo MV et al.; A chemiluminescence fiber-optic biosensor system has been developed for determining glutamine in hybridoma cell cultures producing monoclonal antibodies against viral surface antigens . Glutaminase and glutamate oxidase (GLO) were immobilized onto aminopropyl glass beads via glutaraldehyde activation separately and packed in a column . Two separate columns containing immobilized GLO and catalase were placed upstream to eliminate endogenous glutamate . In the presence of ferricyanide, luminol reacted with hydrogen peroxide released from the enzymatic reactions to produce a chemiluminescence (CL) light signal which was detected and quantitated with a fiber-optic system . In combination with flow injection analysis it was possible to process samples virtually identically, thus avoiding difficulties in reproducing the CL signal . There was an excellent linear relationship between the CL response and standard glutamine concentration in the range 10(-6) to 10(-3) M . A complete analysis could be performed in 2 min including sampling and washing . Each immobilized enzyme column was stable for at least 300 repeated analyses without any loss of activity . When the biosensor system was used for the determination of glutamine in spent mammalian cell cultures, the values obtained compared well with those of high-performance liquid chromatography, thus validating the applicability of the CL fiber-optic system. Prog Brain Res, 1992, 91, 117 - 21 Development of an in vitro cell culture system to mimic the blood-brain barrier; Rauh J et al.; Primary cultures of brain capillary endothelial cells (BCECs) cocultured together with astroglia cells were used to investigate the induction of blood-brain barrier (BBB) characteristics in vitro . By immunofluorescence, histochemical staining, two-dimensional gel electrophoresis and enzyme activity tests we are able to show that BCECs in vitro loose typical blood-brain barrier properties but not their common endothelial phenotype . Astrocytes induce the expression of the blood-brain barrier characteristic enzymes gamma-glutamyltranspeptidase and alkaline phosphatase but only in a coculture system with direct cell to cell contact between BCECs and astroglia cells . C6-glioma cells also re-establish the BBB phenotype but were less effective compared to astrocytes . The susceptibility of the BCECs to an astroglial stimulus depends on the proliferative state of the BCECs. Biull Eksp Biol Med, 1992 Jan, 113(1), 72 - 4 {Characterization of cloned alpha-satellite sequence from the extrachromosomal DNA of HeLa cell culture}; Krokhina TB et al.; We have obtained the alphoid DNA clones, pK1 and pK2, from the extrachromosomal DNA of Hela cells treated by cycloheximide (30 micrograms/ml) . Nucleotide sequences of the clones were aligned . The sides of the pK1 and pK2 are 390 and 184 bp, respectively . The marked RELP for the clones was not observed . The results of in situ hybridization have shown an approximately equal distribution of Ag-grains over major part of human chromosomes, with a slight preference for chromosomes 1, 5 and 19 (the 1-st group of alpha-satellite DNA) . Therefore, the obtained alphoid sequences seem to be rather conservative and non-chromosome-specific . We suppose that increase of the alphoid DNA content in the fraction of the extrachromosomal DNA under the cycloheximide treatment is a result of the sporadic statistical processes rather then consequence of the specific excision. Biosens Bioelectron, 1992, 7(5), 329 - 34 Monitoring glutamine in mammalian cell cultures using an amperometric biosensor; Cattaneo MV et al.; An amperometric biosensor has been developed for monitoring glutamine in the pulsed-batch cultivation of murine hybridoma cells . Glutamine oxidase was cross-linked with bovine serum albumin (BSA) via glutaraldehyde activation and deposited on a preactivated nylon membrane . Glutaminase was then immobilized on the protein layer and the resulting membrane was attached to the sensing area of a hydrogen peroxide probe (platinum vs silver/silver chloride polarized at +0.7 V) . An orthogonal test was performed to optimize the activity of the membrane for glutamine with respect to the concentrations of glutamate oxidase, BSA, glutaminase and glutaraldehyde . There was an excellent linear relationship between the biosensor's response and glutamine in the range 0.1-3 mM . The determination of glutamine could be performed in 2 min and each membrane was reused for at least 300 consecutive analyses . The data obtained also agreed well with those high-performance liquid chromatography, thus validating the applicability of the biosensor. Neuroscience, 1992, 47(4), 979 - 84 Neuroprotective effects of graded reoxygenation following chronic hypoxia in neuronal cell cultures; Sher PK et al.; The present study was undertaken to investigate the comparative effects of rapid vs graded correction of chronic hypoxia in vitro . Cerebral cortical cell cultures obtained from fetal mice were exposed to 5% O2 for 24 h and returned immediately to room air for the following 24 h (Group I); comparable cultures were exposed to 5% O2 for 24 h followed by 10% O2 for an additional 24 h before return to room air (Group II) . At the conclusion of the experimental protocol (time 0), partial pressure of oxygen in the bathing medium of Group I cultures was significantly higher than that of Group II and non-hypoxic controls (151 mmHg vs 124 and 132 mmHg, respectively; P less than 0.05) . Throughout the recovery period, Group II cultures evidenced improved neuronal survival (e.g . 35,800 vs 17,700 neurons/culture well at time 0, P less than 0.01), decreased lactate dehydrogenase efflux into the bathing medium, relative preservation of neuronal morphology, as well as higher specific and clonazepam-displaceable benzodiazepine binding and GABA uptake . Glutamate binding was not differentially affected and glutamine synthetase activity, a predominantly glial marker, was only modestly increased after graded reoxygenation . These results demonstrate that gradual reoxygenation after prolonged hypoxia in vitro (i) improves neuronal survival compared to rapid reoxygenation and (ii) delays the manifestations of metabolic dysfunction even though the length of hypoxic exposure is increased . The findings are also consistent with the concept that a period of relative hyperoxia may contribute to hypoxia-induced neuronal injury. J Neurosci, 1992 Jan, 12(1), 56 - 61 Glutamate uptake disguises neurotoxic potency of glutamate agonists in cerebral cortex in dissociated cell culture; Rosenberg PA et al.; The pharmacological properties of glutamate agonists were compared in astrocyte-rich and astrocyte-poor cultures derived from embryonic rat cerebral cortex . The object of this investigation was to determine the extent to which glutamate uptake might influence the receptor-mediated neurotoxic actions of these compounds . In astrocyte-rich cultures, using 30 min exposures, we observed that the potencies of the poorly transported agonists NMDA (35 microM) and D-glutamate (89 microM) were higher than that of L-glutamate (205 microM) . In astrocyte-poor cultures, L-glutamate was much more potent, with an EC50 of 5 +/- 4 microM (3-12 microM), for a 30 min exposure, whereas the potencies of NMDA and D-glutamate were essentially unchanged . L- and D-aspartate were also more effective in astrocyte-poor cultures, again with EC50 values of approximately 6-10 microM, as compared with 130 and 108 microM, respectively, in astrocyte-rich cultures . In other experiments, blocking sodium-dependent glutamate uptake in astrocyte-rich cultures, by using a sodium-free medium, made glutamate as potent an agonist as in astrocyte-poor cultures . Finally, we directly assessed the glutamate uptake system in astrocyte-rich and astrocyte-poor cultures and found that uptake was reduced approximately 25-fold in the astrocyte-poor cultures . These results show that in the presence of abundant astrocytes the neurotoxic potencies of L-glutamate, L-aspartate, and D-aspartate are substantially under-estimated. Acta Microbiol Hung, 1992, 39(2), 137 - 47 Combined effects of flavonoids and acyclovir against herpesviruses in cell cultures; Mucsi I et al.; The combined antiviral effects of some flavonoid compounds and acycloguanosine (acyclovir, Zovirax) were studied on the multiplication of herpes simplex virus types 1 and 2 in HEp-2 cells and on pseudorabies (Aujeszky) virus in chick embryo fibroblast cells by the yield reduction method . The flavonoids quercetin, quercitrin (quercetin-3-L-rhamnoside) and apigenin exhibit antiviral activity against these herpesviruses, and acyclovir is currently one of the most effective antiherpetic agents . In these studies, the simultaneous application of flavonoids with acyclovir resulted in an enhanced antiviral activity . A mathematical formula was used to interpret the drug interaction, resulting in FIC (fractional inhibitory concentration) indices . Meaning a synergic interaction, all combinations exhibited synergy, FIC values of 0.6-0.8 being commonly observed. Basic Res Cardiol, 1992, 87 Suppl 2, 19 - 31 Sympathetic modulation of the cardiac myocyte phenotype: studies with a cell-culture model of myocardial hypertrophy; Long CS et al.; Myocardial hypertrophy is the common endpoint of many cardiovascular stimuli such as hypertension, myocardial infarction, valvular disease, and congestive failure . Catecholamines have long been implicated in the pathogenesis of myocardial hypertrophy, however, it is very difficult to sort out catecholamine mechanisms in vivo . We have developed a cell-culture model which excludes hemodynamic effects and allows the assignment of receptor specificity to catecholamine effects . Utilizing this system, we have shown that stimulation of the alpha 1 adrenergic receptor leads to the development of myocardial hypertrophy and results in the selective up-regulation of the fetal/neonatal mRNAs encoding skeletal alpha-actin and beta-MHC, a pattern similar to that seen with hypertrophy in-vivo . Utilizing a co-transfection assay, we have also obtained data that suggest that the beta-PKC isozyme is in a pathway regulating transcription of the beta-MHC isogene . Beta adrenergic stimulation of the cultured cardiac myocytes also results in a modest degree of hypertrophy, however, this effect may be dependent upon myocyte contractile activity and may involve, at least in part, the non-muscle cells present in the culture system. Genetica, 1992, 87(2), 65 - 73 Evidence for de novo rearrangements of Drosophila transposable elements induced by the passage to the cell culture; Di Franco C et al.; The genomic distribution and the number of elements of eleven transposon families have been compared by the Southern technique between permanent cultured cells, larval salivary glands and the brains and whole flies of an inbred Drosophila line (inb-c) from which the cells were established . In cultured cells, changes in restriction patterns consistent with various types of rearrangements such as amplification, transposition and excision of the elements of copia, 1731, 412, 297 and mdg-4 transposon families are detected whereas B 104, G and blood elements appear stable . In previous reports these rearrangements were not detected among individuals of the inb-c line or among samples of somatic tissues, or in samples spanning years of maintenance of cultured cells . Hence, we believe that they have been induced de novo during the passage to the cell culture. Vaccine, 1992, 10 Suppl 1, S36 - 9 Molecular basis of virulence and growth of hepatitis A virus in cell culture; Emerson SU et al.; The ability of engineering variants of hepatitis A virus (strain HM175) to replicate in cell culture or to cause disease in marmosets was evaluated . Virus variants were encoded by chimeric genomes constructed from infectious cDNA clones of two viruses (wild type and cell-culture-adapted) which differed in their ability to grow in vitro and to cause acute hepatitis in marmosets . Transfection and infectivity assays indicated that virus growth in vitro could be enhanced by subcloning the cell substrate prior to infection or by introducing multiple combinations of two or more mutations into the wild type genome . Various chimeric viruses induced liver enzyme elevations in marmosets, indicating that attenuation of virulence also required multiple mutations. Acta Ophthalmol Suppl, 1992, (205), 29 - 33 Anterior capsule opacification: a cell culture model; Ohara K et al.; We cultured the human lens epithelial cells with attaching anterior lens capsule using surgically removed capsular flaps obtained in intraocular lens implantation . The capsular flap was placed with cell side down in a small culture well . In phase contrast microscopy, an anti-meniscus plate was placed on the bathing medium to avoid optical aberration . The human lens epithelial cells showed outgrowth which ceased after 3 to 4 weeks of incubation . The cells under the capsule lost distinctive cell margin . The cells outgrew from the capsular edge both onto the anterior capsular surface and the well bottom . The outgrowth length and Bromodeoxyuridine uptake indicated a low proliferative potency of the human lens epithelial cells. Neurotoxicology, 1992 Spring, 13(1), 179 - 84 Glia toxicity in dissociation cell cultures induced by cyclosporine; Stoltenburg-Didinger G et al.; Intravenously applied cyclosporine is the most effective and best analyzed immunosuppressive agent to date . Most frequently, this drug shows nephrotoxic or hepatotoxic side effects, which have already been investigated in detail . In addition to these adverse effects, there is also clear clinical evidence for toxic damage to the central nervous system . On the basis of magnetic resonance tomography and computed tomography studies, the white matter seems to be primarily affected . A systematic approach to neurotoxicity has been established in the following model . Mixed in-vitro cell cultures of dorsal root ganglia (DRG) and of the central nervous system were prepared from 6 to 14 day old chick embryos (E6-E14) . For cultivation of nerve and glia cells we used beta NGF, a soluble trophic factor, and NTF B 82 as a matrix factor . Differentiated cultures were incubated with cyclosporine for intravenous application . Within a period of several days up to two weeks the cultures were analyzed using phase contrast microscopy, light microscopy, scanning and transmission electron microscopy . Glia cells and fibroblasts showed the most pronounced toxic effects . Their cytoplasm was infiltrated with smaller and larger vesicles which contained neutral lipids . Control cultures remained unaffected . Because of the close correlation between the in-vitro damage of glia cells and the clinically observed alteration of the white matter, we think our in-vitro model is helpful for the investigation of the neurotoxic effects of cyclosporine and immunotherapeutic drugs. Neurotoxicology, 1992 Spring, 13(1), 171 - 7 Neuronal cell cultures as a model for assessing neurotoxicity induced by encephalitic viruses; Shahar A et al.; Primary dispersed and organotypic cultures were prepared from selected brain areas and spinal cords of rat (Sprague-Dawley) and mouse (SJL/OLA(F) Ness-Ziona) fetuses and neonates . Following fiber regeneration, synapse formation and myelination, cultures were infected with one of the following viruses: Rabies CVS-21 strain, Sindbis Alphavirus, West-Nile Flavivirus and Theiler Murine Encephalomyelitis virus . Light and electron microscopical studies showed clear differences in the target cells for virus infection; time of viral replication and in the intensity and specificity of the cytopathic effects induced by these viruses . Thus, Sindbis and Theiler viruses induced severe cytotoxicity and demyelination due to rapid viral replication in both neurons and all glial cell types . Rabies and West-Nile viruses, on the other hand, replicated mainly in neurons and at a much slower rate, causing only mild damage to the cells and the myelin sheath . A very specific alignment of West-Nile virions was observed along the interperiod lines of the myelin sheath in several myelinated axons . This peculiar arrangement of the virions, entrapped between the myelin lamellae may lead to a novel concept in the understanding of viral infection. Arch Virol, 1992, 125(1-4), 251 - 9 Heterologous resistance to superinfection by louping ill virus persistently infected cell cultures; Venugopal K et al.; Louping ill virus, a tick-borne arbovirus readily established a persistent infection in porcine kidney (PS) cells after initially inducing minor cytopathic changes . Nucleotide sequence analysis of the envelope glycoprotein of the viral RNA recovered from the persistently infected cells showed no changes as compared with the virus used to establish persistent infections . More than 80 per cent of the cells contained virus specific antigen when analysed by indirect immunofluorescence microscopy . This persistently infected cell line resisted superinfection with either homologous or most heterologous flaviviruses . However, the yellow fever French neurotropic virus (YF FNV) multiplied in the persistently infected cells and evidence of dual infections in these cells was obtained using specific monoclonal antibodies in double labelling immunofluorescence tests . The relevance of these observations is discussed in the light of other evidence that tick-borne viruses can survive for long periods in wild animal species. Am J Ind Med, 1992, 21(6), 807 - 23 In vitro activity of silicon carbide whiskers in comparison to other industrial fibers using four cell culture systems; Johnson NF et al.; Silicon carbide whiskers (SiCW) and continuous glass filaments are important components of composite materials having potentially widespread use in the automotive, aerospace, and power generation industries . We determined the in vitro activity of three well-characterized samples of silicon carbide whiskers and a continuous glass filament sample in four different cellular assays and compared this to the activities of UICC crocidolite, JM Code 100 glass microfiber, and erionite in the same assay systems . The SiCW had a diameter range of 0.32-0.75 microns and a length range of 4.5-20.1 microns . The SiCW was significantly toxic; on a mass basis, one SiCW sample was more toxic than crocidolite; however, JM Code 100 glass microfiber, which is not toxic in vivo (i.e., it does not cause fibrogenesis or carcinogenesis when inhaled), was also more toxic than crocidolite . The glass filament sample was the least cytotoxic of all the samples tested . On a fiber number basis, all three SiCW samples were more toxic than crocidolite . The results of our study showed that SiCW exhibits significant in vitro biological reactivity . Thus, despite the caution that must be exercised in extrpolating the results of in vitro studies to conclusions about in vivo health effects, SiCW should be considered toxic until further toxicological data are available. Neuroscience, 1992, 48(3), 631 - 9 Serotonergic regulation of type II corticosteroid receptor binding in hippocampal cell cultures: evidence for the importance of serotonin-induced changes in cAMP levels; Mitchell JB et al.; The development of the hypothalamic-pituitary-adrenal response to stress is profoundly altered by environmental events . One target for environmental regulation within the hypothalamic-pituitary-adrenal axis is the hippocampal type II corticosteroid (or glucocorticoid) receptor system, which mediates the negative-feedback effects of glucocorticoids on hypothalamic-pituitary-adrenal activity . Thus, adult rats handled early in life show increased hippocampal type II corticosteroid receptor density and increased sensitivity to the inhibitory effects of circulating glucocorticoids on post-stress hypothalamic-pituitary-adrenal activity . Both effects persist throughout life . The effects of handling on type II corticosteroid receptor development are, at least in part, mediated by changes in hippocampal 5-hydroxytryptamine turnover . Moreover, 5-hydroxytryptamine can regulate type II corticosteroid receptor density in cultured hippocampal cells, providing a paradigm for examining the neurochemical mechanisms by which environmental stimuli might regulate neural differentiation . In the present studies, we examined the intracellular mechanisms underlying the effects of 5-hydroxytryptamine on type II corticosteroid receptors ({3H}RU 28362 binding) in hippocampal cell cultures . cAMP, but not cGMP, levels in cultured hippocampal cells were significantly increased by the addition of 5-hydroxytryptamine to the medium . The cAMP response to 5-hydroxytryptamine was biphasic: an initial increase in cAMP levels occurred in response to nanomolar 5-hydroxytryptamine concentrations (EC50 = 7.2 nM), while a second increase was apparent at low micromolar concentrations . 5-Hydroxytryptamine also increased {3H}RU 28362 binding (EC50 = 4.5 nM) with a maximal effect at a concentration of 10 nM . There was no further increase in {3H}RU 28362 binding even with higher, micromolar concentrations of 5-hydroxytryptamine.(ABSTRACT TRUNCATED AT 250 WORDS) Diabetologia, 1992 Jan, 35(1), 63 - 9 Mumps virus infects beta cells in human fetal islet cell cultures upregulating the expression of HLA class I molecules; Parkkonen P et al.; The ability of mumps virus to infect pancreatic Beta cells and cause alterations in their HLA expression was evaluated in cultured human fetal islet cell clusters . Mumps virus could be isolated during the whole culture period (6-8 days) and 60% of cells, including Beta cells, contained viral nucleocapsid protein at the end of the culturing . A minor decrease in insulin secretion was observed in some of the infected cultures . The infection was invariably associated with an increase in the expression of HLA class I molecules . This enhancement was mediated by soluble factors secreted by infected cells . The infection could not induce the expression of HLA-DR molecules . However, external interferon-gamma was able to cause a clear rise in DR-expression which was observed only on non-Beta-cells . Rubella and coxsackie B4 viruses were also able to enhance the expression of class I molecules while herpes simplex virus type 2 was not . The results suggest that certain viruses are able to infect Beta cells and cause alterations in their immunological appearance . Increased HLA class I expression in infected islets may exaggerate the autoimmune process in pre-diabetic individuals by increasing the activity of autoreactive cytotoxic T cells. Arch Virol, 1992, 122(1-2), 163 - 73 Anti-idiotypic antibodies to bovine herpesvirus-1 inhibit virus infection in cell cultures; AbdelMagid OY et al.; A panel of murine monoclonal antibodies (MAbs) to bovine herpesvirus-1 (BHV-1) was prepared . Three of them were neutralizing MAbs and reacted against 130/75/50 kDa, 77 kDa, or 97 kDa glycoproteins (gp) . A fourth non-neutralizing MAb recognized the 97 kDa gp . Competition radioimmunoassay demonstrated that each of the four MAbs reacted against a different virus epitope . Anti-idiotypic antibodies (anti-id) to the four MAbs were produced in rabbits and purified by sequential immunoaffinity chromatography . Each anti-id inhibited the binding of its respective MAb to BHV-1 in competitive ELISA and blocked BHV-1 neutralizing activity of the MAb . This inhibition suggested that the anti-ids were specific for the antigen binding site of the MAbs . Treatment of MDBK cells with anti-ids inhibited BHV-1 infection, which suggested that the anti-ids block a cellular component essential for virus infection . Absence of significant cross-reactivity among the anti-ids for heterologous MAbs indicated that they recognized unique determinants on the antigen binding site of the homologous MAb. Mem Inst Oswaldo Cruz, 1992 Jan-Mar, 87(1), 1 - 7 Brazilian dengue virus type 1 replication in mosquito cell cultures; Barth OM et al.; The development of dengue viruses type 1 obtained from acute human sera and inoculated into mosquito cell cultures, was observed by standard transmission electron microscopy and cytochemical staining . It follows the trans-type mechanism already established of other dengue types . Direct passage of single virus particles across the cell membrane seems to be a pathway of entry and exit in dengue-1 infected cells . The nature of numerous electron translucent vesicles and tubules, produced simultaneously during virus replication inside the rough endoplasmic reticulum, was analyzed by cytochemical tests . The largest amount of virus particles was produced inside cell syncytia. J Biopharm Stat, 1992, 2(1), 31 - 48 Exploratory data analytic techniques to evaluate anticancer agents screened in a cell culture panel; Hodes L et al.; Information theory is used to provide a measure of selectivity, i.e., the degree to which a drug has preferential toxicity or growth inhibition for one or a few cell lines from a large panel . The selectivity measure is intended to complement a measure of differential growth inhibition in evaluating the drug development potential of a new compound . Also, a similarity measure obtained from information theory is used to classify drugs according to their pattern of responses on the panel . Some structure-activity relations emerge . This work is applied to 176 agents selected to be tested by the National Cancer Institute in about 50 cell lines. Chin J Biotechnol, 1992, 8(2), 131 - 7 A basic study on the hybridoma cell culture of microencapsulation; Ning G et al.; We report here that the constitute component of Na alginate is a very important factor on microcapsular strength . We developed a method of estimating viable cell count in batch microcapsular process and the factors influencing the full loading rate in microcapsules . The physical integrity of microcapsule and full loading rate were greater than 95% . The cell line of C3 hybridoma was found to reach maximum intracapsule cell density of 8.8 x 10(6) - -2.1 x 10(7) cells/ml during static culture period of 6-10 days . During this period, the mouse monoclonal antibody accumulated in the intracapsular space to a final concentration of 379 micrograms/ml . Analysis of intracapsular protein by SDS gel electrophoresis during the culture period demonstrated that there was one band or two bands of protein which means its purity is relatively high. Eur Surg Res, 1992, 24(6), 378 - 82 Comparative cell culture effects of shape memory metal (Nitinol), nickel and titanium: a biocompatibility estimation; Putters JL et al.; Nitinol is an equiatomic alloy of nickel and titanium which has been attracting increasing interest in the field of biomedical engineering . To quantify toxicity as a preliminary evaluation of biocompatibility, inhibition of mitosis in human fibroblasts in tissue cultures exposed to test materials is an accepted screening method, although a dose-effect relationship had never been investigated . In this experiment, the effect of an increasing dose exposure to Nitinol, nickel or titanium on human fibroblasts in cell cultures was tested in subgroups in comparison with a control group . The results showed that nickel induces a significant (p < or = 0.05) inhibition of mitosis in human fibroblasts, whereas no significant effects of this kind were found for titanium or Nitinol . According to the results of these studies, Nitinol is to be considered in this respect biocompatible and comparable to titanium, which would seem to justify application as a surgical implant. Folia Parasitol (Praha), 1992, 39(4), 387 - 90 Inhibition of proliferation of tumour cell cultures by biologically active substances isolated from the tissues of Fasciola hepatica and Fasciola hepatica-infected rat liver; Tsocheva N et al.; Biologically active substances (BAS) were isolated from the tissues of Fasciola hepatica L . and from F . hepatica-infected rat liver by ethanol precipitation from aqueous tissue homogenates . A marked inhibiting effect of the newly isolated BAS on hepatoma MC29 cell culture proliferation and a slight inhibiting effect of the newly isolated BAS on myeloma cell culture proliferation was found . The strongest inhibiting effect was by BAS isolated from the tissues of F . hepatica . The inhibiting effect of the BAS isolated from F . hepatica-infected liver was stronger than the effect of the BAS isolated from normal liver tissue. Tumour Biol, 1992, 13(5-6), 358 - 63 Comparison of mitogen- and virus-induced interferon production in whole blood cell cultures of patients with various solid carcinomas and controls; Elsasser-Beile U et al.; Using a sensitive immunoassay, the mitogen-induced production of interferon-gamma (IFN-gamma) and virus-stimulated IFN-alpha production was investigated in whole blood cell cultures of 115 control subjects and 225 untreated patients with various solid carcinomas . In the cultures of the tumor patients, significantly lower levels of IFN-gamma were found as compared to the controls (p < or = 0.001) and the differences were most evident in those tumor groups containing mostly patients with advanced clinical stages . IFN-alpha values were only slightly lower in the cultures of the carcinoma patients, with p values < or = 0.05 . Statistically, there was no correlation between IFN-alpha and IFN-gamma values . Since the IFN-gamma differences between tumor and controls and the correlation to groups with higher tumor stages were much clearer than IFN-alpha differences, we conclude that IFN-gamma measurements after polyclonal induction may be the better parameter for showing a depressed cellular immunological activity in patients with malignancies than virus-induced IFN-alpha secretion. Reprod Toxicol, 1992, 6(6), 467 - 73 The combined effects of cocaine and amphetamine on primary postnatal rat heart cell cultures; Melchert RB et al.; Recent reports demonstrated that perinatal exposure to cocaine (Coc) and amphetamines (Amph) predisposed the infant to adverse cardiovascular consequences . Dose- and time-dependent effects of Coc and Amph on postnatal rat myocardial cell cultures are described . Contractile activity, morphology, lactate dehydrogenase (LDH) release, MTT formazan production, and neutral red (NR) retention were determined . No contractile activity was observed in cultures treated with the highest drug doses . After 24 h, the percentage of areas exhibiting contractile activity was decreased in cultures exposed to the lowest doses of both drugs . When Coc and Amph were combined, beating rates were significantly altered . Morphologic alterations were observed in all treatment groups . LDH release occurred in cultures exposed to the highest doses of both drugs . No significant differences were observed for MTT or NR . These data demonstrate that Coc and Amph doses > or = 1 x 10(-5) M induce adverse effects on morphology and contractile activity of postnatal myocardial cell cultures. Acta Physiol Hung, 1992, 79(1), 65 - 72 Impact of combined hormonal pretreatment (insulin+TSH) on the imprinting of hormones administered in combination to Chinese hamster ovary cell culture; Csaba G et al.; Cultured Chinese hamster ovary (CHO) cells were treated (imprinted) with insulin and with thyrotropin (TSH) related to gonadotropins (FSH+LH) . When one week later the treatment was repeated with one of the hormones, considerable differences could be observed in the binding capacity of the cells . In the hormone combination TSH was able to evoke persistent imprinting only to a markedly lesser degree than insulin, meanwhile the imprintatory effect of insulin was of greater extent even on the cell regarded to be unspecific for insulin . Hormone treatment of one hour duration--when investigated immediately after--did not extinct the binding capacity to TSH but enhanced that to insulin . With the deterioration of the conditions of culturing, the enhanced binding capacity disappeared. Acta Microbiol Hung, 1992, 39(3-4), 207 - 21 The influence of cell culture and storage conditions on HIV-1 infectivity and fusogenic activity; Ongradi J et al.; We have previously demonstrated that acidic medium inhibits the replication of HIV-1 . The present study was designed to examine the effects of other growth conditions and infection of fibroblasts by coculture with HIV infected lymphoid cells . Several lymphoblastoid cell lines normally grown in RPMI-1640 were grown in Eagle's MEM . These cells supported virus replication to higher titres than did RPMI-1640 . Peak viral titres were achieved within 24-48 h after newly infected or chronically infected cells were placed in fresh medium . When virus was stored in liquid medium either frozen or at higher temperatures, virus titres were retained for several months while frozen but decreased upon storage at 4 degrees C or higher . If cells were passaged after trypsinization in Ca(++)-depleted medium, then a decreased susceptibility of cells for HIV-1 by 2 log10 at 24 h post infection was observed . Infectivity of cell-free and cell-associated HIV-1 was measured using syncytium formation, reverse transcriptase activity and p24 antigen . No fusion between HIV-1 infected CD4+ lymphoblasts and CD4- fibroblasts was observed but HIV-1 infected lymphoid cells, even in the absence of syncytium formation, exerted a strong toxic effect on fibroblasts . This study extends previous findings that medium acidity was inhibitory to virus replication and survival . Thus, conditions for study of HIV must be well controlled in buffered medium so that misleading results are not obtained regarding virus multiplication and possibly regarding transmission to and pathogenesis in CD4- cells. Drugs, 1992, 44 Suppl 1, 105 - 110 Use of cell culture for optimisation of direct antiatherogenic therapy with verapamil; Orekhov AN et al.; The potential of verapamil to prevent atherogenesis induced by atherogenic serum in cell culture was investigated . Smooth muscle cells were cultured from human aortic intima and incubated with blood serum from patients with coronary artery disease . Only serum causing a significant rise in total cholesterol content in cultured cells during a 24-hour incubation period was used . The addition of verapamil to cells decreased serum-induced cholesterol accumulation . The maximum antiatherogenic effect of verapamil in vitro was recorded at 10(-5) to 10(-4) mol/L . Blood serum obtained from patients before and after oral verapamil administration was added to cultured cells . The atherogenic potential of serum obtained after verapamil administration was significantly lower than the predose value . To optimise direct antiatherogenic therapy with verapamil, the minimum dose that produced the maximum effect was established as 30 to 40mg . Using a 40mg dose, the maximum effect of oral verapamil was observed 3 hours after administration . A second 40mg dose was given 5 hours after the first dose, when the blood serum atherogenic potential was about 40% of the predose value and rising . This second dose maintained the atherogenic potential of serum at about 40% . Thus, to decrease atherogenic potential of serum and to maintain it at a low level, verapamil should be administered at a dose of 40mg 5 times daily with a 4- to 5-hour interval between doses. Blood Cells, 1992, 18(2), 187 - 93; discussion 194-5 A simple method for hemoglobin measurement in cell culture system; Baliga BS et al.; A simple method of hemoglobin analysis in a cell culture system is described . Hemoglobins synthesized in cell cultures are labeled with radioactive amino acids . The cell extract containing radiolabeled hemoglobin is mixed with A, F, S, C, hemoglobin markers and separated by cellulose acetate electrophoresis . Individual bands of hemoglobin are cut from the gel and analyzed for radioactivity . This method is especially useful for determination of newly synthesized minute amount of hemoglobin in cell extracts that are difficult to visualize by staining procedure. Biochem Biophys Res Commun, 1991 Dec 31, 181(3), 1164 - 72 Dystrophin: a sensitive and reliable immunochemical assay in tissue and cell culture homogenates; Ho-Kim MA et al.; A modified polyacrylamide gel electrophoresis system is described which provides excellent resolution of very high molecular weight proteins . This system has been successfully applied to the immunochemical detection of dystrophin in mouse and rat skeletal muscle, mouse myotubes in cell culture, and in human muscle-biopsy specimens . The mass of total homogenate protein (3-12 micrograms) and the relative quantity of dystrophin detected immunologically were found to be strongly correlated (r = 0.970 - 0.995) . The method described here requires minute quantities of tissue or cells to accurately evaluate the relative amount of dystrophin present . The entire procedure for the detection of dystrophin is simple, rapid and cost efficient compared to other available techniques. Brain Res Dev Brain Res, 1991 Dec 17, 64(1-2), 145 - 54 Dissociated cell culture of rat cerebral cortical neurons in serum-free, conditioned media: GABA-immunopositive neurons; Stichel CC et al.; The gamma-aminobutyric acid (GABA)ergic properties of embryonic (E15d) rat cortical neurons were studied in dissociated serum-free culture by immunohistochemical methods . GABA-like immunoreactivity was found in a subpopulation of neurons from the first day onwards . The number of GABA-positive neurons reached mature values (10.5-12.6%) within the first week, while their morphological differentiation was not found to be fully completed until the 11th day of culture and was characterized by several discrete developmental stages . First, GABA-positive neurons gained their mature complement of neurites at 3 days in vitro (DIV) . Three days later somal maturation became evident, followed at least by the maturation of the neuritic arbor . Double-labelling studies revealed the coexpression of GABA and tyrosine hydroxylase within the same cells . The similarities of relative number, morphology, time course of development and biochemistry of cultured GABAergic neurons compared with those in situ suggest that the applied culture system is a useful model to investigate several aspects of GABAergic neurotransmission at the cellular level. Biochem Pharmacol, 1991 Dec 11, 42 Suppl, S151 - 6 Accumulation of amiodarone and desethylamiodarone by rat alveolar macrophages in cell culture; Antonini JM et al.; Amiodarone is a clinically effective antiarrhythmic drug shown to cause lung damage in humans and animals . While the mechanism of this pulmonary toxicity is unknown, it may be associated with the accumulation of amiodarone and its principal metabolite, desethylamiodarone, by alveolar macrophages . In the present study, characteristics of the uptake of these drugs by rat alveolar macrophages in vitro were examined . The alveolar macrophages were collected by pulmonary lavage from male Fischer 344 rats . Amiodarone and desethylamiodarone were incubated separately (2.5 microM) with the cells in culture for 1, 2, 4 and 18 hr . High performance liquid chromatography was used to measure drug uptake . At 1 and 2 hr, the uptake of desethylamiodarone by alveolar macrophages was significantly greater (P less than 0.05) than that of amiodarone, but over time, the accumulation of amiodarone began to approach that of desethylamiodarone and was not significantly different by 4 hr . To simulate a more physiological situation, plasma levels achieved in the adult male rat after 1 week of amiodarone treatment (150 mg/kg) were used . Amiodarone (1.95 micrograms/mL) and desethylamiodarone (0.80 microgram/mL) were added together into the cell culture . At 1 and 18 hr, the ratio of desethylamiodarone/amiodarone uptake was significantly greater (P less than 0.05) than in incubation medium containing no cells, indicating an enhanced uptake of desethylamiodarone . Metabolic inhibitors (KCN, 2,4-dinitrophenol, and ouabain) and other cationic, amphiphilic drugs (chlorcyclizine, chlorphentermine, and imipramine) were added individually to the cell cultures containing amiodarone or desethylamiodarone . During 1 hr of incubation, these agents had no effect in blocking the accumulation of amiodarone and desethylamiodarone in the cells . The efflux of amiodarone or desethylamiodarone was measured from cells following incubation for 4 hr with each drug . After this time, the medium was replaced with drug-free medium, and the cells were incubated for another 24 hr . Sixty-three percent of amiodarone was lost as compared to only 31% of desethylamiodarone over the 24-hr period (P less than 0.05) . The results of this study are suggestive of a preferential uptake and retention of desethylamiodarone as compared to amiodarone . The accumulation of the drugs appears not to be due to active transport or associated with any carrier protein involved in the transport of other structurally-related compounds. Proc Natl Acad Sci U S A, 1991 Dec 1, 88(23), 10740 - 3 Inhibition of the activation of Hageman factor (factor XII) by human vascular endothelial cell culture supernates; Ratnoff OD et al.; The supernatant fluid (conditioned medium) of cultured human vascular endothelial cells inhibits activation of Hageman factor (factor XII), whether by ellagic acid, bovine brain sulfatides, or bismuth subgallate; inhibition appears to be a property of one or more proteins in the culture supernates . This phenomenon may contribute to maintaining the fluidity of circulating blood by inhibiting surface activation of the intrinsic pathway of coagulation. Lipids, 1991 Dec, 26(12), 1086 - 92 Biotransformation of alkylglycerols in plant cell cultures: production of platelet activating factor and other biologically active ether lipids; Mangold HK et al.; Plant cells in culture are capable of incorporating exogenous 1-O-alkyl-sn-glycerols into various neutral and ionic ether lipids . 1-O-Alkyl-2-acyl-sn-glycero-3-phosphocholines, the major class of compounds thus formed, are used for the preparation of platelet activating factor (PAF) in high yields . Similarly, the prochiral 2-O-alkyl-sn-glycerols are transformed to chiral 2-O-alkyl glycerophospholipids from which compounds can be obtained that exhibit antiviral activity in plant and animal cells . Reaction of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholines with phospholipase D in the presence of ethanolamine leads to 1-O-alkyl-2-acyl-sn-glycero-3-phosphoethanolamines, which serve as starting material for the preparation of 1-O-alkyl-2-acyl-sn-glycero-3-phospho-(N-acyl)ethanolamines, compounds known to have antitumor activity. Chin Med Sci J, 1991 Dec, 6(4), 230 - 2 Inhibition of HIV replication by baicalin and S . baicalensis extracts in H9 cell culture; Zhang X et al.; Crude extracts of Scutellia baicalensis and a particular low molecular weight substance known as Baicalin were shown to inhibit HIV antigen expression in H9 cell culture, with 50% inhibitory doses of 0.6 microgram/ml and 3.3 micrograms/ml, respectively . They also inhibited P24 antigen production in H9 cells, with IC50's of 1.94 and 4.74 micrograms/ml, respectively . Cytoxicity was tested in H9 and HUT78 cells: Both exhibited relatively low levels of cytoxicity . These results indicated that S . baicalensis and Baicalin strongly inhibit HIV replication in cell culture. J Spinal Disord, 1991 Dec, 4(4), 428 - 36 Cell culture of the intervertebral disc of rats: factors influencing culture, proteoglycan, collagen, and deoxyribonucleic acid synthesis; Ichimura K et al.; To establish cell culture of the nucleus pulposus and anulus fibrosus of rat intervertebral disc, the effects of culture conditions on the growth of cells and the synthesis of DNA, proteoglycan, and collagen were studied . For cell culture of the nucleus pulposus, the use of 3-week-old rats and a medium adjusted to pH 7.0 was optimal . There was almost no difference in growth between cells in Ham's F12 medium and those in Dulbecco's Modified Eagle Medium . In cells isolated from the anulus fibrosus, a medium adjusted to pH 7.0-7.6 was preferable, but irrespective of rat age . Culture cells of the nucleus pulposus were composed of large cells with vacuoles and small polygonal cells . These cells had a slight growth activity and a fair capability of proteoglycan and collagen synthesis . Culture cells of the anulus fibrosus were composed of polygonal and spindle-shape cells, and the growth was more vigorous with the potentials for proteoglycan and collagen synthesis than the nucleus cells. Asian Pac J Allergy Immunol, 1991 Dec, 9(2), 121 - 4 Comparative study of respiratory syncytial virus in nasopharyngeal aspirates using conventional cell culture, shell viral centrifugation culture, immunofluorescence and biotin-avidin enzyme linked immunosorbent assays; Woodtayakorn J et al.; 133 nasopharyngeal aspirates (NPA) were simultaneously tested for the presence of respiratory syncytial virus (RSV) by conventional cell culture (CCC), shell vial centrifugation culture (SVC), immunofluorescence assay (IFA) and biotin-avidin enzyme linked immunosorbent assay (B-A ELISA) . These yielded positive results in 32(24%), 45(33.8%), 36(27%) and 40(30%) of specimens, respectively . Specimens positive by IFA and B-A ELISA were all also positive by SVC . The sensitivity of CCC, IFA, and B-A ELISA comparing to SVC was 71%, 80%, and 88.9%, respectively . For rapid detection of RSV, we recommend the SVC method where a cell culture laboratory is available and the B-A ELISA method where a cell culture laboratory is not available. Gaoxiong Yi Xue Ke Xue Za Zhi, 1991 Dec, 7(12), 614 - 21 The effects of epidermal growth factor and chondroitin sulfate on the animal corneal endothelial cell culture; Lee HJ et al.; In order to investigate the effects of different culture media containing various concentrations of epidermal growth factor (EGF) and chondroitin sulfate (CDS) on the growth of cultured corneal endothelial cell, pig corneal endothelial cells were used for this study . The cells were divided into 7 groups and each group was cultured with a medium containing: 1 . Eagle's minimal essential medium with Earle's salt (EMEM) only; 2 . EMEM + EGF (10 ng/ml); 3 . EMEM + EGF (100 ng/ml); 4 . EMEM + CDS (1 mg/ml); 5 . EMEM + CDS (25 mg/ml); 6 . EMEM + EGF (10 ng/ml) + CDS (1 mg/ml); 7 . EMEM + EGF (100 ng/ml) + CDS (25 mg/ml), respectively . The results are shown below: (1) High concentration of EGF (100 ng/ml) stimulated the growth of pig corneal endothelial cells, shortened doubling time and the cells reached confluence earliest in group 3 . But a low concentration of EGF (10 ng/ml) showed no effects on the growth of pig corneal endothelial cells . (2) A high concentration of CDS (25 mg/ml) might retard the growth of pig corneal endothelial cell, but a low concentration of CDS (1 mg/ml) has no retarding effects on the growth of pig corneal endothelial cells . (3) A high concentration of EGF could "antagonize" the CDS induced growth retarding effects of endothelial cells . (4) Those cells cultured with a medium containing high concentrations of EGF had more mitotic activity, including many prominent binuclear and polynucleolar cells . (5) The cells cultured with a medium containing high concentrations of CDS showed a more flat-shaped morphology under observation with a phase contrast microscope.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Cell Endocrinol, 1991 Dec, 82(2-3), 265 - 73 Effects of recombinant human inhibin and testosterone on gonadotropin secretion and subunit mRNA in superfused male rat pituitary cell cultures stimulated with pulsatile gonadotropin-releasing hormone; Jakubowiak A et al.; Effects of recombinant human inhibin (rh inhibin) and testosterone on follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secretion and mRNA levels of gonadotropin subunits were investigated in superfused male rat pituitary cell cultures . During superfusion, the cells were stimulated with gonadotropin-releasing hormone (GnRH) pulses (10 nM, 6 min/h) and exposed to rh inhibin (2 ng/ml) and/or testosterone (10 nM) for up to 20 h . The concentrations of FSH and LH were measured in effluent media by radioimmunoassay (RIA), and subunit mRNAs were determined by Northern blot hybridizations using rat FSH beta, LH beta and alpha genomic and cDNA probes . Rh inhibin suppressed the secretion of FSH (30-40% of control) and the secretion of LH to 50-60% of control, but inhibited only FSH beta mRNA (to non-detectable levels) . Testosterone alone suppressed the release of LH to 50% of control, whereas FSH release was increased to 130-160% (P less than 0.05) of control . This increase was due to higher interpulse values without significant changes in the pulse amplitude . Also FSH beta mRNA level was increased (1.5-fold, P less than 0.05) but only after 17-20 h of treatment . On the other hand, testosterone had no effect on LH beta and alpha subunit mRNA levels . Testosterone in combination with rh inhibin showed an inhibitory effect on LH beta mRNA; however, the pattern of LH release was not significantly different from that observed with rh inhibin or testosterone alone . Combined effects of testosterone and rh inhibin on FSH secretion and FSH beta mRNA were similar to those observed with rh inhibin alone.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Endocrinol, 1991 Dec, 5(12), 1880 - 6 Single amino acid substitutions in recombinant bovine prolactin that markedly reduce its mitogenic activity in Nb2 cell cultures; Luck DN et al.; Three amino acid residues of bovine PRL (bPRL) have been examined for their roles in the mitogenic activity of the hormone in Nb2 lymphoma cell cultures . The residues of interest, R21, R177, and K187, are conserved in eight pituitary PRLs, but not in the related, nonlactogenic bGH . Using site-specific mutagenesis, a number of recombinant methionyl bPRL variants have been prepared, each of which contained a single amino acid substitution of one of the three residues; a variety of amino acids was used for substitution . Twelve exchanges of R177 (to A, L, N, K, D, E, Y, G, S, Q, H, and F) all led to marked decreases in mitogenic activity . Even the conservative change, R177K, led to a decrease in mitogenic activity of about 90%; all the other R177 substitutions led to even more marked decreases; there was essentially complete loss of activity when the positively charged R177 was replaced by the negatively charged aspartate . Exchanges of R21 (to A, L, N, and K) were less dramatic, with the greatest decrease (79%) occurring in the case of R21A . Exchanges of K187 (to A, L, N, and R) had a relatively minor effect on the mitogenic activity of the hormone . Residues R21 and R177 in bPRL are located in putative helices 1 and 4, respectively; in the three-dimensional structure of the hormone these residues are predicted to be quite closely apposed . The results suggest that R177 and, to a lesser degree, R21 have important roles in the mitogenic activity of bPRL. Biull Eksp Biol Med, 1991 Dec, 112(12), 615 - 7 {Effects of growth factors on growth of stromal CFU-f in mouse bone marrow cell cultures}; Gorskaia IuF et al.; Purified mouse IL-1 at doses 15-100 mu/ml inhibits the growth of stromal clonogenic cells /CFU-f/ both in full bone marrow cell cultures /F-cultures/ and in adherent bone marrow cell cultures /A-cultures/ . Rec . human TNF-alpha inhibits growth of these cells at doses greater than 50 u/ml, but stimulates it /in 1.5 fold increase/at low doses /0.1-20 u/ml/ in cultures of both types . Rec . mouse IL-3 at doses 0.8-50 mu/ml slightly increases/in 1.6 fold increase/the in vitro growth of CFU-f and inhibits it at low doses in F-cultures . In A-cultures this factor stimulates CFU-f growth at all doses tested, but this stimulating effect takes place if only explantation density of mouse bone marrow cells in sufficiently high. J Am Soc Nephrol, 1991 Dec, 2(6), 1153 - 7 Heparin increases hillock formation in mesangial cell cultures; Ayo SH et al.; Mesangial cells in culture develop hillocks, which are composed of aggregates of cells, necrotic cellular debris, and extracellular matrix material . The significance and mechanism of their formation are unknown . To determine whether a proliferative component is involved in hillock formation, cells were treated with heparin or irradiated to inhibit proliferation . Heparin caused a 50% inhibition of mesangial cell growth and stimulated hillock formation three-fold to fourfold . Irradiated cells developed hillocks to the same extent as did nonirradiated cells, and the addition of heparin also increased hillock formation threefold to fourfold . Dextran sulfate and chondroitin B sulfate had no effect on mesangial cell hillock formation . Mesangial cells cultured in the presence of 50 micrograms/mL of heparin were less tightly adhered than nontreated cells, as assessed by a trypsin adhesion assay (control cells, 12% detached; heparin-treated cells, 72% detached) . Thus, it appears that heparin, a glycosaminoglycan with potent antimitogenic activity, stimulates mesangial cell hillock formation, possibly by decreasing cell adhesion. Am J Physiol, 1991 Dec, 261(6 Pt 1), C1196 - 203 A spectroscopic method for assessing confluence of epithelial cell cultures; Jovov B et al.; We describe a convenient nonelectrophysiological technique for assessing cell proliferation and subsequent tight junction formation for epithelial monolayers grown on permeable supports . The method involves the use of phenol red (PR), a standard pH indicator in most cell culture media . In addition, we report a systematic error in a commercially available system for measuring transepithelial electrical properties . Briefly, the flux of PR across the epithelium was measured from the serosal solution into the mucosal solution . The mucosal solution was first replaced with a PR-free solution and then collected at timed intervals . The PR concentration was measured using a spectrophotometer set at the isosbestic point for PR (479 nm) . PR flux was then calculated and used as an index of the permeability of the epithelium to PR . This method was tested using the renal epithelial cell line A6 . After cell seeding, PR flux decreased in two phases: an initial large decrease, associated with cell growth and monolayer confluence, and a second decrease associated with tight junction formation {assessed by measuring transepithelial conductance (Gt)} . In addition to monitoring tight junction formation, PR flux measurements were also used to estimate the net movement of solution by the epithelial cells between the mucosal and serosal compartments . For convenience, Gt was initially measured in culture dishes using a commercially available "chopstick" electrode system . However, the chopstick system yielded Gt values that were on average 51% lower than values for the same preparations when measured in standard Ussing-type chambers . The discrepancy was due to a nonuniform current field produced by the chopstick electrodes. Pharmacol Toxicol, 1991 Dec, 69(6), 416 - 20 Inhibition of dye transfer in rat liver WB cell culture by polychlorinated biphenyls; Hemming H et al.; In the present investigation the scrape loading/dye transfer assay and microinjection technique are used in order to investigate inhibition of cell-cell communication induced by different polychlorinated biphenyl (PCB) congeners . In these in vitro assays, inhibition of intercellular communication is directly measured as decreased transfer of a fluorescent dye (Lucifer Yellow CH) from donor cells loaded with the dye to surrounding recipient cells . The results show that substitution in the ortho position from the carbon bridge is essential and at least one chloro substituent in ortho position is necessary for the ability to inhibit intercellular communication . The results also suggest that an increase in the number of ortho substituted chlorine atoms in the PCB molecule enhances the ability to inhibit intercellular communication . On the other hand, the total number of substitutions may not be crucial for the ability to inhibit intercellular communication . Our results suggest that PCB-induced down-regulation of intercellular communication is a result of a specific mechanism and not due to unspecific membrane perturbation. Aviat Space Environ Med, 1991 Dec, 62(12), 1159 - 65 Vector-averaged gravity alters myocyte and neuron properties in cell culture; Gruener R et al.; To investigate whether changes in the gravitational field of developing neurons and myocytes affect cellular development, we rotated cultures of embryonic spinal neurons and myocytes in a horizontal clinostat . Rotation in the clinostat produces, from the cells' perspective, a "vector-free" gravity environment by continuous averaging of the vector . In this way, rotation in the clinostat simulates the microgravity of space where the gravity vector is substantially reduced . At rotation rates of 1-50 rpm, cellular and nuclear areas of myocytes were significantly enlarged and the number of presumptive nucleoli increased . In neurons, frequent and large swellings appeared along neuritic shafts . Some of these changes were reversible after cessation of rotation . Since our data are generally consistent with findings from other cell types subjected to spaceflight, we suggest that the vector-free gravity environment of the clinostat appears to simulate, at least in part, the microgravity of space . Our data further show that cellular processes are sensitive to altered gravity and suggest that cell development in the microgravity of space may be significantly altered. Int Immunol, 1991 Dec, 3(12), 1223 - 9 Neuroimmune modulation of lymphocyte function--I . Substance P enhances immunoglobulin synthesis in lipopolysaccharide activated murine splenic B cell cultures; Pascual DW et al.; Murine B cells have been shown to possess substance P (SP) receptors, but their functional and biological significance remains unresolved . While previous studies have suggested that SP can induce B cells to secrete Ig, the effect could be indirect since mixed cultures were used . In order to assess directly the ability of SP to trigger normal B cells, we have studied the effects of this neuropeptide on purified splenic B cells in vitro . Although an activation, e.g . lipopolysaccharide (LPS), was required, the functionality of the B cell SP receptors was clearly shown by the ability of subnanomolar concentrations of this neuropeptide to augment antibody secretion in a dose-dependent fashion . Specifically, IgM and IgG levels, determined by an isotype-specific sandwich ELISA, were greatly enhanced at 10(-10) M SP by as much as 500 and 572% respectively, while IgA levels were only modestly affected . Even picomolar concentrations of SP could significantly increase IgM levels . This observed enhancement of Ig production was SP specific since B cells co-cultured in the presence of excess SP antagonist were reduced to basal LPS-stimulated Ig levels . Furthermore, this synergistic stimulation by SP and LPS upon normal B cells could not be attributed to SP-induced cell proliferation since stimulatory concentrations of SP were not mitogenic and at high concentrations could inhibit cell proliferation . Rather, it was observed that the increased IgM and IgG secretion was in part attributable to a greater number of B cells secreting antibodies as demonstrated with an ELISPOT assay.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Chem Neuropathol, 1991 Dec, 15(3), 217 - 33 Paradoxical potentiation by low extracellular Ca2+ of acute chemical anoxic neuronal injury in cerebellar granule cell culture; Verity MA et al.; Acute chemical anoxic injury was produced in primary cerebellar granule cell cultures incubated with iodoacetate (IAA) alone or IAA combined with potassium cyanide (KCN) . Cytotoxicity was assessed using Trypan blue exclusion or LDH release . Four millimolars of KCN induced approx 30% neuron death at 3 h, whereas greater than 50% cell death was produced by 0.2 mM IAA . No potentiation of cytotoxicity was observed by IAA + KCN . A total of 0.2 mM IAA produced an early major reduction of intracellular ATP prior to the onset of neuron injury or reduction in intracellular glutathione (GSH) . Medium Na+ replacement by choline, K+, or methylglucamine protected against IAA-induced neuronal injury, reduced the rate of decline of intracellular ATP but had no effect on intracellular GSH . Some 80% neuronal survival was obtained when Na+ was deleted from the medium even after the intracellular ATP had been reduced to less than 10% of control . Removal of Ca2+ from the medium had no effect on control culture, Trypan blue exclusion, GSH, or ATP, but potentiated the onset and magnitude of IAA-induced cytotoxicity . ATP and GSH decline . Loading of granule cells with the Ca2+ chelator Fura-2 did not influence IAA-induced cytotoxicity in control or low Ca2+ media . Addition of 50 microM glutamate had a minimal cytotoxic effect over 3 h and the combined addition of 0.2 mM IAA plus 50 microM glutamate did not potentiate IAA-induced injury . The glutamate receptor antagonists, D-2-amino-5-phosphonovaleric acid (APV) or kynurenate did not block IAA-induced injury in control medium but inhibited the potentiation of toxicity seen in the low Ca2+ medium . This study suggests the use of IAA as a chemical anoxic agent in cerebellar granule cell culture . The early, dose-dependent decline in ATP may be dissociated from GSH change . Acute IAA-induced injury is Na+/Cl- dependent but paradoxically potentiated in low Ca2+ medium . The low Ca2+ potentiated component was sensitive to glutamate/NMDA receptor antagonists and associated with reduction of intracellular GSH. J Cell Biol, 1991 Dec, 115(6), 1783 - 90 Development of Phodopus sungorus brown preadipocytes in primary cell culture: effect of an atypical beta-adrenergic agonist, insulin, and triiodothyronine on differentiation, mitochondrial development, and expression of the uncoupling protein UCP; Klaus S et al.; A new cellular model for the study of brown adipocyte development and differentiation in vitro is presented . Preadipocytes isolated from brown adipose tissue (BAT) of the djungarian dwarf hamster Phodopus sungorus are able to proliferate and differentiate in vitro into true brown adipocytes able to express the BAT marker protein the uncoupling protein (UCP) . Whereas basal UCP expression is very low, its mRNA levels as well as the UCP detected by immunoblotting are highly increased by beta-adrenergic stimulation . The novel, atypical beta-adrenergic compound D7114 (ICI Pharmaceuticals, Macclesfield, Cheshire, England) was found to increase the number of adipocytes as well as UCP mRNA and UCP content of mitochondria, indicating the involvement of an atypical or beta 3 receptor . Insulin was found to play an important role in brown adipocyte differentiation and mitochondrial development, whereas T3 seemed to be implicated more directly in UCP expression . In a defined, serum-free medium a synergistic stimulatory action of insulin and T3 on UCP expression was found, which seems to involve a pathway different from that of beta-adrenergic UCP stimulation. Fiziol Zh SSSR Im I M Sechenova, 1991 Dec, 77(12), 86 - 90 {The action of parathyroid hormone on a cell culture of the Vero line}; Barabanova VV et al.; The effect of parathyroid hormone (PTH) involves activation of the fibroblast-like cells proliferation at the phase of active division alone . The PTH increases the resistance of the vero line cells against the change of the pH. J Clin Microbiol, 1991 Dec, 29(12), 2789 - 93 Detection of rodent coronaviruses in tissues and cell cultures by using polymerase chain reaction; Homberger FR et al.; A polymerase chain reaction (PCR) method was developed for the detection of rodent coronaviruses in biological material by using reverse transcriptase and two primers which flanked an M gene sequence of 375 bp . PCR detected all of 11 different strains of mouse hepatitis virus (MHV) as well as rat sialodacryoadenitis virus but not bovine coronavirus or human coronavirus strains OC43 and 229E . The M gene sequences of bovine coronavirus and human coronavirus OC43 are homologous to that of MHV, but minor differences exist in the primer regions, preventing annealing of the primers . For detecting MHV-Y in tissue samples, PCR was faster than and at least as sensitive as either of the two bioassays (infant mouse bioassay and mouse antibody production test) currently used for MHV diagnostic purposes. J Cell Biol, 1991 Dec, 115(6), 1725 - 35 A cell culture model of the blood-brain barrier; Rubin LL et al.; Endothelial cells that make up brain capillaries and constitute the blood-brain barrier become different from peripheral endothelial cells in response to inductive factors found in the nervous system . We have established a cell culture model of the blood-brain barrier by treating brain endothelial cells with a combination of astrocyte-conditioned medium and agents that elevate intracellular cAMP . These cells form high resistance tight junctions and exhibit low rates of paracellular leakage and fluid-phase endocytosis . They also undergo a dramatic structural reorganization as they form tight junctions . Results from these studies suggest modes of manipulating the permeability of the blood-brain barrier, potentially providing the basis for increasing the penetration of drugs into the central nervous system. Am J Clin Nutr, 1991 Dec, 54(6 Suppl), 1247S - 1251S Ascorbate stabilizes the differentiated state and reduces the ability of Rous sarcoma virus to replicate and to uniformly transform cell cultures; Schwarz RI; In primary avian tendon cells, Rous sarcoma virus can coexist or completely take over the cell . Infection, at high multiplicity or under conditions that promote high virus production (no ascorbate and high serum concentrations), results in almost complete oncogenic transformation of the culture . This is indicated in part by a radical change in morphology, growth at high cell density, and a dramatic drop in the production of procollagen from approximately 50% to approximately 3% of total protein synthesis . In contrast, infection at low multiplicity, infection with a replication defective virus, or the presence of ascorbate restrict the ability of the virus to transform the culture . Thus, there appears to be a balance between the normal and transformed states of the cell that can be shifted depending on the cellular environment and the level of infection . Ascorbate stabilizes the normal state by reducing virus production and promoting the synthesis of differentiated proteins. Brain Res Dev Brain Res, 1991 Nov 19, 63(1-2), 1 - 12 Differences in transmitter release, morphology, and ischemia-induced cell injury between cerebellar granule cell cultures developing in the presence and in the absence of a depolarizing potassium concentration; Peng LA et al.; Release of glutamate and aspartate was measured in mouse cerebellar granule cells in primary cultures grown for 4-16 days in serum-containing tissue culture medium with either a partially depolarizing (25 mM) or a physiological concentration of potassium (5.4 mM) . The cells migrated to form aggregates connected by a network of processes during the first week in culture and both groups of cultures survived for at least 2 weeks . In cultures grown in the presence of 25 mM potassium for at least 8 days there was a large (approximately 10 nmol/min/mg protein), calcium-dependent glutamate release and a smaller aspartate release during superfusion with 50 mM potassium . This response was not present in cultures grown in the physiological medium . Nevertheless, exposure to an elevated potassium concentration caused a normal, or even enhanced calcium entry into the cells . Phase contrast microscopy showed a similar appearance of the cellular aggregates under each of the two conditions . Electron microscopy revealed that the aggregates consisted of a centrally located neuropil and peripherally located granule cell bodies . The morphology of the cell bodies and the neuropil in the cells grown at the high potassium concentration closely resembled that of cerebellar granule cells in vivo . In the cells grown at the low potassium concentration, cell bodies, axons and synaptic vesicles looked normal, but the remainder of the neuropil, especially dendrites, showed massive degeneration . Immunochemical measurements demonstrated similar amounts of synaptophysin under each of the two culturing conditions, thus confirming our impression that there were similar numbers of synaptic vesicles and hence presynaptic elements in the two types of cultures . Fluorescence microscopy, using fluorescein diacetate to stain living cells and propidium iodide to stain dead cells, indicated a much greater resistance to ischemic cell injury in the cells cultured at the low potassium concentration . Possible reasons for this difference are discussed. FEMS Microbiol Lett, 1991 Nov 15, 68(2), 129 - 34 Ultrastructure of Chlamydia pneumoniae in cell culture; Popov VL et al.; The electron microscopic appearance of Chlamydia pneumoniae elementary bodies with pear-shaped, loose outer membrane has been suggested as one criterion of its classification as a new chlamydial species . The study of the original strain TW 183 in LCL 929 and HL cells and a low-passage isolate of Kajaani-6 isolate in HL cells revealed spherical compact elementary bodies common to other chlamydia. Eur J Pharmacol, 1991 Nov 13, 208(3), 239 - 47 Comparison of calcium ionophore and receptor-activated inositol phosphate formation in primary glial cell cultures; Wigginton SA et al.; The possible role of Ca2+ influx in alpha 1-adrenoceptor-stimulated {3H}inositol phosphate {( 3H}InsP) formation was examined in primary cultures of glial cells from 1-day-old rat brain . The Ca2+ ionophore A23187 caused a concentration- and time-dependent increase in {3H}InsP formation similar in magnitude to that caused by norepinephrine (NE) . Responses to A23187 and NE were both completely dependent on extracellular Ca2+, with a similar concentration dependence . However, cadmium was more potent in blocking the response to A23187 than to NE . Lanthanum (1 mM) blocked the response to NE, although cobalt (5 mM) did not . The {3H}InsP response to A23187 was not additive with the response to NE or to the muscarinic agonist carbachol, although responses to NE and carbachol were addictive Both A23187 and ionomycin inhibited the additive stimulation caused by a combination of NE and carbachol, and this inhibition was potentiated by cadmium . Ionomycin stimulated {3H}InsP formation at concentrations lower than those inhibiting receptor-mediated responses, and this stimulation was not additive with responses to NE or carbachol . High-performance liquid chromatography separation showed similar patterns of {3H}InsPs formed in response to both Ca2+ ionophore and receptor agonists . These results raise the possibility that receptor-activated Ca2+ influx may be involved in stimulation of {3H}InsP formation in these cells. J Pharmacol Methods, 1991 Nov, 26(3), 211 - 22 Rabbit aortic smooth muscle cell culture . A model for the pharmacological study of diabetes-induced alterations in cell proliferation; Alipui C et al.; Atherosclerotic vascular disease is the most common complication of diabetes mellitus . Enhanced vascular smooth muscle cell proliferation plays a central role in atherosclerotic lesion formation . Studies using explant cultures have demonstrated that aortic smooth muscle cells from rats with experimental or genetic diabetes have enhanced rates of proliferation when compared to controls . However, this method of culture may select for cells with enhanced migratory potential . In the present studies, aortic smooth muscle cells were successfully cultured from control and diabetic rabbits after enzymatic and mechanical dispersion from thoracic aortic segments . The proliferative patterns of control cells were characterized and growth rates of diabetic cells were compared to controls . Primary cultures from control rabbits grew after an initial 5-day lag period to achieve threefold increases in cell number by 9 days . Subcultures of aortic smooth muscle cells entered the logarithmic phase of growth after 2 days, reaching the plateau phase of growth in 5-7 days and achieving three to fourfold increases in cell number . The final density to which cultures grew was not affected by the number of cells attached on day 1 for the range studied . Cells from diabetic rabbits displayed shorter doubling times and reached greater densities at confluence than did cells from controls . These data support the hypothesis that diabetes induces an atherogenic response . The dissociated rabbit aortic smooth muscle cell culture provides a model in which to study diabetes-induced modulation of cell proliferation that is amenable to pharmacological manipulation to investigate agonist and growth factor-induced responses. J Thorac Cardiovasc Surg, 1991 Nov, 102(5), 666 - 72 Prolonged hypothermic cardiac storage with University of Wisconsin solution . An assessment with human cell cultures; Fremes SE et al.; Hypothermic storage of cardiac allografts is routinely used for transplantation but is associated with an increased mortality when ischemic times are greater than 4 hours . The ideal storage conditions (solution and temperature) could extend the current limits of cold ischemia . Human endothelial cells and ventricular myocytes were studied to screen various solutions and temperatures for organ preservation . Four solutions (modified Euro-Collins, phosphate-buffered saline, Stanford cardioplegia, and University of Wisconsin) were evaluated . Endothelial cells were evaluated after prolonged hypothermic storage consisting of 0 degree, 4 degrees, and 8 degrees C for 36 hours, and ventricular myocytes were stored at 0 degree and 8 degrees C for 24 hours . Cell viability was determined by morphology (10 dishes per group), and trypan blue exclusion (5 dishes per group) in addition to a cell adhesion assay (endothelial cells 5 dishes per group) and adenine nucleotide analysis with high-performance liquid chromatography techniques (ventricular myocytes 5 dishes per group) . Endothelial cell morphology was best preserved by University of Wisconsin solution (p less than 0.001, chi 2) and at 0 degree C (p less than 0.01, chi 2) . Endothelial cells stored with University of Wisconsin solution excluded trypan blue better (1.0% +/- 0.5% cells stained, p less than 0.001 . Analysis of variance {ANOVA}) . Cell adhesion was poorly protected with Stanford cardioplegia (p less than 0.001, ANOVA) . Myocyte morphology was preserved best with University of Wisconsin solution at 0 degree C (p less than 0.001, chi 2) . According to trypan blue staining, Euro-Collins and University of Wisconsin solutions were superior to Stanford cardioplegia or phosphate-buffered solutions (p less than 0.001, ANOVA) . Temperature did not influence the trypan blue results . Adenosine triphosphate was maintained best with University of Wisconsin solution at 0 degree C (p less than 0.01, ANOVA) . Myocytes were more sensitive to the effects of prolonged storage compared with endothelial cells by morphologic criteria and trypan blue staining characteristics, irrespective of the shorter preservation times . University of Wisconsin solution was the most effective solution tested . Colder temperatures (0 degree to 4 degrees C) provided better protection than 8 degrees C . Myocytes were more sensitive to prolonged preservation than endothelial cells . Furthermore, the technique used appears helpful as a model of prolonged hypothermic storage and could be expanded to assess other interventions. J Gen Virol, 1991 Nov, 72 ( Pt 11), 2653 - 60 Selection kinetics during serial cell culture passage of mixtures of wild-type Autographa californica nuclear polyhedrosis virus and its recombinant Ac360-beta-gal; Huang YS et al.; Detailed analysis of the selection process in serial co-infections of cell cultures by wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) strain E2 (AcNPV/E2) and Ac360-beta-gal, a genetically engineered strain, shows that the unaltered strain was clearly dominant even when it initially constituted the minority component in the inoculum . A method of calculating a selection coefficient that quantifies the relative advantage of one strain of virus over the other under specific culture conditions is described . Calculated selection coefficients were relatively homogeneous and almost exclusively favoured the progenitor . Selection pressure was not influenced by the relative proportions of the two strains in the population . Selection coefficients, as determined in the present study, may be useful for evaluating the effect of a genetic alteration on viral fitness under specified conditions . Unexpected high frequencies of mixed phenotype plaques were observed during infectivity titrations of media from early serial passages of co-infected cultures . Statistical evaluation implicates some non-heritable combinational phenomenon . Virus plated from mixed phenotype plaques show high segregation of phenotypes implying that genetic recombination does not contribute in a major way to the high mixed phenotype frequencies . Electron microscopic examination of virion pellets from infected 72 h cell culture media similarly argue against co-envelopment as a major contributory factor to the high frequency of mixed phenotype plaques . The cause remains undetermined. Fertil Steril, 1991 Nov, 56(5), 997 - 1000 Presence of messenger ribonucleic acid for epidermal growth factor (EGF) and EGF receptor demonstrable in monolayer cell cultures of myometria and leiomyomata; Yeh J et al.; There is a paucity of data concerning the factors involved in the growth of uterine leiomyomata . The expression of mRNAs encoding EGF and the EGF receptor in myometrial and leiomyoma cultures suggest that EGF may be involved in the autocrine/paracrine regulation of human uterine leiomyomata and myometrial growth. Fertil Steril, 1991 Nov, 56(5), 881 - 7 Human ovarian granulosa cell culture: determination of blood cell contamination and evaluation of possible culture purification steps; Beckmann MW et al.; OBJECTIVE: To determine the degree of blood cell contamination in GC preparations; to assess techniques of GC purification; and to determine possible effects of contaminating cells and purification techniques on cultured GC in terms of steroid hormone production . DESIGN: Contamination of GC by white blood cells was assessed by Wright's stain and immunohistochemistry . Purification was attempted by: (1) Ficoll density centrifugation (to remove polymorphonuclear leukocytes {PML}); (2) incubation in tissue culture plastic dishes (to remove adherent monocyte/macrophages); and (3) incubation in the presence of high salt (to remove lymphocytes) . RESULTS: Ficoll density centrifugation reduced PML to 2% of total cells, incubation in plastic dishes reduced monocyte/macrophage contamination from 6% to 7% down to less than 1%, and high-salt incubation reduced lymphocyte contamination from 10% to 12% down to 4% . Granulosa cells plated after preparation with addition of high salt showed increased progesterone production, which could not be entirely explained by the removal of contaminating lymphocytes . CONCLUSION: These results indicate that the human GC culture system is more complex than is often assumed, and methods that remove a majority of these white blood cells are presented. Carcinogenesis, 1991 Nov, 12(11), 2001 - 6 Effects of biochanin A on metabolism, DNA binding and mutagenicity of benzo{a}pyrene in mammalian cell cultures; Chae YH et al.; The search for potential chemopreventive agents from higher plants based upon alteration of benzo{a}pyrene (B{a}P) metabolism in cell cultures resulted in isolation of the isoflavone biochanin A . The mechanisms by which biochanin A inhibits the metabolic activation of B{a}P were investigated in hamster embryo cell cultures . Biochanin A treatment inhibited the metabolism of B{a}P to water-soluble metabolites . B{a}P-9,10-diol and B{a}P-7,8-diol by 44, 60 and 52% respectively . Biochanin A inhibited the formation of glucuronide conjugates from 3-OH-B{a}P and 9-OH-B{a}P . Biochanin A also inhibited, in a dose-dependent manner, oxidation of B{a}P by homogenate (S-9) of Aroclor 1254-induced rat liver . Exposure of hamster embryo cells to biochanin A and {3H}B{a}P resulted in a decrease in the total level of {3H}B{a}P bound to DNA compared with the control groups at all time points studied between 24 and 120 h . This decrease was due to reduction in the formation of DNA adducts from both (+)-anti-B{a}P-diolepoxide and (+)-syn-B{a}P-diolepoxide . In a hamster embryo cell-mediated V79 cell mutation assay, biochanin A treatment resulted in a dose-dependent reduction in the number of B{a}P-induced mutants . These results indicate that biochanin A inhibits metabolic activation of B{a}P to mutagenic intermediates and warrants further investigation as a potential chemopreventive agent. J Protozool, 1991 Nov-Dec, 38(6), 88S - 90S Indicators of Pneumocystis carinii viability in short-term cell culture; Armstrong MY et al.; Growth of P . carinii in culture has been difficult to document in the absence of reliable methods for distinguishing live from dead organisms . We studied three markers of cell function in P . carinii during the course of short-term cell culture, and correlated these with the number of P . carinii present in culture supernatants . The markers were glucan synthase activity, esterase activity and cell membrane integrity . The last two were assessed by double staining with fluorescein diacetate and propidium iodide followed by analysis of fluorescence using flow cytometry . The rise in P . carinii number after 5 to 7 days in culture was associated with increased glucan synthase activity . Flow cytometry analysis of day-6 P . carinii cultures confirmed that over 80% of the organisms catalyzed the conversion of fluorescein diacetate to fluorescein and excluded propidium iodide . The demonstration of three indices of metabolic activity in an expanding P . carinii population has confirmed the efficacy of a culture system as a means of sustaining the continued activity, albeit short-lived, of viable P . carinii. Cesk Epidemiol Mikrobiol Imunol, 1991 Nov, 40(4-5), 229 - 35 {Factors affecting the results of culture of Chlamydia trachomatis in a McCoy cell culture}; Stefanik M et al.; The authors confirmed that the sensitivity of cultivation of the laboratory strain Chlamydia trachomatis, serotype E in a McCoy cell culture is increased by centrifuging of the inoculum and addition of cycloximidine to the cultivation medium . Conversely, postcentrifugation sedimentation of the inoculum and addition of glucose to the medium has no effect . To meet the needs of the parasite and the host cells the glucose content in the basic p component of the medium--MEM is sufficient . Exchange of the basic constituent of the medium--MEM for RPMI 1640 did not prove useful in the cultivation of the laboratory strain of chlamydia . In cell cultures where medium with RPMI 1640 was used chlamydia inclusions were not found. J Protozool, 1991 Nov-Dec, 38(6), 64S - 65S Comparison of pulsed field gel electrophoresis karyotypes of Pneumocystis carinii derived from rat lung, cell culture, and ferret lung; Weinberg GA et al.; Pulsed field gel electrophoretic karyotypes of Pneumocystis carinii derived from three sources were compared: immunosuppressed virus-free rats transtracheally inoculated with Pneumocystis-infected rat lung; WI-38 cell/Cytodex bead cell cultures inoculated with the same material; and immunosuppressed ferrets which reactivated latent Pneumocystis pneumonia . Karyotypes of DNA from Pneumocystis trophozoites or cysts from rat lung, and trophozoites from cell culture were identical . In contrast, ferret Pneumocystis DNA karyotypes were distinctly different . Rat Pneumocystis gene probes reacted with Southern- transferred rat Pneumocystis DNA but not with ferret Pneumocystis DNA . We concluded that neither the source nor life stage of rat Pneumocystis carinii influenced genomic karyotype, and that rat and ferret Pneumocystis are genetically diverse. Horm Metab Res, 1991 Nov, 23(11), 539 - 44 Platelet stimulation for prostacyclin production in aortic endothelial cell cultures: alteration in diabetes mellitus; Inoguchi T et al.; We evaluated the effect of platelets on prostacyclin (PGI2) production in bovine aortic endothelial cell cultures . Human platelet extract significantly stimulated PGI2 production by cultured aortic endothelial cells in a time- and dose-dependent manner, suggesting that platelets contain PGI2-stimulatory activity (PSA) . Supernatant fluid separated from platelets activated by collagen also exhibited PSA . The factor(s) causing the PSA of platelets was non-dialysable and heat-stable (56 degrees C for 30 min or 100 degrees C for 3 min), was completely inhibited by trypsin pretreatment, and exhibited an affinity to heparin agarose . Furthermore, gel filtration chromatography showed that the factor(s) responsible for the platelet PSA was eluted at three different peaks with approximate molecular weights of 50,000, 25,000 and 11,000 . The PSA of platelet extract from patients with non-insulin-dependent diabetes mellitus (NIDDM) (n = 10) was compared to that from age-matched control subjects (n = 10) . Platelet extract from patients with NIDDM stimulated cultured aortic endothelial cells to produce greater amounts of PGI2 than did that from control subjects . These data suggest that the increased PSA of platelets isolated from diabetic patients may contribute to the abnormal interaction between platelets and the vascular wall in diabetic patients. Matrix, 1991 Nov, 11(5), 367 - 72 A controlled precursor add-back model of elastogenesis in smooth muscle cell cultures; Faris B et al.; Neonatal rat aortic smooth muscle cell cultures are capable of synthesizing and accumulating relatively large amounts of insoluble elastin in the extracellular matrix . There are two major soluble elastin molecules in these cultures, one of 77 kDa (protropoelastin) and the other of 71 kDa (tropoelastin) . We examined the ability of the cell culture system to insolubilize exogenously added soluble elastin precursor moieties into the elastin matrix . To accomplish this, cultures were allowed to develop an enriched elastic fiber matrix for approximately two weeks in first passage . This accumulated matrix then served as the "substrate" for the exogenously added precursor elastin molecules . Culture-derived radioactive soluble elastin was added to the "substrate" cultures and the presence of radioactivity in the insoluble elastin as well as in the lysine-derived crosslinks unique to elastin (desmosines) was measured . When purified {3H}-valine radiolabeled protropoelastin was used, more than 15% of the radioactivity added was detected in the alkali-resistant insoluble elastin within 24 hours . After an initial 4-hour incubation of the cells with {3H}-lysine-labelled soluble elastin, most of the radioactivity in the insoluble elastin was associated with the lysine and only a negligible amount was detected in the desmosines . However, during a 16-day chase period, the ratio of radioactive desmosines to lysine increased dramatically, suggesting that not only insolubilization, but crosslinking occurs as well . The add-back system described herein should provide a means to probe the molecular properties of protropoelastin and increase our understanding of the mechanisms of elastic fiber formation. Gig Sanit, 1991 Nov, (11), 66 - 7 {The comparative toxicity of inorganic mercury compounds for a cell culture and the whole organism}; Ivanova LA et al.; In experiments with two tissue culture lines it was shown, that cytotoxicity of mercury (1) deriva