Cell cultures

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) derivatives (measured by CL50) is lesser two times, than it was for mercury (2) derivates . No significant differences in cytotoxicity were noted by the cell morphology and monolayer mitosis activity coefficient studies . This demonstrates the equal toxicity and dangerous to both derivates.

J Microsc, 1991 Nov, 164 ( Pt 2), 169 - 74
Microscopy of cell cultures through thin plastic films; Moroz PE; Microscopy of cells growing in vessels containing plastic films as a substrate or as a transparent window is facilitated by a contact cap on the objective or the contact objectives for intravital microscopy . When applied to microscopical examination of living cells through a thin film, the cap considerably improves the conditions of observation with high-power dry objectives and makes it possible to use water- and oil-immersion objectives.

Cell Regul, 1991 Nov, 2(11), 927 - 38
Tenascin variants: differential binding to fibronectin and distinct distribution in cell cultures and tissues; Chiquet-Ehrismann R et al.; In the chicken, three tenascin variants have been characterized that are generated by alternative splicing of 3 of its 11 fibronectin type III repeats . Using monoclonal antibodies that react with common regions versus extra repeats of tenascin, we could distinguish and separate tenascin variants and investigate their interaction with fibronectin using multiple experimental procedures . Interestingly, in all assays used the smallest tenascin variant bound more strongly to fibronectin than the larger ones . These biochemical data were paralleled by the observation that in chick embryo fibroblast cultures only the smallest form of tenascin could be detected in the fibronectin-rich extracellular matrix network laid down by the cells . Furthermore, each tissue present in adult chicken gizzard contained a distinct set of tenascin variants . Those tissues particularly rich in extracellular matrix, such as the tendon, contained the smallest tenascin only . Intermediate-sized tenascin was present in smooth muscle, whereas the largest form was exclusively detectable underneath the epithelial lining of the villi . Thus it appears that cell type-specific forms of tenascin exist that are appropriate for the functional requirements of the respective extracellular matrices.

Acta Virol, 1991 Nov, 35(6), 503 - 10
Analysis of Coxiella burnetii isolates in cell culture and the expression of parasite-specific antigens on the host membrane surface; Roman MJ et al.; Coxiella burnetii isolates may be classified into several groups based on plasmid character . These groups may also be correlated with disease syndrome--chronic or short-term acute . L929 mouse fibroblast cells were exposed, independently, to two members of the three major C . burnetii groups, and their growth/morphological characteristics analysed by light and electron microscopy, including High Voltage Electron Microscopy . The fates of the isolates were followed . Two acute isolates {Nine Mile (RSA 493) and Henzerling (RSA 331), QpH1-type plasmids} and two chronic isolates (S Q217 and L Q216, plasmidless) readily infected L929 cells in static culture . Priscilla (Q177) and F (Q228) isolates (QpRS-type plasmid, and implicated in causing chronic Q fever) took longer to infect cells, and, unlike the members of the other two groups, gradually disappeared when shifted to suspension culture . Cells infected with Q177 and Q228 exhibited a higher degree of vacuolation than cells infected with the other isolates . Parasite-specific antigens on the surfaces of the host cells were analysed by immunofluorescence/flow cytometry . The acute and plasmidless isolates caused the display of significantly more C . burnetii-specific antigen on the host cell membrane than the two QpRS plasmid-containing isolates . This host cell model system clearly reveals biological differences among the C . burnetii groups.

J Nat Prod, 1991 Nov-Dec, 54(6), 1677 - 80
Production of anti-tumor-promoting iridoid glucosides in Genipa americana and its cell cultures; Ueda S et al.; The Genipa americana plant contains geniposide {3} and geniposidic acid {2} in the fruits and geniposidic acid {2} in the leaves . On callus induction, the plant produces tarennoside {1}, geniposidic acid {2}, and gardenoside {4} in high levels . The leaves of Ge . americana plants redifferentiated from the callus tissues produce 2.

Res Virol, 1991 Nov-Dec, 142(6), 443 - 8
Comparison of two methods for rapid detection of cytomegalovirus viraemia in cell culture; Pepin JM et al.; Two methods for rapid CMV viraemia detection in cell culture with immunoperoxidase staining performed 40 h post-inoculation were compared, on 230 blood samples: (1) a 24-well plate centrifugation assay and (2) a 25-cm2-culture flask technique, without centrifugation . The flask technique was significantly more sensitive than the 24-well plate technique . Moreover, the flask assay was more cost-effective and offered other advantages such as easier sterile handling and less damage of the cell monolayer.

J Cell Physiol, 1991 Nov, 149(2), 260 - 8
Time course of IL1 and IL6 synthesis and release in human bronchial epithelial cell cultures exposed to toluene diisocyanate; Mattoli S et al.; We have previously demonstrated that human bronchial epithelial cells release appreciable amounts of interleukin 1 (IL1) and interleukin 6 (IL6) when exposed to toluene diisocyanate (TDI) in vitro . TDI is an inflammatory and asthmogenic stimulus presumed to act at least in part through immunological mechanisms . The epithelial cell-derived IL1 and IL6 can promote T cell activation and proliferation in culture, and if this also happens in vivo they may contribute to the persistence of the inflammatory response of the bronchial mucosa observed in TDI-sensitive asthmatics . In this study, we confirmed the release of biologically active IL1 beta and IL6-like substances from bronchial epithelial cells exposed to isocyanates in vitro, and related the rate and the magnitude of the cytokine secretion with the pattern of IL1 beta and IL6 gene expression and the extent of epithelial cell injury . In the epithelial cell cultures exposed to TDI, there was a parallel, progressive increase in the expression of IL6 mRNA and in the secretion of IL6 protein between 48 hours and 6 days after exposure . By contrast, although increasing amounts of biologically active IL1 beta were detected in the supernatants of TDI-exposed epithelial cells throughout the 6-day period following exposure, augmented levels of IL1 beta mRNA were only evident 6 days after exposure, suggesting that TDI exposure might have initially affected the enzymatic cleavage of the intracellular IL1 beta precursor and the mechanisms which regulate the secretion of mature IL1 beta.

J Endocrinol, 1991 Nov, 131(2), 287 - 93
Studies on the renin-angiotensin system in primary monolayer cell cultures of the rat epididymis; Wong PY et al.; Monolayer cell cultures formed from the rat cauda epididymidis exhibited renin-like and angiotensin-converting enzyme (ACE) activities and contained immunoreactive angiotensin I (AI) and angiotensin II (AII) . Renin-like activity, determined indirectly by radioimmunoassay of generated AI at a near-neutral pH of 6.0, was demonstrated in the cell lysate but was almost undetectable in the serum-free cell culture medium, suggesting that renin expression in epididymal cells is an intracellular phenomenon . In contrast, both AI and AII were detected in the cell lysate and cell culture medium . The level of AI was enhanced by pretreating the cells with the ACE inhibitor captopril (100 nmol/l) . Incubating the cell monolayers with thoroughly washed sperm cells obtained from the intact cauda epididymides of rats increase (P less than 0.01) the AII content of the cell culture medium, with a parallel decline (P less than 0.01) in the AI concentration . However, adrenaline (0.23 mumol/l), which was found to stimulate electrogenic anion secretion by cell monolayers grown on previous supports, was without effect on the renin-like activity or concentration of angiotensins . The ACE activity in cells was confirmed by its strong dependence on chloride ion and its susceptibility to inhibition by captopril (100 nmol/l) . Enzyme activity was significantly (P less than 0.005) higher in the culture medium than in the cell lysate and cell membrane fragments . Angiotensinogen, which is obligatory for an intrinsic renin-angiotensin system, is present in epididymal cells . Presumably, it is synthesized and processed in the cell cytosol by intracellular renin.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurosci Lett, 1991 Oct 28, 132(1), 105 - 8
Prostaglandins sensitize nociceptors in cell culture; Pitchford S et al.; Using the whole cell patch clamp technique, a population of nociceptors were identified, by virtue of their small size and capsaicin responsivity . Response to capsaicin was increased following treatment with the hyperalgesic prostaglandins, PGE2 and PGI2 . Treatment of the cells with the cyclic adenosine monophosphate (cAMP) analogues, 8 bromo cAMP and dibutyryl cAMP, also resulted in an increase in the capsaicin-induced currents . The effects of the cAMP analogues were greater than that produced by prostaglandin treatment . We conclude that PGE2 and PGI2 act directly on nociceptors, with cAMP as second messenger, to sensitize them to noxious stimulation.

Brain Res Dev Brain Res, 1991 Oct 21, 62(2), 281 - 5
Region-specific immunolocalization of atrial natriuretic peptide in mixed fetal rat brain cell cultures; Schipper HM et al.; In adult rodent CNS, atrial natriuretic peptide (ANP) has been localized by immunolabeling and nucleic acid hybridization techniques primarily to hypothalamic neurons and, to a lesser extent, to neurons of the telencephalon and brainstem . In canine brain, ANP immunoreactivity has been reported in neocortical and brainstem astrocytes . Yet, in a recent study using fetal rat tissue, ANP was detected by radioimmunoassay in predominantly neuronal, but not glial, cultures . In the present study ANP was detected by radioimmunoassay on in vitro day 8 in media derived from fetal rat diencephalic and rhombencephalic, but not telencephalic, cultures and in cell homogenates from all 3 regions surveyed . Using indirect immunofluorescence less than one cell per 400x field stained for ANP in the telencephalic cultures and ANP immunopositivity colocalized exclusively to neurons by concomitant anti-neurofilament immunolabeling . In diencephalic monolayers, approximately 1-3 cells per 400x field were ANP-positive; although most of these cells were neurons, small numbers of ANP-positive astrocytes were also detected using anti-glial fibrillary acidic protein (GFAP) immunolabeling . ANP-positive cells were most numerous in rhombencephalic cultures (5-10 cells per 400x field); as in diencephalic cultures, ANP immunoreactivity colocalized to both neurons and astrocytes in this region . In diencephalon and rhombencephalon, less than 1% of all ANP-positive cells were astrocytes and less than 1% of GFAP-positive astrocytes exhibited immunoreactive ANP . In fetal brain, neuronal and astrocytic ANP may play a role in fluid and electrolyte homeostasis . Alternatively, fetal brain cell ANP may subserve entirely different functions (e.g . as trophic factors) as has been suggested for other neuropeptides in the developing nervous system.

Cancer Genet Cytogenet, 1991 Oct 15, 56(2), 203 - 7
In vitro bone marrow cell culture and cytogenetic analysis in a case of myelodysplasia; Maccari A et al.; A case of refractory anemia with sideroblastosis and a number of bone-marrow blasts slightly over the limit which separates the I/II and III FAB-subtypes of myelodysplastic syndromes is described . The leukemic-like type of in vitro growth and the multiple karyotypic changes observed in the bone-marrow cells at presentation were both indicators of the malignant nature of the disorder and underlined the importance of these studies in assessing diagnosis and prognosis in patients with preleukemic disorders . The role that the chromosome aberrations, del(11)(q14) and del(18)(q21), both found in 100% of the bone-marrow metaphases examined, may play in the pathogenesis of the disease is also discussed.

J Histochem Cytochem, 1991 Oct, 39(10), 1385 - 94
Binding and endocytosis of thrombospondin and thrombospondin fragments in endothelial cell cultures analyzed by cuprolinic blue staining, colloidal gold labeling, and silver enhancement techniques; Volker W et al.; We investigated the distribution of thrombospondin-specific binding sites and the uptake of thrombospondin-gold conjugates in cultured porcine endothelial cells by light and electron microscopy . Colloidal gold marker and silver enhancement techniques were applied for cytochemical detection of monomeric thrombospondin and fragments of thrombospondin . Thrombospondin binds to granular and fibrillar structures and to sites of cell-cell contact on the cell surface, as indicated by many proteoglycan-cuprolinic blue precipitates . Cell migration tracks on the culture dish bottom are most heavily stained . Labeling of intact thrombospondin and of proteolytic fragments of thrombospondin with colloidal gold followed by silver intensification enables one to detect its binding and uptake in endothelial cells . Binding to the cell surface and uptake of thrombospondin-gold particles was inhibited by heparin but not by hyaluronic acid or chondroitin sulfate . The heparin binding region at the N-terminal end of the thrombospondin molecule proved to be essential for cell surface binding . Gold-conjugated thrombospondin fragments devoid of the heparin binding region were not internalized . After 60 min incubation at 37 degrees C, thrombospondin-gold particles accumulated in the lysosomal compartment close to the nucleus . In the presence of monensin and ammonium chloride, vesicles in this area are swollen and the concentration of particulate marker is reduced . Binding and uptake of thrombospondin by vascular endothelial cells appears to require linkage of the heparin binding region of the thrombospondin molecule to coated pits and heparan sulfate-rich molecules as receptors . Colloidal gold conjugation of thrombospondin fragments proved to be useful for cytochemical characterization of molecular domains.

Carcinogenesis, 1991 Oct, 12(10), 1791 - 4
The effect of diacylglycerols on fibronectin release and its reversal by retinoic acid in cell culture; Scheidl H et al.; Previous work from our laboratory showed that tumor promoters such as phorbol ester (TPA) stimulated the release of fibronectin (FN) from the surface of several cell types in culture, and that this stimulation was counteracted by retinoic acid . Diacylglycerols (DAGs) are the endogenous ligands of the TPA receptor and can activate and translocate protein kinase C (PKC) in a manner similar to TPA . To show that the release of FN is related to activation of PKC, we tested the action of DAGs on FN release from human lung fibroblasts and its counteraction by retinoic acid . We found that DAGs stimulated the release of FN in a concentration- and time-dependent manner . The stimulation of the release of FN correlated with the translocation-activation of PKC by DAG . Retinoic acid reversed the action of DAG with respect to stimulation of FN release and inhibited this release even in the absence of DAG . These results suggest that the release of FN is in some way related to translocation-activation of PKC.

Am J Physiol, 1991 Oct, 261(4 Pt 2), F571 - 7
High glucose increases diacylglycerol mass and activates protein kinase C in mesangial cell cultures; Ayo SH et al.; We showed previously that glomerular mesangial cells displayed increased fibronectin, laminin, and type IV collagen synthesis and mRNA levels when grown in medium containing 30 mM glucose compared with those cells grown in 10 mM glucose {S . H . Ayo, R . A . Radnik, W . F . Glass II, J . A . Garoni, E . R . Rampt, D . R . Appling, and J . I . Kreisberg . Am . J . Physiol . 260 (Renal Fluid Electrolyte Physiol . 29): F185-F191, 1990} . However, total protein synthesis and actin mRNA were unchanged . In this report, we show that an increase in medium glucose concentration resulted in an increase in diacylglycerol (DAG) mass and transiently increased protein kinase C (PKC) activity as assessed by the translocation of PKC from the soluble to the particulate fraction . Effects of increased glucose on DAG were evident at 30 min and were maintained through 1 wk of growth in medium containing 30 mM glucose . Although total PKC activity (i.e., soluble plus particulate fractions) did not change with high-glucose treatment, the percent activity associated with the particulate fraction (i.e., activated PKC) increased significantly after 60 min in RPMI 1640 medium with 30 mM glucose . The distribution of PKC returned to control values by 24 h . High glucose did not stimulate phosphoinositide hydrolysis, as evidenced by the absence of an increase in the water-soluble inositol phosphates, indicating that DAG was not generated through the action of a phosphoinositide-specific phospholipase C . Cells treated with the cell-permeable DAG analogue 1-oleoyl-2-acetyl glycerol to activate PKC displayed approximately two-fold increases of fibronectin, laminin, and type IV collagen mRNA levels after normalization against actin.(ABSTRACT TRUNCATED AT 250 WORDS)

Endocrinology, 1991 Oct, 129(4), 2110 - 8
Estrogen action on growth hormone in pituitary cell cultures from adult and juvenile macaques; Bethea CL; Basal growth hormone (GH) levels are higher in female than male primates, and estradiol (E) treatment of gonadectomized primates increases serum GH . To determine if the effect of E is mediated at the level of the somatotroph, we verified the effect of E-treatment on serum GH in spayed macaques, and then examined the effect of E on GH secretion in serum-free monkey pituitary cultures . Daily blood samples were obtained from cynomolgus macaques which were spayed upon detection of menstruation and immediately implanted with either empty (n = 5) or E-filled (n = 5) 2-cm silastic capsules . The average level of GH was significantly higher (P less than 0.003) in the E-treated group than in the control group (6.4 +/- 0.7 vs . 3.7 +/- 0.6 ng/ml) . Pituitaries from rhesus monkeys were dispersed and cultured in 48-well plates on extracellular matrix in DME/F12 with insulin, transferrin, selenium . Using pituitary cells from a long-term spayed female, two plates were established with and without phenol red . Each plate was treated with E in a dose-related manner (0.001-10 nM) from days 0-18 (4 wells per dose) . There was a significant dose-related increase in medium prolactin (PRL) in both plates, but E had no effect on GH . Therefore, the effect of E on GH in spayed monkeys cannot be accounted for by a direct action on somatotrophs . Additional phenol red-free pituitary cultures were established from four juvenile males, one adult male, two juvenile females, and two adult females and treated with E in a dose-related fashion for 28 days . Neither the adult male, the adult female, nor the juvenile female cultures exhibited an increase in GH with E treatment . Only the four juvenile male pituitary cultures showed a variable increase in GH with maximal responses ranging from 5 to 40% over control . This data suggested that the juvenile male pituitary contained an E-sensitive GH-secreting cell population which is not present in the other pituitary cultures . PRL and GH double-immunocytochemical staining of pituitary cultures from an adult female and a juvenile male revealed a significant population of rounded and possibly double-labeled cells in the juvenile male culture which were infrequently seen in the adult female culture . Speculatively, this population could represent mammosomatotroph stem cells that corelease GH and PRL upon stimulation.

J Steroid Biochem Mol Biol, 1991 Oct, 39(4A), 501 - 11
Estradiol and progesterone effects on relative luteinizing hormone and follicle stimulating hormone release induced from superfused anterior pituitary cell cultures by defined LHRH pulse regimens; Kellom TA et al.; These studies examined the capacity of estradiol and progesterone to modulate relative luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion from superfused anterior pituitary cells when stimulated with luteinizing hormone releasing hormone (LHRH) pulse regimens of specific amplitude, duration and frequency . There was particular interest in whether such steroid and LHRH treatments induced evidence of divergent LH or FSH secretion . Pituitaries were recovered from adult, 2 week ovariectomized rats and cultured for 48 h with collagen coated Cytodex microcarrier beads . Cultures were preincubated either with or without estradiol (1 or 10 nM) for 48 h and were subsequently incubated for 3,6 or 12 h with 100 nM progesterone . All groups were then pulsed with 1 of 3 LHRH regimens; regimen 1 delivered 8 ng in a single 100 microliters bolus once/h; regimen 2 divided the 8 ng dose of regimen 1 into 3 equal doses administered at 4 min intervals thereby maintaining the 8 ng mass of regimen 1 while extending the duration of exposure; regimen 3 was the same as regimen 2 except that the 3 equal doses were administered at a pulse frequency of 1 per 2 h rather than 1 per h thereby not only maintaining the duration of exposure as in regimen 2 but also reducing the pulse frequency . 1 nM estrogen alone for 48 h had no effect on LHRH stimulated LH release regardless of regimen; however, FSH was increased when hourly pulses of increased duration were applied (regimen 2) . When estrogen was increased to 10 nM, regardless of regimen, LH was predominantly inhibited while FSH was unaffected . When 1 nM estrogen was followed by progesterone, both LH and FSH were elevated at 6 h progesterone in response to regimen 2; with 10 nM estrogen however, a divergent response was observed in that LH release was elevated at 6 h while FSH was elevated at 3 h in response to regimens 2 and 3 . These results first of all confirm that progesterone in combination with estrogen is capable of exerting both inhibitory and stimulatory effects on gonadotropin secretion; secondly, these studies show that, as a direct pituitary effect, the LHRH regimen and the gonadal steroid milieu are capable of interacting to significantly influence the relative secretion of LH and FSH . The data therefore suggest that the divergent gonadotropin secretion seen in various physiological states in vivo is due likely in part to a combination of estrogen and progesterone priming in combination with the hypothalamic LHRH secretory pattern.

J Neurosci Res, 1991 Oct, 30(2), 372 - 81
EGF enhances the survival of dopamine neurons in rat embryonic mesencephalon primary cell culture; Casper D et al.; Epidermal growth factor (EGF) immunoreactive material has been demonstrated to be present in the basal ganglia . In this study, we investigated the effect of EGF on cells cultured from 16-day embryonic rat mesencephalon, which included dopamine neurons that project to the striatum in vivo . EGF receptors were detected in untreated cultures by {125I}-EGF binding . Treatment of the cultures with EGF resulted in up to 50-fold increases in neuronal high-affinity dopamine uptake . Scatchard analysis of uptake kinetics and counting of tyrosine hydroxylase-immunoreactive cells suggest that the effect of EGF on uptake is due to increased survival and maturation of dopaminergic neurons . By contrast, the high-affinity uptake for serotonin was increased only threefold over its controls . There was no significant effect on high-affinity gamma-aminobutyric acid (GABA) uptake . These results suggest that EGF is acting as a neurotrophic agent preferential for dopaminergic neurons in E16 mesencephalic cultures . Immunocytochemistry for glial fibrillary acidic protein demonstrated an increase in astroglia with EGF treatment . Fluorodeoxyuridine, an agent that is toxic to proliferating cells was able to eliminate the effect of EGF on dopamine uptake, suggesting that EGF may be increasing dopaminergic cell survival largely through a population of dividing cells.

Curr Opin Neurobiol, 1991 Oct, 1(3), 360 - 3
The blood-brain barrier in and out of cell culture; Rubin LL; During the past year, the blood-brain barrier has received much attention from neurobiologists and neurologists and real progress has been made in establishing cell-culture model systems that should make future studies more simple . Confusion about the development of the blood-brain barrier, and the exact role of astrocytes in this process persist, but these issues should soon be clarified.

Epilepsy Res, 1991 Oct, 10(1), 24 - 32
Seizure-like activity in cell culture; Furshpan EJ; I will describe experiments with neurons in long-term culture that display seizure-like electrical activity . The neurons are dissociated from the hippocampal formation of newborn rats and then chronically exposed to agents that block synaptic transmission, especially glutamatergic transmission . Seizure-like behavior of the neurons develops as the cultures mature and is revealed when the blocking agents are withdrawn . This spontaneous electrical behavior of the culture has many of the characteristics of seizure activity in intact cortex . It can be very intense and can lead to the death of many neurons . This system allows the familiar experimental advantages of dissociated-cell culture to be applied to the study of seizure-like activities . The experiments to be described were all done with mass cultures containing hundreds or thousands of neurons . However, many of the seizure-like events observed in mass cultures can also be seen in microcultures containing only a few neurons.

NMR Biomed, 1991 Oct, 4(5), 246 - 53
Characterization of a microcarrier cell culture system for 23Na MR spectroscopy studies; Shedd SF et al.; A MR spectroscopy method is described for the simultaneous discrimination and observation of sodium from the three compartments created by an intact cell monolayer . Results are reported for Madin Darby Canine Kidney (MDCK) cells, an epithelial-like continuous cell line, cultured on Cytodex 1 microcarrier beads and perfused with medium containing 6 mM dysprosium (III) tripolyphosphate {Dy(TPP)2(7-)} as shift reagent . The sodium spectrum shows three resonances which are assigned to the shifted intrabead (basolateral) and extrabead (apical) pools and the unshifted intracellular pool . Ouabain inhibition of the Na(+)-K(+)-ATPase cellular pump mechanism was used to demonstrate the sensitivity of the method for monitoring intracellular sodium . The supported MDCK cells in this system remained viable after exposure for 5 h to medium containing Dy(TPP)2(7-) at a concentration of 6 mM, as determined by trypan blue dye exclusion and by comparison of the log growth rate and ability to form domes in subsequent generations of exposed cells vs unexposed controls.

Mech Ageing Dev, 1991 Oct, 60(2), 171 - 87
Comparison of rates of neuronal development and survival in human and rat cerebral cortical cell cultures; Mattson MP et al.; A problem that has not been addressed directly at the cellular and molecular levels concerns the basic mechanisms governing rates of development and aging in the nervous system . We have begun to examine this problem by employing parallel cell cultures of cerebral cortical neurons taken from rat and man, two species with markedly different developmental periods and life spans . Cortical cultures were established from rat and human fetuses and were maintained under identical conditions . Long-term neuronal survival in human cortical cultures was strikingly greater than in rat cortical cultures, and axonal outgrowth was significantly slower in the human neurons . These differences were consistently observed in a variety of culture media . Exchanges of culture medium between cultures of the two species did not alter the differences in neuronal survival and axon outgrowth suggesting that the differences between human and rat neurons were probably not due to factors released into the culture medium . Furthermore, the differences were not related to developmental stage at the time of culture initiation since similar results were obtained in cultures established from fetuses of different gestational ages . These initial data indicate that differences in the developmental time courses and life spans of human and rat cerebral cortex are reflected at the level of the individual neuron, and provide a system in which to explore the mechanisms responsible for these differences.

Clin Otolaryngol, 1991 Oct, 16(5), 493 - 7
The nature of the epithelium in acquired cholesteatoma . Part 2 . Cell culture; Lee RJ et al.; The exact nature and role of the epithelial layer in cholesteatoma remains undetermined . The aim of this study was to investigate cholesteatoma epithelium and normal aural epithelia in common cell culture conditions . Samples of cholesteatoma, external meatal epidermis and middle ear mucosa were obtained, successfully grown in cell culture, and subcultured . No significant morphological differences were found between cholesteatoma and aural epidermis . The only differences noted were delayed onset of colony formation, and the need to subculture prior to the cultures becoming confluent in the cholesteatoma cultures . Further research is required to account for these differences in growth patterns.

J Virol, 1991 Oct, 65(10), 5232 - 6
Zidovudine-resistant human immunodeficiency virus selected by passage in cell culture; Larder BA et al.; Variants of human immunodeficiency virus (HIV) with reduced sensitivity to zidovudine (3'-azido-3'-deoxythymidine) have been selected by passage of virus in cell culture in the presence of drug . Wild-type, sensitive virus became partially resistant to zidovudine by passage 12 (50% inhibitory dose values measured in HeLa CD4+ cells increased from 0.014 to 0.2 microM), and genetic analysis using the polymerase chain reaction revealed that mutations in the reverse transcriptase coding region identical to those seen in clinical isolates from treated individuals had occurred . The order of appearance of these resistance mutations in passaged virus was also similar to that in clinical isolates . The partially resistant strain, HIVRTMC/F, became highly zidovudine resistant by passage 12 (50% inhibitory dose values increased from 0.4 to 2.5 microM during passages 7 to 11) . Nucleotide sequence analysis of the reverse transcriptase from this variant revealed a novel amino acid substitution (Lys----Glu) at codon 219 . A different substitution at this codon (Lys----Gln) had been seen previously in clinical isolates . When this mutation was created in HIVRTMC/F by site-directed mutagenesis, the resulting partially resistant virus became highly resistant, thus confirming the significance of this change . In view of the possibility that this mutation might occur in HIV isolates during treatment of patients, we adapted our selective polymerase chain reaction procedure to enable screening for this change in clinical samples . The virus passage procedure described here may be useful for gaining further insight into the mutational events occurring during the development of resistance to zidovudine and other HIV inhibitors.

J Neurochem, 1991 Oct, 57(4), 1185 - 90
Activation of high-affinity uptake of glutamate by phorbol esters in primary glial cell cultures; Casado M et al.; The effects of 12-O-tetradecanoylphorbol 13-acetate (TPA), a potent activator of protein kinase C, on high-affinity Na(+)-dependent glutamate transport were investigated in primary cultures of neurons and glial cells from rat brain cortex . Incubation of glial cells with TPA led to concentration- and time-dependent increases in the glutamate transport that could be completely suppressed by the addition of the protein kinase C (PKC) inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine . The TPA effects could be mimicked by oleoylacetylglycerol and by the diacylglycerol kinase inhibitor R59022 . The effects of TPA were potentiated by the Ca2+ ionophore A23187 . Under the chosen experimental conditions TPA had no effect on glutamate transport in neurons . We conclude that PKC activates the sodium-dependent high-affinity glutamate transport in glial cells and that it has dissimilar effects on neurons and glial cells.

Brain Res Mol Brain Res, 1991 Oct, 11(3-4), 355 - 8
Production of thrombin and antithrombin III by brain and astroglial cell cultures; Deschepper CF et al.; There is increasing evidence that some proteases and protease inhibitors are produced within the central nervous system . It has been proposed that the balance between these two classes of proteins may be an important modulator of brain cell growth and differentiation . Here we report that antithrombin III (ATIII) is produced in brain and primary astroglial cultures . In addition, we show that human astroglial cultures contain prothrombin mRNA, and secrete a thrombin-like protein that makes complexes with antithrombin III.

J Virol, 1991 Oct, 65(10), 5184 - 9
A 15-kilobase-pair region of the human cytomegalovirus genome which includes US1 through US13 is dispensable for growth in cell culture; Kollert-Jons A et al.; The genome of a temperature-sensitive, DNA-negative mutant of human cytomegalovirus was cloned in cosmids and analyzed by restriction endonuclease mapping and Southern blotting . The data presented show that in the mutant genome, nearly half of the short segment was deleted (14.3 to 15.1 kb; map position, 0.83 to 0.9), including the genes for a potential immediate early protein (US3) and a structural glycoprotein of 47 to 52 kDa (US6 through US11) . The deleted DNA region was replaced by a 20.8- to 21.6-kb fragment that represented an inverted repetition of the retained portion of the short segment (map position, 0.92 to 1.0), suggesting that US20 through US36 were duplicated in the mutant . Northern (RNA) blots with appropriate probes of total cell RNA extracted from mutant-infected cells confirmed the absence of mRNAs originating from US3 or from US8 through US11 . It is concluded that the deleted genes are dispensable for human cytomegalovirus replication in cell culture.

Biochem Biophys Res Commun, 1991 Sep 16, 179(2), 939 - 44
The effect of beta-aminopropionitrile on elastin gene expression in smooth muscle cell cultures; Jackson LE et al.; When beta-aminopropionitrile (BAPN) is added to neonatal rat aortic smooth muscle cell cultures there is a decrease in insoluble elastin accumulation with a concomitant increase in tropoelastin and tropoelastin fragments in the culture medium . The experiments described here examine the biological significance of this fragmentation . BAPN, as well as purified tropoelastin fragments isolated from spent medium of cells grown in the presence of BAPN, were added to cultures . A decrease in elastin mRNA was observed in cultures grown in the presence of BAPN and also in those cultures to which the purified tropoelastin moieties were added . These studies indicate that the inhibition of lysyl oxidase by BAPN prevents elastin crosslinking which results in an increase in tropoelastin moieties, thus leading to a down regulation of the steady state levels of elastin mRNA.

Eur J Biochem, 1991 Sep 15, 200(3), 751 - 7
Pterocarpan phytoalexin biosynthesis in elicitor-challenged chickpea (Cicer arietinum L.) cell cultures . Purification, characterization and cDNA cloning of NADPH:isoflavone oxidoreductase; Tiemann K et al.; NADPH:isoflavone oxidoreductase (IFR) is the first soluble enzyme of the pterocarpan-specific part of phytoalexin biosynthesis in chickpea (Cicer arietinum L.) . The enzyme was purified to apparent homogeneity by a five-step procedure from chickpea cell cultures treated with yeast extract as elicitor . Analysis by gel filtration and SDS/PAGE showed that the enzyme consists of a single polypeptide with a molecular mass of 36 kDa . Km values for the substrates 2'-hydroxyformononetin, 2'-hydroxypseudobaptigenin and NADPH were 6, 6 and 20 microM, respectively . The IFR showed pronounced specificity for the substitution pattern of isoflavones . We found a 2'-hydroxy group and a 4',5'-methylenedioxy or 4'-methoxy function to be essential for acceptance as substrate . The isoelectric point of the protein was determined as 6.3 by IEF and there is no evidence for the existence of isoenzymes . Partial amino acid sequences of IFR were determined from internal peptides obtained by tryptic digestion of the protein and corresponding oligonucleotides were synthesized . A lambda gt10 cDNA library was constructed using poly(A)-rich RNA isolated from chickpea cell cultures treated with Ascochyta rabiei elicitor . 150 positive clones were obtained by screening 2 x 10(5) clones with an IFR-specific oligonucleotide . The identity of sequenced clones was confirmed by comparison of the deduced amino acid sequence with the internal peptide sequences of purified IFR . The sequence of a 1183-bp clone contained a continuous open reading frame of 954 bases encoding a polypeptide of 318 amino acids with a calculated molecular mass of 35.4 kDa, indicating that a full-length cDNA coding for IFR was isolated.

Proc Natl Acad Sci U S A, 1991 Sep 15, 88(18), 7998 - 8002
Replication of human immunodeficiency virus type 1 in primary dendritic cell cultures; Langhoff E et al.; The ability of the human immunodeficiency virus type 1 (HIV-1) to replicate in primary blood dendritic cells was investigated . Dendritic cells compose less than 1% of the circulating leukocytes and are nondividing cells . Highly purified preparations of dendritic cells were obtained using recent advances in cell fractionation . The results of these experiments show that dendritic cells, in contrast to monocytes and T cells, support the active replication of all strains of HIV-1 tested, including T-cell tropic and monocyte/macrophage tropic isolates . The dendritic cell cultures supported much more virus production than did cultures of primary unseparated T cells, CD4+ T cells, and adherent as well as nonadherent monocytes . Replication of HIV-1 in dendritic cells produces no noticeable cytopathic effect nor does it decrease total cell number . The ability of the nonreplicating dendritic cells to support high levels of replication of HIV-1 suggests that this antigen-presenting cell population, which is also capable of supporting clonal T-cell growth, may play a central role in HIV pathogenesis, serving as a source of continued infection of CD4+ T cells and as a reservoir of virus infection.

J Immunol, 1991 Sep 15, 147(6), 1810 - 5
Granulocyte-macrophage colony-stimulating factor is a major macrophage fusion factor present in conditioned medium of concanavalin A-stimulated spleen cell cultures; Abe E et al.; In 1983, we reported that the conditioned medium (CM) of spleen cell cultures treated with Con A greatly induced fusion of mouse alveolar macrophages within 2 to 3 days at a very high rate of more than 80% (Proc . Natl . Acad . Sci . USA 80:5583, 1983) . In the course of examining macrophage fusion factors (MFF) present in Con A-CM, we found that IL-4 induced fusion of alveolar macrophages with a time course similar to that induced by Con A-CM . However, the maximal fusion rate induced by IL-4 (4 ng/ml) was about 35% . Furthermore, the fusion induced by Con A-CM was blocked only partially by adding IL-4 antibody, indicating that there are unknown MFF other than in Con A-CM . Of several other cytokines produced by Con A-stimulated spleen cells, IL-6 (20 ng/ml), IFN-gamma (45 ng/ml) and granulocyte-macrophage (GM)-CSF (10 ng/ml) greatly potentiated the fusion induced by 4 ng/ml of IL-4 . The assay of these cytokines in Con A-CM proved that it contained 0.44 +/- 0.04 ng/ml of IL-4, 1.0 +/- 0.24 ng/ml of IL-6, 9.1 +/- 0.07 ng/ml of IFN-gamma, and 11.6 +/- 1.66 ng/ml of GM-CSF . When the potentiating effects of IL-6, IFN-gamma and GM-CSF on macrophage fusion were examined in the presence of 0.4 ng/ml of IL-4, only GM-CSF increased the fusion rate to the maximal level induced by Con A-CM at its physiologic concentration (10 ng/ml) . The macrophage fusion induced by Con A-CM was greatly suppressed by adding antibody against GM-CSF . GM-CSF had a biphasic effect on growth and fusion, depending on its dose levels used: 0.01 to 0.1 ng/ml increased proliferation without inducing fusion and 10 ng/ml preferentially induced fusion . There was a negative relationship between macrophage growth and fusion . IL-4 was a potent inhibitor of proliferation of macrophages induced by GM-CSF . These results clearly indicate that GM-CSF is a major MFF present in Con A-CM.

Proc Natl Acad Sci U S A, 1991 Sep 15, 88(18), 8222 - 6
Ferritin gene transcription is regulated by iron in soybean cell cultures; Lescure AM et al.; Iron-regulated ferritin synthesis in animals is dominated by translational control of stored mRNA; iron-induced transcription of ferritin genes, when it occurs, changes the subunit composition of ferritin mRNA and protein and is coupled to translational control . Ferritins in plants and animals have evolved from a common progenitor, based on the similarity of protein sequence; however, sequence divergence occurs in the C termini; structure prediction suggests that plant ferritin has the E-helix, which, in horse ferritin, forms a large channel at the tetrameric interface . In contemporary plants, a transit peptide is encoded by ferritin mRNA to target the protein to plastids . Iron-regulated synthesis of ferritin in plants and animals appears to be very different since the 50- to 60-fold increases of ferritin protein, previously observed to be induced by iron in cultured soybean cells, is accompanied by an equivalent accumulation of hybridizable ferritin mRNA and by increased transcription of ferritin genes . Ferritin mRNA from iron-induced cells and the constitutive ferritin mRNA from soybean hypocotyls are identical . The iron-induced protein is translocated normally to plastids . Differences in animal ferritin structure coincide with the various iron storage functions (reserve for iron proteins and detoxification) . In contrast, the constancy of structure of soybean ferritin, iron-induced and constitutive, coupled with the potential for vacuolar storage of excess iron in plants suggest that rapid synthesis of ferritin from a stored ferritin mRNA may not be needed in plants for detoxification of iron.

Presse Med, 1991 Sep 14, 20(28), 1327 - 9
{Trisomy 20 mosaicism in amniotic cell culture . Genetic counselling during the prenatal diagnosis}; Rabineau D; The author presents a new case of trisomy 20 mosaicism in an amniotic fluid culture and emphasizes the special aspects of this chromosomal abnormality . The prenatal diagnosis of "pseudo-mosaicism" is easier when in situ culture techniques are used . Controls of the results on a new sample of amniotic fluid and/or on foetal blood are useful to reinforce the diagnosis and to allow pregnancy to be continued.

Biochim Biophys Acta, 1991 Sep 3, 1094(2), 153 - 60
Effect of rat plasma high density lipoprotein with or without apolipoprotein E on the cholesterol uptake and on the induction of the corticosteroid biosynthetic pathway in newborn rat adrenocortical cell cultures; Hammami M et al.; High density lipoprotein (HDL) has been shown to induce the cellular accumulation of cholesterol esters and the biosynthetis of 21-hydroxysteroids (corticosteroids) newborn rat adrenocortical cells cultivated in serum-free medium . In order to identify the component(s) of HDL responsible for these effects, we investigated the ability of rat HDL subfractions and HDL with or without apolipoprotein E to deliver cholesterol to cells and to stimulate the steroid biosynthetic pathways in adrenal cultured cells . The total cholesterol uptake from HDL2 was greater than that observed with HDL rich in apolipoprotein E (HDL1 and HDLc) . Furthermore, the increase of the ratio between 21-hydroxysteroids and reductive metabolites of progesterone was higher with HDL2 than with HDL1 or HDLc . The results of competitive studies between LDL and HDL subfractions indicate that adrenal cells take up cholesterol from HDL2 and LDL by separate mechanisms but that LDL and HDL containing apolipoprotein E share the same uptake processes . In experiments with various concentrations of HDLc or HDL without apolipoprotein E, the adrenal cells displayed a higher affinity for rat HDLc than for rat HDL without apolipoprotein E . However, HDL without apolipoprotein E produced a higher enhancement of the cholesterol cell content and was 3-fold more effective in stimulating 21-hydroxylated steroid production than rat HDLc . Although these findings suggest a participation of HDL with apolipoprotein E in the HDL interaction with rat adrenal cells, the predominant effect on these cells is devoluted to HDL containing mainly apolipoprotein A.

Antimicrob Agents Chemother, 1991 Sep, 35(9), 1914 - 6
Activity of pentamidine and pentamidine analogs against Toxoplasma gondii in cell cultures; Lindsay DS et al.; The capabilities of pentamidine and nine pentamidine analogs to inhibit the development of Toxoplasma gondii were examined in vitro . Treatment of infected cultures with pentamidine and five of its analogs caused a significant (P less than 0.05) reduction in the numbers of tachyzoites produced . Analogs of pentamidine may be useful agents in the treatment of toxoplasmosis.

JPEN J Parenter Enteral Nutr, 1991 Sep-Oct, 15(5), 540 - 5
Effect of methionine-deprived nutrition on cell growth and cell kinetics in cell cultures and experimental tumors; Usami M et al.; The effect of methionine-deprived nutrition on cell growth and cell kinetics was investigated in cell cultures and in tumor-bearing rats using the total parenteral nutrition (TPN) technique . A simultaneous flow cytometric measurement of the cellular DNA content and the amount of 5-bromodeoxyuridine incorporated into cellular DNA was performed for analysis of cell kinetics . The methionine-free medium demonstrated a cytocidal effect on the growth of SLC cells after 6 hours of culturing . It decreased viability from 80% in the control medium to 23%, and it decreased the S phase and increased the G0/G1 phase of the cell cycles . The methionine-deprived medium showed a concentration-dependent inhibition in cellular growth . Methionine-deprived TPN was seen to inhibit AH109A and SLC tumor growth compared with conventional TPN and decreased the S phase and increased the G0/G1 phase of cell cycles . These results confirm that methionine deprivation blocks cells from processing into the G1 phase and recycling, and that it is effective in inhibiting tumor growth in cultures and in vivo.

Exp Eye Res, 1991 Sep, 53(3), 375 - 87
Cell cultures of human ciliary muscle: growth, ultrastructural and immunocytochemical characteristics; Tamm E et al.; Primary ciliary muscle cell cultures derived from human donors (16-91 years) were established and characterized by comparing them with ciliary muscle in tissue sections using immunocytochemical and ultrastructural methods . Monoclonal antibodies against desmin, vimentin, alpha-actinin, smooth muscle (sm) specific alpha-actin and von Willebrand factor were used . In tissue sections of the ciliary body, ciliary muscle cells, vascular muscle cells, pericytes, endothelial cells and fibroblasts stain for vimentin . Both types of muscle cells and the pericytes stain for alpha-sm-actin, but only ciliary muscle cells stain for desmin . For tissue cultures, explants of the meridional and partly the reticular portion of the ciliary muscle were dissected and grown directly or after digestion of the explant with collagenase . Ten primary cell cultures with a typical hill-and-valley growth pattern similar to smooth muscle cells and two with a growth pattern similar to fibroblasts were established . All cultures could be subcultured up to the fifth passage . In fibroblast-like cultures 5-10% of the cells stained for alpha-sm-actin . Staining for desmin was not observed . In smooth muscle-like cultures, all cells stained positive for alpha-sm-actin . Desmin staining was not seen in growing non-confluent smooth muscle-like cultures . In confluent cultures, about 10% of the cells stained positive for desmin, preferentially in areas where the cells had formed hills . No culture stained for von Willebrand factor . Staining for alpha-actinin in smooth muscle-like cultures showed that the dense bands of the myofilaments were arranged in register, similar to the typical ciliary muscle cell morphology seen in tissue sections . Ultrastructurally, the smooth muscle-like cultures showed the typical morphology of cultured smooth muscle cells . We conclude that the smooth muscle-like cultures consist of ciliary muscle cells.

Thymus, 1991 Sep, 18(2), 67 - 77
Age-dependent changes in the proportion of macrophages in primary thymus non-lymphoid cell cultures; Jones KH et al.; Primary murine thymus cell cultures consist of thymus epithelial cells, macrophages and interdigitating dendritic cells . Results of the present study indicated that the proportion of macrophage-like cells in cultures was directly related to the age of the thymus donors through 7 weeks of age . We standardized procedures to compare primary thymic cultures derived from mice, 1 day to 115 days of age . Cultures were grown 6 to 8 days and then tested for nonspecific esterase, acid phosphatase, phagocytosis of latex beads, Fc receptors, and MAC-1 staining . Less than 10% macrophage-like cells were identified in cultures from mice 1 to 3 days-old, but the proportion increased to 50% macrophage-like cells in cultures from 7 week-old mice . Thereafter the proportion plateaued although there was considerable variation among older mice . Further studies showed that the proportions of macrophage-like cells was not affected by the number of cells initially seeded into culture, nor by the amount of serum added to the culture medium . These data suggest that macrophage/interdigitating cell precursors immigrate into the thymus for 4 to 6 weeks after birth and support the hypothesis that these precursors may be influenced to differentiate into thymus macrophages or interdigitating cells by the thymic microenvironment.

Oncogene, 1991 Sep, 6(9), 1609 - 15
Activated Val-12 ras p21 in cell culture fluids and mouse plasma; Hamer PJ et al.; Activation of ras oncogenes has been associated with a variety of cancers as well as their precursor lesions . Ras proteins activated by substitutions at amino acid positions 12, 13 or 61 have not been identified in normal tissues and therefore their detection may have clinical value . In this study our objective was to determine whether activated ras proteins could be released into the extracellular environment . To test this hypothesis, we used ras-transformed NIH3T3 cells that express an activated p21 containing valine (Val-12 p21) at position 12 instead of the normal glycine (Gly-12 p21) and a monoclonal antibody (mAb) designated DWP that is specific for the activated Val-12 ras proteins . Culture fluids collected from NIH3T3 cells transformed by the activated Val-12 p21 were shown, using mAb DWP in a sandwich ELISA format, to contain the activated Val-12 p21 . In contrast, culture fluids from non-Val-12-containing cells were unreactive with mAb DWP . PSV-LM-EJ cells which overexpress the activated Val-12 p21 were injected subcutaneously (SQ) into nude mice to produce tumors . At the time of gross tumor appearance (14-21 days after tumor cell inoculation), plasma was collected from the PSV-LM-EJ tumor-bearing mice as well as from a series of control mice . Employing mAb DWP as a detection reagent in the sandwich ELISA format, we were able to detect the Val-12 p21 in the plasma of the PSV-LM-EJ tumor-bearing mice . Activated Val-12 p21 was not present in the plasma of non-tumor-bearing mice, or in the plasma of mice bearing SQ tumors composed of non-Val-12 p21 ras-transformed cells . This report is the first description of an activated ras protein (Val-12 p21) in the plasma of tumor-bearing mice and demonstrates that the results of the Val-12 p21-specific ELISA could be validated with Western blot format.

Endocrinology, 1991 Sep, 129(3), 1489 - 96
Regulation of transepithelial electrical resistance in two-compartment Sertoli cell cultures: in vitro model of the blood-testis barrier; Janecki A et al.; The effects of FSH, testosterone (T), and incubation temperature on the development of inter-Sertoli cell (Sc) tight junctions were investigated in vitro by using repetitive measurements of transepithelial electrical resistance (TER) . Control cultures developed stable TER of 100-145 omega cm2 during the initial 3-4 days of incubation at either 33 or 36.5 C, suggesting the formation of simple but continuous tight junctions . The presence of FSH (200 ng/ml) at 33 C delayed the onset of TER development by 3-5 days . The addition of FSH at the time of stable TER (day 5) resulted in a rapid (24 h) decrease of TER to 35-40 omega cm2, which returned to the control level during the subsequent 5-7 days . T alone (0.001-10 microM) caused an early and dose-dependent increase in TER to 165-240 omega cm2 . In mono-layers incubated at 36.5 C, the continuous presence of FSH resulted in a dose-dependent increase in TER, which stabilized at 260-380 omega cm2 after 4-6 days . At this temperature, the addition of FSH on day 5 caused a rapid drop of TER similar to that observed at 33 C . This drop could not be prevented by antiproteases (aprotinin, epsilon-aminocaproic acid, or 10% fetal bovine serum) and was followed by an increase in TER up to 260-300-omega cm2 . The Sc monolayers developed FSH-induced TER of 230-280 omega cm2 at 33 C, but only after several days of culture at 36.5 C . The effects of T at 36.5 and 33 C were similar, but the maximal TER values were significantly higher (290-380 omega cm2) at 36.5 C . The concomitant presence of T and FSH at 36.5 C resulted in the highest TER levels (580-1200 omega cm2) within 4-6 days, suggesting the synergistic effect of the two hormones on TER development . Dihydrotestosterone was more effective than T when used together with FSH, whereas estradiol had no effect . The different patterns of TER did not result from differences in Sc number or metabolic activity and probably reflected developmental and/or maturational changes in the inter-Sc tight junctions . It is concluded that FSH, T, and temperature play a role in the development of high TER by Sc monolayers (formation of tight junctions) in vitro . FSH and T appear to regulate TER via separate pathways and to cooperate by a yet unknown synergistic mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

Am J Physiol, 1991 Sep, 261(3 Pt 1), G407 - 16
Electric properties of rat liver cell cultures on gas-permeable membranes; Wehner F et al.; In rat hepatocytes grown on gas-permeable membranes (Petzinger et al . In Vitro Cell . Dev . Biol . 24: 491-499, 1988), cellular and canalicular potentials as well as input resistances were measured using two-channel microelectrodes . In HCO3(-)-containing solutions, we found -30.9 +/- 0.4 (SE) (n = 141) and -13.9 +/- 1.4 mV (n = 22) for cell and canalicular membrane potentials, respectively . There was no dependence of these parameters on the age of the primary culture . Canalicular input resistance, however, increased from 13.3 +/- 2.0 M omega (n = 4) at day 1 after seeding to 36.1 +/- 5.0 M omega (n = 9) at day 2 and stabilized thereafter, while cell input resistance continuously decreased from 37.0 +/- 3.3 M omega at 1 h (n = 6) to 5.2 +/- 2.1 M omega (n = 27) at 3 days after preparation . In ion substitution experiments there were no changes in the transference numbers for K+, Na+, or Cl- that could account for this effect . Cable analysis, however, revealed that the decrease in input resistance reflects a time-dependent increase in electrical coupling between cells . We conclude that rat liver cells on gas-permeable membranes are highly suited for the quantitative analysis of cell-to-cell interaction . In addition, cells and canaliculi are readily accessible with two-channel microelectrodes, making this preparation a promising tool for electrophysiological analysis of hepatocellular transport mechanisms.

Cancer Res, 1991 Sep 1, 51(17), 4588 - 93
Biological efficacy of boronated low-density lipoprotein for boron neutron capture therapy as measured in cell culture; Laster BH et al.; Low-density lipoproteins (LDLs) are known to be internalized by the cell through receptor-mediated mechanisms . There is evidence that LDLs may be taken up avidly by tumor cells to provide cholesterol for the synthesis of cell membranes . Thus, the possibility exists that LDLs may provide an ideal vehicle for the transport of boron to tumor cells for boron neutron capture therapy . A boronated analogue of LDL has recently been synthesized for possible application in boron neutron capture therapy . The analogue was tested in cell culture for uptake and biological efficacy in the thermal neutron beam at the Brookhaven Medical Research Reactor . It was found that boron concentrations 10 times higher than that required in tumors for boron neutron capture therapy were easily obtained and that the amount of uptake was consistent with a receptor-mediated binding mechanism . The measured intracellular concentration of approximately 240 micrograms 10B/g cells is significantly higher than that obtained with any other boron compound previously evaluated for possible clinical application.

Vopr Virusol, 1991 Sep-Oct, 36(5), 356 - 61
{The strain features of the HIV-1 circulating in the USSR based on data from a study of its properties in cell cultures}; Marennikova SS et al.; Both variants of HIV-1 reported in the literature: slow/low and rapid/high types, were detected among the strains isolated from the subjects examined in 4 foci of HIV-1 infection in the south of the RSFSR and Byelorussia . All the 17 strains isolated in the southern RSFSR foci belonged to the slow/low type and had a low and unstable replication potential in donor peripheral blood mononuclear cells and in MT-4 cell line . All of them were isolated from subjects with asymptomatic infection and from children with initial clinical manifestations of the disease . Only one strain isolated in Byelorussia belonged to the rapid/high type . Its replicative activity was very similar to that of the classical HIV-1--HTLV-IIIB strain . Long-term (up to 7 months) propagation of slow/low strains did not result in any increase of their replicative activity . The capacity to form syncytia was found not only in the rapid/high type strains but also in the majority of slow/low strains under study.

Vopr Virusol, 1991 Sep-Oct, 36(5), 384 - 6
{The effect of different factors on the reproduction of influenza viruses and reassortants in cell cultures}; Podcherniaeva RIa et al.; The influence of the maintenance medium, polyethylene glycol (PEG), DEAE-dextran, and low temperature on reproduction of influenza A, B, and C viruses and their reassortants in diploid and continuous cell cultures was determined . Lowering of pH in the maintenance medium to 6.5 was found to decrease reproduction of influenza A (H1N1) and A (H3N2) viruses and increase that of influenza B viruses . Treatment of cells with PEG solution increased the yield of influenza B and C but not A viruses . However, influenza A virus strains proved to be capable of producing infectious progeny in nonpermissive cell lines treated with PEG . Addition of DEAE-dextran to the medium exerted no effect on the infectivity of influenza A and B reassortants . Moreover, infection of MDCK cells after a "cold shock" led to an increase in hemagglutinin titres in influenza A reassortants.

J Clin Microbiol, 1991 Sep, 29(9), 2086 - 8
Comparison of the Clearview Chlamydia test, Chlamydiazyme, and cell culture for detection of Chlamydia trachomatis in women with a low prevalence of infection; Skulnick M et al.; Two antigen detection systems, Clearview Chlamydia (Unipath Ltd., Bedford, United Kingdom) and Chlamydiazyme (Abbott Laboratories, North Chicago, Ill.), were compared with culture for the diagnosis of chlamydia infection in women attending gynecological clinics . Chlamydia trachomatis was isolated from 43 (4.5%) of the 965 women tested . In comparison with tissue culture, the Clearview Chlamydia and Chlamydiazyme tests had sensitivities of 79.0 and 74.4%, respectively, and both had a specificity of 99.6% . The results show that the Clearview Chlamydia test is comparable to Chlamydiazyme for the detection of C . trachomatis from endocervical specimens in a population with a low prevalence of infection.

Lipids, 1991 Sep, 26(9), 684 - 8
ApoA-I secretion by rabbit intestinal mucosa cell cultures; Carlson TL et al.; Lipid and apolipoprotein (apo) A-I concentrations in different density fractions of New Zealand White (NZW) and Watanabe (WHHL) rabbit plasma were studied . Aside from the low plasma apoA-I and high density lipoprotein (HDL) cholesterol levels in WHHL rabbits, the distribution of apoA-I was also different between the two rabbits . ApoA-I was concentrated in both the HDL2 and HDL3 fractions of NZW rabbits but was found primarily in the HDL3 fraction of WHHL rabbits . ApoA-I secretion in these two rabbits was further studied in vitro by using intestinal and hepatocyte cell cultures . ApoA-I secretion was highest from cultures of the duodenum and the proximal end of the jejunum; whereas, cell cultures of the distal end of the small intestine secreted very little apoA-I into the medium . Intestinal cell cultures from WHHL rabbits secreted less, but significant amounts of, apoA-I compared to that of NZW rabbits . ApoA-I was most concentrated in the density range of 1.12-1.21 (HDL3) fraction in medium containing 10% fetal calf serum (FCS) . Serum-free medium promoted apoA-I secretion by intestinal cell cultures that was mostly found in the d greater than 1.21 (lipoprotein-deficient) fraction . Hepatocytes isolated from the same rabbits by collagenase perfusion secreted little apoA-I, and it was found only in the d greater than 1.21 fraction . The addition of oleic acid into the culture medium with 10% FCS decreased the secretion of total apoA-I and HDL by intestinal cell cultures and increased the secretion of very low density lipoprotein (VLDL) and intermediate density lipoproteins (IDL).(ABSTRACT TRUNCATED AT 250 WORDS)

Biomaterials, 1991 Sep, 12(7), 690 - 4
Cytocompatibility of two coating materials, amorphous alumina and silicon carbide, using human differentiated cell cultures; Naji A et al.; The cytocompatibility of two coating materials, amorphous alumina and silicon carbide deposited by radio-frequency sputtering, was studied using alveolar bone osteoblasts and gingival fibroblasts from human healthy tissues . Cytocompatibility was assessed at the level of both the basic (attachment, proliferation and cell protein content) and the specific features (intracellular alkaline phosphatase activity and the cytoskeleton) of the cells in direct contact with the coating . Titanium was used as the reference material . The results showed that both silicon carbide and amorphous alumina are cytocompatible for human fibroblasts and osteoblasts, whereas titanium appears the least cytocompatible of all the three substrates . Moreover, the amorphous alumina coating seems slightly bioactive . It seems that these coatings, particularly amorphous alumina, could be used to protect alloys against corrosion, and consequently combine the good mechanical properties of the alloys with the good biocompatibility of the coatings . These coatings seem to perform more suitably than titanium if the strength of the bond between the coating and the underlying alloys is strong enough to give a stable composite material.

Proc Natl Acad Sci U S A, 1991 Sep 1, 88(17), 7481 - 5
Phosphorylation of insulin-like growth factor (IGF)-binding protein 1 in cell culture and in vivo: effects on affinity for IGF-I; Jones JI et al.; The insulin-like growth factors (IGF-I and IGF-II) are present in extracellular fluids bound to specific IGF-binding proteins (IGFBPs) . We and others have reported varying biologic activity of different preparations of IGFBP-1 that appeared to have identical amino acid sequences and molecular sizes . This observation prompted us to determine whether IGFBP-1 undergoes posttranslational modifications . Immunoprecipitation was used to show that Chinese hamster ovary cells (transfected with a human IGFBP-1 cDNA construct) and human hepatoma (HepG2) cells secrete 32P-labeled IGFBP-1 following incubation with {32P}orthophosphate . Phospho amino acid analysis of 32P-labeled IGFBP-1 revealed only phosphoserine residues . A method was developed that could separate nonphosphorylated IGFBP-1 from four or five phosphorylated isoforms . Using this technique we demonstrated that human amniotic fluid and human fetal serum contain a large proportion of nonphosphorylated IGFBP-1, as well as phosphorylated forms . In contrast, HepG2 cells and human decidual cells secrete predominantly the phosphorylated isoforms . These observations suggest that IGFBP-1 is secreted as a phosphoprotein and is subsequently dephosphorylated in vivo . Binding studies showed that the phosphorylated IGFBP-1 secreted by HepG2 cells has a 6-fold higher affinity for IGF-I than it does after dephosphorylation . We conclude that IGFBP-1 is phosphorylated and that this phosphorylation is a physiologically important posttranslational modification.

Proc Natl Acad Sci U S A, 1991 Sep 1, 88(17), 7834 - 8
Excitatory and inhibitory autaptic currents in isolated hippocampal neurons maintained in cell culture; Bekkers JM et al.; Individual rat hippocampal neurons, grown in isolation from other neurons on small spots of permissive substrate, were studied in order to characterize the electrical properties of the synapses that such cells formed with themselves (autapses) . Excitatory (probably glutamatergic) or inhibitory (probably type A gamma-aminobutyratergic) autapses were frequently found . Excitatory autaptic currents reversed near the potential expected for monovalent cations were blocked by the glutamatergic antagonist kynurenic acid, and possessed a slow component with the pharmacological profile of N-methyl-D-aspartate-type channels . These currents also exhibited trial-to-trial statistical fluctuations in their amplitudes, this being well-described by quantal analysis . Inhibitory autaptic currents reversed at hyperpolarized potentials, as expected for chloride-permeable pores and were blocked by picrotoxin, a type A gamma-aminobutyric receptor antagonist . It is concluded that autaptic currents in culture are identical to those found at synapses.

Biol Reprod, 1991 Sep, 45(3), 387 - 94
Soluble laminin and arginine-glycine-aspartic acid containing peptides differentially regulate type IV collagenase messenger RNA, activation, and localization in testicular cell culture; Sang QX et al.; Rat testicular cells in culture produce several metalloproteinases including type IV collagenases (Sang et al . Biol Reprod 1990; 43:946-955, 956-964) . We have now investigated the regulation of testicular cell type IV collagenase and other metalloproteinases in vitro . Soluble laminin stimulated Sertoli cell type IV collagenase mRNA levels . However, three peptides corresponding to different domains of the laminin molecule (CSRAKQAASIKVASADR, FALRGDNP, CLQDGDVRV) did not influence type IV collagenase mRNA levels . Zymographic analysis of medium collected from these cultures revealed that neither soluble laminin nor any of the peptides influenced 72-kDa type IV collagenase protein levels . However, peptide FALRGDNP resulted in both, a selective increase in two higher molecular-weight metalloproteinases (83 kDa and 110 kDa and in an activation of the 72-kDa rat type IV collagenase . Interleukin-1, phorbol ester, testosterone, and FSH did not affect collagenase activation . Immunocytochemical studies demonstrated that the addition of soluble laminin resulted in a redistribution of type IV collagenase from intracellular vesicles to the cell-substrate region beneath the cells . Peptide FALRGDNP induced a change from a vesicular to peripheral plasma membrane type of staining pattern . Zymography of plasma membrane preparations demonstrated triton-soluble gelatinases of 76 kDa, 83 kDa, and 110 kDa and a triton-insoluble gelatinase of 225 kDa . These results indicate that testicular cell type IV collagenase mRNA levels, enzyme activation, and distribution are influenced by laminin and RGD-containing peptides.

Res Virol, 1991 Sep-Oct, 142(5), 381 - 5
Kinetic study of the replication of a cell-culture-adapted hepatitis A virus; Zou S et al.; The kinetics of replication of hepatitis A virus (LCDC-01) was studied in foetal rhesus monkey kidney cells (FRhK-4) . Cells infected at a multiplicity of infection (MOI) of 2.0 showed no viral antigen production until 12 h post-infection using radioimmuno assay (RIA); however, at 48 h post-infection a logarithmic increase in antigen concentration began, which peaked by day 7 . Similar patterns were observed with cultures infected with lower MOI (0.20 and 0.02) but events were delayed by about 24 h . In contrast, detection of antigen by fluorescence antibody methods occurred at only 72 h after inoculation, with either 2.0 or 0.02 MOI, and peaked by day 9 . The production of infectious virus did not begin until 24 h post-infection as measured by RIA and gradually peaked by day 6 . Viral RNA was first detected 24 h post-infection by hybridization assay . The amount of viral RNA in the infected cells increased significantly between days 4 to 7 . Restriction in the synthesis of RNA or infectious virus was not observed.

Neuroreport, 1991 Sep, 2(9), 541 - 3
Binding interaction of gamma aminobutyric acid A and B receptors in cell culture; Kardos J et al.; We have examined the effects of 0.1 mM baclofen and pertussis toxin treatment on gamma aminobutyric acid A (GABAA) receptor binding in whole cells and in membrane of cells from cerebellar primary culture . Baclofen, a GABAB receptor ligand, caused a marked decrease in the bicuculline sensitive (GABAA) binding of {3H}muscimol on the whole cells, but failed to inhibit specific {3H}muscimol binding to cells pretreated with pertussis toxin . The effect of baclofen was only seen in whole cells and not in cell membranes . These findings suggest that activation of GABAB receptor reduces GABAA binding sites through a GTP binding protein.

Zhonghua Yi Xue Za Zhi, 1991 Sep, 71(9), 505 - 7, 36
{Monocyte chemotactic factor produced from cell culture of rabbit aortic smooth muscle}; Zhu Y; In the present studies conditioned media from cultured rabbit aortic smooth muscle cells (SMCs) were collected, and the chemotactic activity for rabbit peripheral blood monocytes was investigated by micropore filter assay . The results showed that the conditioned media were significantly chemotactic but not chemokinetic for monocytes . Chemotactic activity was heat stable at 80 degrees C for 30 min, but vanished upon boiling and after incubation with trypsin . It still existed after dialysis . Thus we believe that cultured rabbit aortic SMCs can secrete a protein which is chemotactic for peripheral blood monocytes.

Toxicol Lett, 1991 Sep, 58(1), 85 - 95
Detection of the Ah receptor in rainbow trout: use of 2-azido-3-{125I}iodo-7,8-dibromodibenzo-p-dioxin in cell culture; Swanson HI et al.; The Ah receptor was detected in RTG-2 cells (rainbow trout embryonic gonad cells) following the addition of the photoaffinity ligand, {125I}2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, to cells in culture . Cytosolic and nuclear extracts were prepared and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and revealed one radiolabeled band . Very little non-specific binding was observed under the conditions employed when compared to photoaffinity labeling RTG-2 cytosolic extracts in vitro . The photoaffinity-labeled Ah receptor in RTG-2 cytosol was analyzed by sucrose density centrifugation . The cytosolic form was observed to sediment at approximately 9.8S and the high salt nuclear extract form at approximately 7.5S . The relative molecular weight of the Ah receptor was determined to be 145 kDa under denaturing conditions and is considerably larger than the Ah receptor from mammalian sources . Inhibition of photoaffinity ligand binding to the RTG-2 cytosolic Ah receptor by competing ligands revealed the same rank order of ligand affinity as that previously demonstrated for the mouse Ah receptor.

J Virol, 1991 Sep, 65(9), 5105 - 10
Human herpesvirus 6 induces interleukin-1 beta and tumor necrosis factor alpha, but not interleukin-6, in peripheral blood mononuclear cell cultures; Flamand L et al.; The human herpesvirus 6 (HHV-6) is known to interact intimately with cells of the immune system . Here we report that HHV-6 is a potent inducer of interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) in cultures of peripheral blood mononuclear cells . In contradistinction, HHV-6 has no effect on IL-6 synthesis . Maximal IL-1 beta and TNF-alpha gene transcription, as detected by polymerase chain reaction amplification analysis, is observed at 12 and 6 h postinfection, respectively . Release of IL-1 beta and TNF-alpha into the culture supernatants peaked at 24 h and gradually decreased with time . Heat-inactivated virus was unable to stimulate IL-1 beta and TNF-alpha syntheses, whereas UV-irradiated virus retained the full monokine-inducing potential of the native particle . Preincubation of viral preparation with neutralizing anti-HHV-6 antibody resulted in the abrogation of this cytokine-inducing effect, whereas treatment of cells with phosphonoacetic acid (an inhibitor of viral DNA polymerase activity) had no effect on the ability of the virus to stimulate monokine release . These results indicate that HHV-6 can exert a strong immunomodulatory effect by stimulating the cells of myeloid lineage to produce these cytokines.

J Virol, 1991 Sep, 65(9), 4882 - 6
Mutations responsible for adaptation of hepatitis A virus to efficient growth in cell culture; Emerson SU et al.; Chimeric genomes of hepatitis A virus strain HM-175 were constructed from cDNA clones of the wild-type virus and its cell culture-adapted variant . RNA transcribed in vitro from each construct was assayed for infectivity by transfection of cultured cells . RNA transcribed from the wild-type cDNA clone was minimally infectious and produced virus that grew inefficiently in vitro, whereas that transcribed from certain chimeric genomes consistently produced virus that grew efficiently in cultured cells . Mutations in the P2 region were found to be necessary for efficient virus growth in vitro, while mutations in the 5' noncoding region imparted a conditional enhancement of growth in vitro.

Cytotechnology, 1991 Sep, 7(1), 25 - 32
Optimization of cell culture conditions for G-CSF (granulocyte-colony stimulating factor) production by genetically engineered Namalwa KJM-1 cells; Hosoi S et al.; An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained . Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized . The maximal production of G-CSF was at the most 1.8 micrograms/ml/day using a 25 cm2 tissue culture flask, even though the cell number was above 7 x 10(5) cells/ml . The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control . The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers . ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively . Under the control of pH at 7.4 and DO at 4 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1 x 10(7) cells/ml), and the amount of G-CSF reached 41 micrograms/ml . These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.

J Biotechnol, 1991 Sep, 20(2), 131 - 9
Expression of recombinant calf prochymosin in mammalian cell culture; Kolmer M et al.; The calf preprochymosin cDNA was cloned into an extrachromosomal mammalian cell expression vector containing Epstein-Barr virus sequences using polymerase chain reaction . Transfection of HeLa cells yielded Hygromycin B resistant cell clones, expressing immunoreactive prochymosin, which was quantitatively secreted into the culture medium . Based on Western blotting we estimated that selected cell clones produced about 10-20 mg prochymosin per liter in 20 h . The biological activity of the secreted chymosin was confirmed by milk clotting assay.

Cytotechnology, 1991 Sep, 7(1), 15 - 24
Growth limitation in hybridoma cell cultures: the role of inhibitory or toxic metabolites; Ronning OW et al.; Hybridoma cells usually grow to fairly low cell densities in batch cultures (1-3 x 10(6) cells/ml) . The reason for this is either that essential nutritional components of the medium are consumed, or that the cells produce some kind of inhibitory or toxic metabolite . This investigation presents evidence for the latter . Spent medium from cultures of hybridoma cells did not support growth of cells at lower cell densities (1-3 x 10(5) cells/ml) . The ability to support cell growth could not be restored by adding additional serum, energy sources (glucose, pyruvate) or L-glutamine . Furthermore, the consumption of amino acids could not account for this growth inhibition . On the contrary, the spent medium contained a substance that inhibited cell growth . This substance or metabolite was found in a fraction eluted from a gel filtration column when spent medium was applied to the column . This substance was found in the spent medium from all hybridoma and myeloma cell lines that were tested . The molecular weight of the substance was about 5 kD . Spent medium from two hybridoma cell lines also contained a substance that was eluted in the same fraction as albumin (67 kD) . It is likely that this (or these) substance(s) is responsible for the growth limitation in hybridoma cell cultures.

Biochem Pharmacol, 1991 Aug 8, 42(5), 1129 - 35
Endotoxin-inducible cytotoxicity in liver cell cultures--I; Hartung T et al.; It is known that rodents challenged with a combination of galactosamine and endotoxin develop a fulminant hepatitis within several hours . Until now, no in-vitro correlate for this organ-specific lesion has been described . Here, in-vitro conditions have been developed which allow examination of lipopolysaccharide (endotoxin)-inducible cell injury to hepatocytes . Under these in-vitro conditions (RPMI 1640 supplemented with 10% calf serum, 40% oxygen tension) which require the presence of functionally intact Kupffer cells, a concentration-dependent lactate dehydrogenase release is inducible by different lipopolysaccharides in hepatocyte cultures from Fischer rats . It can be abrogated by polymyxin B . These co-cultures secreted tumor necrosis factor-alpha into the medium upon a lipopolysaccharide stimulus . The presence of a tumor necrosis factor-alpha antiserum reduced the major part of the endotoxin-inducible cytotoxicity . Similarities in vitro and in vivo of the cytotoxic potency of various endotoxin species and the different responsiveness of hepatocytes from two different rat strains support that this co-culture system might be useful for studying endotoxin-inducible lesions in vitro.

Exp Cell Res, 1991 Aug, 195(2), 401 - 11
Localization of hyaluronate in primary glial cell cultures derived from newborn rat brain; Asher R et al.; We have devised a technique that enables one to localize hyaluronate in cultured cells . Cells were probed with the glial hyaluronate binding protein (GHAP) which was itself then visualized by conventional indirect immunofluorescence . The hyaluronate binding properties of this protein have been established . This technique was applied to the study of hyaluronate synthesis in glial cells . These cells do not themselves produce GHAP . O-2A progenitor cells were obtained from the cerebral hemispheres of newborn rats . These cells are bipotential in that they are able to differentiate into either oligodendrocytes or type 2 astrocytes depending on the composition of the culture medium . In cultures of O-2A progenitor cells maintained in the absence of serum, in which large numbers of oligodendrocytes appeared, very little hyaluronate was produced . The galC+ cells were invariably hyaluronate negative . Cultures of the same cells, maintained in the presence of 10% FCS, contained large numbers of hyaluronate producing cells . The hyaluronate producing cells were typically small, process-bearing, and GFAP+ . Some, but not all, were A2B5+ and could, therefore, be identified as type 2 (GFAP+, A2B5+) astrocytes . Type 1 (GFAP+, A2B5-) astrocytes were also active in the synthesis of hyaluronate, to the extent that they were able to coat their substrate with hyaluronate . Among cells of the O-2A lineage, then, hyaluronate production would appear to be restricted to astrocytes . This may have some bearing on the origin of hyaluronate in the extracellular matrix of CNS white matter.

J Virol Methods, 1991 Aug, 33(3), 305 - 10
Comparison of rapid immunofluorescence assay to cell culture isolation for the detection of influenza A and B viruses in nasopharyngeal secretions from infants and children; Spada B et al.; In the hospital setting it is often critical to isolate patients appropriately in order to prevent nosocomial infection . This is especially true with respiratory infection in infants and young children . At the present time a rapid immunofluorescence assay (IFA) for respiratory syncytial and parainfluenza viruses is routinely carried out in our laboratory . During January and February of 1990 we used monoclonal antibodies specific for influenza A and B viruses (Baxter-Bartels, Bellevue, WA) in this rapid IFA . 152 samples of NPS were tested by cell culture isolation (CCI) and IFA for the presence of influenza antigens . Twenty-seven samples were positive by both methods, and 114 were negative by both . Three samples were positive by IFA and negative by CCI, while eight samples were positive by CCI and negative by IFA . Five of these eight samples were not positive until 10 to 14 days after inoculation into cell culture, suggesting that the virus inoculum was small . Using CCI as the 'gold' standard, IFA was 90% sensitive and 93% specific . Because of its turn-around time (2-4 h) and acceptable sensitivity and specificity, IFA for influenza viruses will be a routine test in our diagnostic laboratory during the influenza season.

J Virol Methods, 1991 Aug, 33(3), 283 - 9
Advantages of multiple cell culture systems for detection of mixed-virus infections; Fong CK et al.; Simultaneous infections by two or more viruses occur frequently, especially in immunosuppressed patients . In order to detect more than one viral agent in a single specimen, multiple cell systems have been employed in our laboratory . Specimens are routinely inoculated into four different cell cultures, namely: MRC-5, a human diploid lung fibroblast cell strain; A549, a human continuous cell line; primary guinea pig embryo (GPE) cell culture, and primary rhesus monkey kidney (RhMK) cell culture . For rapid detection of cytomegalovirus (CMV) antigen, MRC-5 cells grown in shell vials containing coverslips are also inoculated with the same specimens followed by centrifugation . During 1989, nine cases of multiple-virus isolations were obtained in this laboratory . In all nine patients, CMV was detected in MRC-5 cells . Five of the nine cases were co-infected with HSV-1, three were co-infected with adenovirus, and one was co-infected with both HSV-1 and adenovirus . All four adenovirus isolates were obtained in A549 cells . Of the six HSV-1 isolates, one was detected in all three cell cultures, e.g . MRC-5, A549 and GPE; one was detected in both MRC-5 and A549 cells, and four were isolated in a single-cell type only . For nine CMV-positive cases, five were obtained by both conventional and centrifugation cultures, two each were detected by centrifugation or conventional culture only . Thus for a maximum detection of viruses present in a single specimen, it is suggested that multiple-cell-culture systems, together with more than one technique, should be employed.

Eur J Clin Microbiol Infect Dis, 1991 Aug, 10(8), 656 - 9
Comparison of different culture media for isolation of Chlamydia trachomatis by cell culture on HeLa cells; Herbrink P et al.; The isolation yield and number of Chlamydia trachomatis inclusions was compared using DEAE-dextran pretreated HeLa cells cultured in four different media: Eagle's minimal essential medium (EMEM) with and without cycloheximide and Dulbecco's modification of EMEM (DMEM) with and without cycloheximide . Using DMEM without cycloheximide the number of inclusions was significantly higher than using EMEM without cycloheximide . In addition, the size of the inclusions was greatly enhanced . Use of DMEM or EMEM with cycloheximide yielded results comparable to those obtained with DMEM without cycloheximide.

Neuropathol Appl Neurobiol, 1991 Aug, 17(4), 299 - 308
Multiplication of rubella and measles viruses in primary rat neural cell cultures: relevance to a postulated triggering mechanism for multiple sclerosis; Atkins GJ et al.; Rubella virus multiplied to low titre and produced a partial cytopathic effect in rat glial cell cultures . Anti-galactocerebroside staining showed that this cytopathic effect involved the disintegration of oligodendrocytes . A similar effect was produced following infection of myelinating neural cell cultures with rubella virus, but virus multiplication could not be detected in pure neuron cultures . Measles virus was found to multiply and produce a cytopathic effect in primary cultures of both neurons and glial cells . These results are discussed in relation to the ability of measles and rubella viruses to trigger human multiple sclerosis.

J Physiol, 1991 Aug, 439, 579 - 604
Multiple effects of tetraethylammonium on N-methyl-D-aspartate receptor-channels in mouse brain neurons in cell culture; Wright JM et al.; 1 . The mechanisms of tetraethylammonium (TEA) antagonism of N-methyl-D-aspartate (NMDA) responses were investigated in cultured mouse cortical neurons by analysing single-channel and whole-cell currents from patch clamp recordings . TEA (1-5 mM) decreased whole-cell NMDA responses . Kainate and quisqualate receptor-mediated responses were unaffected at these TEA concentrations . 2 . In whole-cell recordings, increasing the NMDA concentration while keeping the TEA concentration constant resulted in greater inhibition by TEA . Thus, TEA-mediated inhibition of NMDA responses was not due to competitive antagonism, and the greater inhibition by a single dose of TEA as NMDA concentration was elevated indicated some form of non-competitive inhibition . In single-channel recordings, two inhibitory effects were seen in 1-5 mM-extracellular TEA: single-channel conductance (gamma) was decreased, and the frequency of channel events was decreased . These effects were not accompanied by any change in average channel open time . 3 . Single-channel current-voltage (I-V) curves obtained in 2, 5, 10 and 30 mM-TEA indicated the decrease in NMDA channel conductance was voltage dependent with larger reduction occurring as patches were hyperpolarized . The data were well fitted by the Woodhull model with the dissociation constant (KD) showing an e-fold increase in inhibition for a 43-45 mV change in membrane potential . The 0 mV KD was 45 mM-TEA decreasing to about 11 mM at -60 mV . The TEA block site appeared to sense approximately 60% of the transmembrane potential field (delta = 0.6) for extracellular application of TEA . 4 . The decrease in channel opening frequency seen in TEA was concentration dependent and generally more sensitive to extracellular TEA than the channel block effect . There was a 50% reduction in the number of NMDA channel openings observed in 5 mM-TEA . Increasing either NMDA or glycine concentrations in constant TEA concentration caused an additional decrease in the frequency of NMDA channel opening . In contrast to extracellular TEA, intracellular TEA had no noticeable effect on open-state probability . 5 . NMDA single-channel currents were observed at positive potentials after completely replacing pipette Cs+ by 140 mM-TEA-Cl indicating TEA could serve as a current carrier through NMDA channels . Single channel I-V curves obtained with pipettes containing 70 or 140 nM-TEA in place of equivalent amounts of Cs+ were fitted by the Goldman-Hodgkin-Katz (GHK) equation over the range of -80 to +70 mV assuming a permeability of 0.45 compared with a Cs+ permeability of 1.0.(ABSTRACT TRUNCATED AT 400 WORDS)

J Neurochem, 1991 Aug, 57(2), 615 - 21
K(+)-evoked depolarization stimulates cyclic AMP accumulation in photoreceptor-enriched retinal cell cultures: role of calcium influx through dihydropyridine-sensitive calcium channels; Iuvone PM et al.; The effect of membrane depolarization on cyclic AMP synthesis was studied in glia-free, low-density, monolayer cultures of chick retinal photoreceptors and neurons . In photoreceptor-enriched cultures prepared from embryonic day 6 retinas and cultured for 6 days, elevated K+ concentrations increased the intracellular concentration of cyclic AMP and stimulated the conversion of {3H}adenine to {3H}cyclic AMP . The K(+)-evoked increase of cyclic AMP accumulation was blocked by omitting CaCl2 from the incubation medium, indicating a requirement for extracellular Ca2+ . Stimulation of cyclic AMP accumulation was also inhibited by nifedipine, methoxyverapamil, Cd2+, Co2+, and Mg2+, and was enhanced by the dihydropyridine Ca2+ channel agonist Bay K 8644 . The enhancement of K(+)-evoked cyclic AMP accumulation by Bay K 8644 was antagonized by nifedipine . Thus, Ca2+ influx through dihydropyridine-sensitive channel is required for depolarization-evoked stimulation of cyclic AMP accumulation in photoreceptor-enriched cultures.

Zhonghua Shen Jing Jing Shen Ke Za Zhi, 1991 Aug, 24(4), 242 - 3, 254
{Neural complications of cell culture rabies vaccine prepared from hamster kidney . Clinicopathological report of a case}; Chen Y; A case died of cell culture rabies vaccine prepared from hamster kidney was reported . Autopsy showed invasion of both the peripheral and the central nervous systems . In the CNS there were not only acute disseminated encephalomyelitis, but also changes similar to acute viral encephalitis.

Sci China B, 1991 Aug, 34(8), 947 - 62
Studies on preparation of inactivated vaccines from cell cultures of mink enteritis virus (MEV) and their immunity; Zhang DL; In this paper, superhigh reproductive rate strains of MEV with titre more than HA8192x or TCID50 log10(9.7) have been achieved both by cultivation in cell lines with different susceptibility to MEV and by isolating and identifying in field by the author . The systematic tests proved that S18 and L12 strains of MEV are the best strains for vaccine preparation . In this study, the best means for the tissue cultivation of MEV and the most advanced technological process for the production and detection of serum-free cell-cultured MEV fluids with super-high HA titre in batches in large quantities have been established for the first time . Optimum conditions for MEV inactivation were determined, and safe and effective inactivated vaccines with mineral oil or Al(OH)3 gel adjuvant were successfully prepared with serum-free cell-cultured MEV fluids . Both vaccines with different adjuvants can be manufactured in batches in large quantities and have been widely used all over China since 1986 . The change laws of the immune response kinetics of the MEV vaccines have been determined by the tests, and the indexes and laboratory animal models for monitoring humoral immune efficiency and immune protective rate of parvovirus vaccines have been established for the first time . The manufacturing and monitoring operation rules of the MEV vaccines with different adjuvants have been laid down by the author.

J Bone Miner Res, 1991 Aug, 6(8), 869 - 81
Bone cell culture in a three-dimensional polymer bead stabilizes the differentiated phenotype and provides evidence that osteoblastic cells synthesize type III collagen and fibronectin; Majmudar G et al.; We report a novel method to culture chick embryo osteoblasts in vitro . Primary cells were grown from explants of calvaria and then cultured within alginate polymer beads . Enriched cultures of primary osteoblasts were obtained because these cells grow readily within alginate beads but other cell types present in the initial outgrowth from calvarial fragments, such as fibroblasts, do not . A reproducible bone cell phenotype was observed in calvarial cells cultured in the alginate polymer for as long as 8 months . Alginate is a uronic acid monomer that reversibly polymerizes based on the presence or absence of divalent cations . Osteoblasts derived from the alginate beads elaborated and mineralized an extracellular matrix in vitro that contained fibronectin, type III collagen, and type I collagen . The synthesis and deposition of these matrix molecules was also demonstrated in the chick embryo calvaria in vivo . Together, these in vitro and in vivo observations provide the first evidence that type III collagen and fibronectin colocalize with type I collagen during the development of avian membranous bone . They also indicate that the phenotype of chick embryo osteoblasts can be expanded to include the synthesis of fibronectin and type III collagen.

J Histochem Cytochem, 1991 Aug, 39(8), 1009 - 16
Identification of peroxidase-positive astrocytes by combined histochemical and immunolabeling techniques in situ and in cell culture; Schipper HM et al.; A subpopulation of astrocytes in the vertebrate brain and in cysteamine-treated brain cell cultures contain cytoplasmic granules which exhibit an affinity for Gomori stains, orange-red autofluorescence, and non-enzymatic endogenous peroxidase activity . Visualization of these cells at the light microscopic level is confounded by the nonspecificity of the various histochemical methods routinely employed . In an attempt to circumvent this problem, we assayed for peroxidase-positive astrocytes using various combinations of diaminobenzidine (DAB) histochemistry and immunolabeling for the astrocyte-specific marker glial fibrillary acidic protein (GFAP) . We determined that (a) DAB histochemistry in conjunction with avidin-biotin-immunoperoxidase labeling for GFAP specifically detects peroxidase-positive astrocytes in situ and (b) DAB histochemistry combined with indirect immunofluorescence for GFAP effectively demonstrates these cells in cysteamine-treated brain cell cultures.

Virology, 1991 Aug, 183(2), 821 - 4
Defective interfering particles of human parainfluenza virus type 3 are associated with persistent infection in cell culture; Moscona A; CV-1 cell lines persistently infected with human parainfluenza virus type 3 (HPF3) contain one or more distinct subgenomic RNAs in addition to standard viral genomes . These RNAs are shown to be the genomes of defective-interfering (DI) particles of the virus; they are present in particles in the culture fluid, and they interfere with the growth of wild-type virus . Removal of the particles from the culture fluid by ultracentrifugation yields a supernatant fluid free from inhibitory activity, demonstrating that the anti-viral effect is not mediated by soluble factors . A role for the DI particles in persistence of HPF3 is considered.

Gene, 1991 Jul 22, 103(2), 155 - 61
Use of sulfonated primers to detect and type papillomavirus in cell cultures and cervical biopsies; Paper T et al.; Human papillomavirus (HPV) was detected by using two sets of deoxyribonucleotide primers for differentiating between 'low-risk' types (HPV11 and HPV6) and 'high-risk' (hri) types (HPV16, HPV18 and HPV33) . A new application of the Chemiprobe method for labeling DNA was used to detect products of the polymerase chain reaction (PCR) from 36 cervical biopsies . This method, first demonstrated by Uchimura et al . (submitted), is based on the sulfonation of a polycytidylic acid tail of 5-20 monomers attached to the 5' end of either one or both of the PCR primers . This procedure can increase the sensitivity of detection of PCR products more than 100-fold with respect to ethidium bromide (EtdBr) staining . Various methods were used to detect hri HPV DNA in the 36 clinical samples . The number of positive results obtained was as follows, two by Southern-blot hybridization; five by PCR amplification followed by electrophoresis and detection of products by EtdBr staining; six by PCR amplification using one or two sulfonated C-tailed primers followed by electroblotting and immunoenzymatic visualization; and five by hybridization of sulfonated genomic viral recombinant with a PCR product immobilized on a membrane . The yield of the PCR product was significantly greater when one of the primers was C-tailed than when both or neither of the primers were C-tailed . PCR employing sulfonated C-tailed oligo primers is very specific and sensitive, and the entire procedure can be employed as a nonradioactive substitute for radioactive dot-blot or Southern-blot hybridization procedures, routinely used for detection of HPV in clinical samples.

Brain Res, 1991 Jul 12, 553(2), 331 - 5
Cell migration along glial fibers in dissociated cell culture of the frog optic tectum; Becker T et al.; Migration of neurons along radial glial fibers is associated with the development of laminated regions in the mammalian brain . We examined cell interactions in dissociated cell cultures of the frog optic tectum, which is well laminated . Using time-lapse photography, we observed active migration of neuron-like cells along strands of radial glia-like cells, which were identified by indirect immunocytochemical staining of the glial fibrillary acidic protein . The migration patterns we observed in our cultures are strikingly similar to those found in mammalian cultures . We hypothesize that this type of neuron-glia interaction is involved in the constitution of laminae in the frog optic tectum.

Am J Clin Pathol, 1991 Jul, 96(1), 127 - 9
Early use of indirect immunofluorescence for the detection of respiratory syncytial virus in HEp-2 cell culture; Bromberg K et al.; Respiratory syncytial virus is detected in cell culture by the presence of cytopathic effect . To detect RSV before cytopathic effect is usually seen, slides were evaluated retrospectively from 482 HEp-2 cell cultures on days 2-4 after inoculation . Indirect immunofluorescent staining detected RSV in 57 of 94 cultures that eventually were found positive by cytopathic effect . In an additional 19 cases that ultimately showed no cytopathic effect, RSV also was detected . In 15 of the latter cases, the presence of RSV was confirmed in the original specimen . Use of indirect immunofluorescence can be used to augment the sensitivity of cell culture for the detection of RSV because cytopathic effect may not always be evident.

Appl Biochem Biotechnol, 1991 Jul, 30(1), 29 - 41
Growth inhibition in animal cell culture . The effect of lactate and ammonia; Hassell T et al.; Eight independent cell lines accumulated ammonia in culture to concentrations between 1.3 and 2.9 mM . The growth inhibition of such concentrations of ammonium chloride when added to culture medium was variable . The cell lines tested could be divided into 3 groups depending on their growth response to 2 mM added NH4Cl . In the first group (293, HDF, Vero, and PQXB1/2) little (less than 14%) or no growth inhibition occurred . In the second group (McCoy and MDCK) a reduction in final cell yield of 50-60% was observed . The third group (HeLa and BHK) was most sensitive to the effects of NH4Cl with growth inhibition (greater than 75%) compared to controls . The growth inhibitory effect of added lactate up to 20 mM was negligible (less than 10%) for 3 cell lines, although one cell line (PQXB1/2) showed greater sensitivity . The interactive effects of ammonia and lactate were determined in a matrix experiment . At lactate (greater than 12 mM) and ammonia (1-4 mM), the growth inhibitory effects of the two components were synergistic . However, at low concentrations of lactate (less than 12 mM) the toxic effect of ammonia was reduced . A proposed mechanism for the sparing effect of lactate on ammonia toxicity is discussed . This may have importance in developing strategies for the optimal growth of ammonia-sensitive cell lines.

J Pharm Sci, 1991 Jul, 80(7), 700 - 4
Effects of nitrendipine on cocaine-induced toxicity evaluated in primary myocardial cell cultures; Melchert RB et al.; Currently, there is no uniform treatment for abnormal cardiac events precipitated by cocaine use . However, clinical strategies include use of calcium channel antagonists for cardiovascular emergencies . In experimental situations using rats, simultaneous administration of nitrendipine (NIT) with cocaine to the whole animal (1.46 x 10(-3) mg/kg/min of NIT; 2 mg/kg/min of cocaine) and isolated retrograde perfused hearts (Langendorff; 1 x 10(-7) M NIT; 1 x 10(-7) to 1 x 10(-4) M cocaine) normalized cocaine-induced abnormalities in heart rhythm and provided protection from acute cocaine-induced morphological lesions . Using similar concentrations of NIT and cocaine, the purpose of this study was to evaluate the direct cardiac cellular effects of NIT on cocaine-induced alterations in beating activity, morphology, and lactate dehydrogenase (LDH) release in a controlled in vitro system of primary myocardial cell cultures . Cultures were established from hearts of 3-5-day-old Sprague-Dawley rats . After the cells had been maintained in culture for 4 days, evaluation of drug effects were made with exposure to 1 x 10(-3) and 1 x 10(-5) M cocaine alone and combinations of these two concentrations of cocaine with simultaneous exposure to 1 x 10(-7) M NIT for 1 to 24 h . Those cells exposed to 1 x 10(-5) M cocaine alone maintained some beating activity after 1, 4, and 24 h . Beating activity was significantly depressed after treatment with 1 x 10(-3) M cocaine alone and with both combinations of cocaine and NIT . Morphological integrity was maintained in all treatment groups for 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)

J Microencapsul, 1991 Jul-Sep, 8(3), 307 - 16
Semi-permeable microcapsules for cell culture: ultra-structural characterization; Shimi SM et al.; Microcapsules made from alginate-poly(L-lysine)-alginate membranes have been studied as vehicles for cell culture in a number of laboratories . We have examined their permeability, robustness and ultrastructure in detail . Permeability to globular proteins could be controlled by using poly-lysine of different mean MW in their construction . However, this parameter also affected the degree to which microencapsulated living cells leaked out of the capsules during and after preparation . Poly-lysine of low MW produced a relatively permeable and robust membrane whereas a high MW produced capsules with the reverse characteristics . A MW of 22,000 appears to be optimal in forming robust capsules which are relatively impermeable to high MW species such as immunoglobulins . The structure of the semipermeable membrane was investigated by electron microscopy and found to be complex but entirely consistent with the data on protein permeability and cell leakage . Microcapsules were not disrupted by gentle treatment with trypsin or chelating agents but dissolved with the addition of heparin, sodium dodecyl sulphate or sodium hydroxide . Empty microcapsules implanted into the peritoneal cavity of rats elicited a host cellular reaction but remained intact for at least three months.

Cell Biol Int Rep, 1991 Jul, 15(7), 581 - 94
Effect of canatoxin on cell cultures; Campos MM et al.; Canatoxin, a toxic protein isolated from Canavalia ensiformis, was shown to inhibit DNA synthesis and to produce a cytolytic effect when added in vitro to various cells, in doses ranging from 50 to 500 nM . In this case no selectivity was found for a certain cell type, as both normal and transformed cells could be affected by the toxic protein . The cytostatic effect was irreversible upon removal of the toxic protein from the medium and could be fully attained after exposing the cells to canatoxin for only 30 min . The use of an in vitro cell culture system may allow for a better insight on the mode of action of canatoxin.

J Comp Pathol, 1991 Jul, 105(1), 17 - 26
The use of fluorescein-conjugated monoclonal antibodies, cell culture and transmission electron microscopy to detect Chlamydia psittaci and associated lesions in experimentally infected mice; Biolatti B et al.; An immunofluorescence test based on a monoclonal antibody (mAb) was used to demonstrate chlamydiae in formalin-fixed and paraffin wax-embedded tissues from 10 adult mice experimentally infected by the oral route with Chlamydia psittaci isolated from the fetal membranes of an aborted ovine fetus . Samples of lung, jejunum and spleen were examined by bright-field microscopy, immunofluorescence and transmission electron microscopy, and were cultured for chlamydia in McCoy cells . These tissues were compared with those of two control mice . All infected mice had splenic hyperplasia and two had pneumonia . The lung appeared to be the target organ for C . psittaci administered by the oral route . Chlamydiae were identified in the lungs of five mice by immunofluorescence, bright-field and transmission electron microscopy . Chlamydiae were cultured from the jejunum of two mice and the spleen of one, but could not be identified at these sites by other methods . Immunofluorescence with an anti-chlamydia mAb was useful for detecting chlamydial antigen in formalin-fixed paraffin wax-embedded samples.

J Clin Microbiol, 1991 Jul, 29(7), 1333 - 8
Simplified microtiter cell culture method for rapid immunotyping of Chlamydia trachomatis; Suchland RJ et al.; Serotyping of Chlamydia trachomatis strains usually requires three to six serial passages in shell vials to attain sufficient antigen for typing procedures . To circumvent this problem, we developed a rapid low-passage method for serotyping of C . trachomatis clinical isolates . Isolates with an inclusion count of greater than or equal to 500 per well in primary isolation were inoculated directly onto cell culture monolayers in microtiter plates for typing . Primary isolates with a lower initial inclusion count were passed one to two times in shell vials until there were greater than or equal to 20 inclusions per well and were then inoculated onto plates for typing . Inclusions were grown to maturity and reacted with a panel of 17 C . trachomatis-specific monoclonal antibodies in pools . Wells were then reacted with a fluorescein isothiocyanate conjugate and read by FA microscopy, and the reaction patterns were compared with prototype strain reaction patterns to determine the serotype . By the microtiter method, we successfully typed 1,711 consecutive C . trachomatis isolates; 1,215 isolates (71%) were typed with no or with one passage . The first 209 isolates typed by the microtiter method were also typed by the dot-enzyme-linked immunosorbent assay serotyping method; 100% agreement was demonstrated among strains that were typeable by both methods . We conclude that the microtiter method is extremely useful for accurate serotyping of large numbers of isolates and requires greatly reduced technician time.

J Anim Sci, 1991 Jul, 69(7), 2855 - 64
Effects of the beta-adrenergic agonist isoproterenol on protein accretion, synthesis, and degradation in primary chicken muscle cell cultures; Ji SQ et al.; Seven-day-old primary myotube cultures derived from embryonic chicken limb muscles were used to determine the effects of the beta-adrenergic agonist isoproterenol (ISO) on muscle protein metabolism in vitro . Isoproterenol increased (P less than .05) total protein accumulation after 2 h of acute exposure and after chronic exposure for 24 and 48 h . Isoproterenol did not consistently retard rate of protein degradation of the total protein (TP), myofibrillar protein (MFP) pools, and myosin heavy-chain subunit (MHC); degradation of these protein pools tended to be slowed by inclusion of ISO in the culture medium . After acute treatment of 1 X 10(-4) M ISO for 2 h, TP, but not MFP and MHC, synthesis rate was increased, and after chronic exposure to 1 X 10(-4), 1 X 10(-5), and 1 X 10(-6) M ISO, TP, MFP, and MHC synthesis rates and net accumulation of TP, cytoplasmic protein, and MHC fractions were enhanced (P less than .05) . The beta-adrenergic antagonist propranolol (1 X 10(-5) M) blocked chronic stimulatory effects of ISO . Furthermore, after 48 h of exposure to ISO, effects on protein synthesis were less pronounced than those observed after 24 h of exposure . Isoproterenol imparted a more pronounced effect on protein synthesis than on protein degradation, indicating that increased muscle protein accretion observed in animals after ISO treatment is likely a function of enhanced protein synthesis.

Cytokine, 1991 Jul, 3(4), 292 - 8
Altered cytokine release in peripheral blood mononuclear cell cultures from patients with the chronic fatigue syndrome; Chao CC et al.; Chronic fatigue syndrome (CFS) is an idiopathic illness associated with a variety of immunologic abnormalities . To investigate potential pathogenetic mechanisms, we evaluated serum levels and peripheral blood mononuclear cell (PBMC) production of selected cytokines and immunoglobulins . Serum bioactive transforming growth factor beta (TGF-beta) levels were higher (P less than 0.01) in patients with CFS (290 +/- 46 pg/mL) than in control subjects (104 +/- 18 pg/mL), but levels of other cytokines tested were not different . Lipopolysaccharide-stimulated release of interleukin 1 beta (IL-1 beta), IL-6, and tumor necrosis factor-alpha was increased (P less than 0.05) in PBMC cultures from patients with CFS versus control subjects; enhanced (P less than 0.01) IL-6 release to phytohemagglutinin was also observed . In contrast, TGF-beta release in response to lipopolysaccharide was depressed (P less than 0.01) in PBMC cultures derived from patients with CFS . No differences in IL-2 and IL-4 or immunoglobulin production were observed . The enhanced release of inflammatory cytokines by stimulated PBMC from patients with CFS suggests that these cells are primed for an increased response to immune stimuli . These data also suggest an association between abnormal regulation of TGF-beta production in vivo and in vitro with the immunologic consequence of CFS.

Bone Miner, 1991 Jul, 14(1), 41 - 54
Mineralized nodule formation in rat bone marrow stromal cell culture without beta-glycerophosphate; Satomura K et al.; Rat bone marrow stromal cells were cultured in the presence of 10 nM dexamethasone and various concentrations of beta-glycerophosphate . At day 12-15, some nodules consisting of polygonal cells were formed in all culture conditions, and these nodules were mineralized 2-3 days later . beta-Glycerophosphate significantly enhanced nodule formation at concentrations of not less than 5 mM . The mineralized nodules formed in the absence of beta-glycerophosphate were examined using phase-contrast microscopy, undemineralized and demineralized tissue histology, histochemistry for alkaline phosphatase, immunohistochemistry for type I, II, and III collagen, energy dispersive X-ray microanalysis, electron diffraction, and Fourier transform infrared spectroscopy (FT-IR) . Mineralized nodules had histological characteristic similar to bone . Cells associated with nodules exhibited high alkaline phosphatase activity, and extracellular matrix of the nodules predominantly consisted of type I collagen . X-Ray microanalysis showed the presence of Ca and P in the mineralized area, and electron diffraction pattern showed the mineral to have apatite crystal structure . Moreover FT-IR indicated that the mineral was a mixture of hydroxyapatite and carbonateapatite . From these observations, it is concluded that the mineralized nodules formed in our culture system are truly bone-like.

Arch Biochem Biophys, 1991 Jul, 288(1), 39 - 47
Partial purification, properties, and kinetic studies of UDP-glucose:p-hydroxybenzoate glucosyltransferase from cell cultures of Lithospermum erythrorhizon; Bechthold A et al.; A glucosyltransferase, which catalyzed the transfer of glucose from UDP-glucose (UDPG) to p-hydroxybenzoate (PHB) in cell cultures of Lithospermum erythrorhizon Sieb . et Zucc., Boraginaceae, was purified 219-fold by ammonium sulfate fractionation and chromatography on DEAE-Sephacel, Sephadex G-150, and phenyl-Sepharose Cl-4B . p-Hydroxybenzoic acid O-beta-D-glucoside (PHB-glc) was identified as a product of the enzymatic reaction . This glucosyltransferase has a molecular weight of 47,500 Da, an isoelectric point at pH 5.0, and a pH optimum of 7.8 . The enzyme does not sediment at 100,000g . Enzyme activity did not require metal cofactors . The enzyme was highly specific for p-hydroxybenzoate (Km 0.264 mM) and UDP-glucose (Km 0.268 mM) . Initial velocity studies suggest that the enzyme reaction mechanism is a sequential rather than a ping-pong mechanism . Product inhibition patterns are consistent with an ordered sequential bi-bi mechanism, where UDPG is the first substrate to bind to the enzyme and UDP the final product released . The data indicate the formation of a dead-end complex between PHB-glc and the enzyme . Uncompetitive inhibition by the substrate PHB can be put down to the formation of an abortive complex between E-UDP and PHB.

Zhonghua Kou Qiang Yi Xue Za Zhi, 1991 Jul, 26(4), 233 - 6, 254
{In vitro toxicity evaluation of 12 kinds of dental materials using a modified cell culture technique}; Ding N; In view of the fact that up to date the various cell toxicity tests could not come to an identical result and compare with each other . This paper, referring to the covering documents of F.D.I . and I.S.O., summing up the principles and characteristics of various cell culture techniques, establishes a modified cell toxicity test . This method, with more respects of wide observations and tangible results, is easy to grasp, well to repeat and can be reserved for checking the results, meanwhile, it can be avoided the contradiction caused by the different experiments on one kind of material . Therefore, it is very worthy of appraising the biological safety of the materials . The study of 12 kinds of dental materials expressed that the toxicity order of copper alloy was mean value of 0 (Jiao Tong University), copper alloy tridium was mean value of 3 (U.S.A.), copper alloy T was mean value of 2 (Jiao Tong University) and the others were mean value of 0-1 . This paper also discussed several problems with common concerns in studying the cell toxicity of the biomaterials.

Ann Acad Med Singapore, 1991 Jul, 20(4), 493 - 7
Human keratinocyte cell culture for the burns patients--a preliminary report; Fei X et al.; One of the major problems in extensive burns is the relative lack of available donor sites for skin grafting . Keratinocyte cell culture in the laboratory was carried out successfully in Singapore General Hospital and shows promise as an alternative source for skin replacement . Our experience further proved that a fibroblast base is necessary for keratinocyte cell culture . It is observed that heterogenous cell source can grow concurrently and become confluent . The colony forming efficiency from trypsinized skin is about 1-4% in primary cultures and 35-40% in secondary cultures . The time taken to reach confluency are 20-21 days and 10-12 days respectively . The thickness of cultured skin is estimated as 0.5 mm (5-6 layers) under light microscope . The size of the harvested cultured skin is approximately one third of the cultured area due to contraction . The expansion ratio before shrinkage is estimated to be approximately 6000-fold based on our data.

Z Naturforsch {C}, 1991 Jul-Aug, 46(7-8), 706 - 7
Growth inhibitions on human cancer cell cultures with the indole sulphur-containing phytoalexins and their analogues; Tempete C et al.; Cell growth inhibitions on human cancer cell cultures were determined for the indole sulphur-containing phytoalexins cyclobrassinin, brassilexin (previously isolated from vegetables of the Cruciferae family) and their synthetic analogues 5-methoxybrassilexin and homocyclobrassinin . The most biologically active of these products is brassilexin (LD50 = 8 micrograms/ml).

Biochem Int, 1991 Jul, 24(5), 951 - 7
Deoxycorticosterone, 18-OH-deoxycorticosterone and corticosterone determination by high performance liquid chromatography in monolayer adrenal cell culture; Matilla MJ et al.; Reversed-phase HPLC offers a rapid, qualitative as well as quantitative method for separation and determination of deoxycorticosterone, 18-OH-deoxycorticosterone and corticosterone in primary culture of adrenocortical cells . The resolution is sufficient for the purpose of quantitative determination . The limitation of this method lies in its sensitivity for serum steroid determination, but is perfectly applicable at cell cultures where the concentration of these steroids is highest.

J Clin Microbiol, 1991 Jul, 29(7), 1295 - 8
Serotyping of Chlamydia trachomatis by indirect fluorescent-antibody staining of inclusions in cell culture with monoclonal antibodies; Wang SP et al.; A new method for determining the serovars of Chlamydia trachomatis isolates by utilizing fluorescent-antibody staining of inclusions in cell culture is described . Monoclonal antibodies which have been successfully used previously for serotyping in the microimmunofluorescence test were employed . The cell culture method offers two advantages over the microimmunofluorescence test for many laboratories . It requires less antigen of the new isolate, about 10% cell culture infectivity versus 50% for the microimmunofluorescence test . Although fewer isolates can be typed at one time in cell culture, the technical requirements of the test are less rigorous.

J Neurosci Res, 1991 Jul, 29(3), 355 - 61
Glutamine stimulates growth in rat cerebral endothelial cell culture; Dux E et al.; Endothelial cells were isolated from rat cerebral cortices using combined enzymatic digestions and Percoll gradient centrifugation . Primary cultures were subsequently grown on collagen-covered dishes in a medium containing 20% fetal calf serum and 0.6 mmol glutamine . The majority of cultures became confluent by day 7 or 8, but some could not reach confluence . The cells were fusiform in shape and exhibited immunoreactivity to factor VIII-related antigen and binding to the lectin Griffonia simplicifolia . Exposure of cultures to media containing 2.6 mmol glutamine resulted in accelerated growth (in cultures were confluent at days 3-4) and change in culture morphology, namely the formation of circular, cell-free areas . However, this treatment did not restore gamma-glutamyl transpeptidase activity that was lost during cultivation . As for other amino acids, asparagine was less potent, glycine and phenylalanine failed to mimic the glutamine effect . In summary, glutamine stimulates growth of cerebral endothelial cells in vitro and so it may supplement for other growth factors in the culture media.

Res Virol, 1991 Jul-Aug, 142(4), 261 - 70
Antiviral activity of carrageenan on hepatitis A virus replication in cell culture; Girond S et al.; Sulphated polysaccharides such as iota-, lambda- and kappa-carrageenans showed a potent inhibitory effect on the replication of hepatitis A virus (HAV) in the human hepatoma cell line PLC/PRF/5 . No cytotoxic effects were detected with concentrations of carrageenans up to 200 micrograms/ml . The selectivity indices of these substances, calculated as the ratio of the dose that reduced the number of viable cells to 50% (CD50) to the effective dose that inhibited 50% of viral antigen expression (ED50), were greater than 400 with iota-carrageenan, greater than 222 with lambda-carrageenan and greater than 10 with kappa-carrageenan . The selectivity index of ribavirin (reference substance) was only 5 . The 3 types of carrageenans resulted in concentration-dependent reduction of HAV-antigen expression and HAV infectivity . lota-and lambda-carrageenan emerged, from the present study, as promising candidates for chemotherapy of acute hepatitis A.

Biull Eksp Biol Med, 1991 Jul, 112(7), 98 - 9
{Effects of propranolol on mitosis-inhibiting activity of G2-chalone and adrenaline in cell culture of Ehrlich's ascites tumor}; Antokhin AI et al.; Chalone-containing preparation from ascite Ehrlich's tumour blocks these cell transition from G2-phase to mitosis and its motion in mitosis in vitro (G2-block, M-block) . Adrenaline blocks these cell transition from G2-phase to mitosis . Propranolol raises G2-block of chalone or adrenaline . Consequently this way of chalones action on cell division includes beta-adrenergic receptors influence of preparation on cell motion in mitosis doesn't change with addition of propranolol . Consequently this way of chalone system action on mitosis doesn't include beta-adrenergic receptors.

Avian Dis, 1991 Jul-Sep, 35(3), 579 - 84
Pathogenesis of marble spleen disease in bursectomized and non-bursectomized ring-necked pheasants following oral inoculation with cell-culture-propagated virus; Fitzgerald SD et al.; Seventy-two 13-week-old ring-necked pheasants were inoculated orally with 5.0 x 10(2) tissue-culture infective dose (TCID) of cell-culture-propagated marble spleen disease virus . Inoculated birds exhibited neither mortality nor clinical disease . Gross and histologic lesions were typical of marble spleen disease . The mean splenic weight was significantly (P less than 0.02) higher in inoculated birds than in controls between 6 and 10 days postinoculation (PI) . The histologic splenic lesions, which consisted of reticuloendothelial cell hyperplasia, intranuclear inclusions within reticuloendothelial cells, and lymphoid depletion, were most prominent between 6 and 10 days PI . In a second experiment, 1-day-old pheasants were chemically bursectomized by dosing birds with 1.2 mg cyclophosphamide on 3 consecutive days . At 7 weeks of age, 54 bursectomized birds were inoculated orally with 5.0 x 10(2) TCID of marble spleen disease virus . Gross and histologic lesions were detected in one of the inoculated pheasants, but the mean splenic weight was not significantly different from control birds at any time PI . These results are evidence of the role of the bursa of Fabricius in the pathogenesis of marble spleen disease.

Infect Control Hosp Epidemiol, 1991 Jul, 12(7), 435 - 8
Efficacy of glove combinations in reducing cell culture infection after glove puncture with needles contaminated with human immunodeficiency virus type 1; Johnson GK et al.; OBJECTIVE: To study the effect of various latex and treated glove combinations in reducing the frequency of human immunodeficiency virus (HIV) infection of tissue culture cells after puncture by surgical needles contaminated with infectious human immunodeficiency virus type 1 (HIV-1) . DESIGN: One, two, or three layers of sterile latex glove material, or two latex layers with intermediate cotton or Kevlar (with or without the virucidal compound nonoxynol-9) were used to cover 24-well cell culture dishes containing MT2 cells in cell culture medium . Surgical needles wet with cell culture medium containing HIV-1 (HTLV IIIA strain) were passed through the glove materials into the culture medium in the wells of the culture dishes . The culture medium in each well was then assayed biweekly for HIV-1 p24 antigen as a test for infection of cells in the well . RESULTS: The rate of HIV-1 infection of cell cultures after glove puncture was greater than 90% with a single latex surgical glove barrier, 23% to 60% with double or triple layers of latex gloves, less than 8% with an intermediate cotton glove impregnated with 4% nonoxynol-9, 6% with an intermediate Kevlar glove, and 0% with an intermediate Kevlar glove impregnated with nonoxynol-9 . CONCLUSIONS: An intermediate glove of Kevlar or of Kevlar or cotton impregnated with virucidal compound nonoxynol-9 between standard latex gloves may improve surgical glove safety, compared with latex gloves alone with respect to needlestick transmission of HIV-1 . The experimental model used may permit rapid investigation of other glove systems as barriers to the transfer of infectious agents through gloves by needlestick.

J Gen Virol, 1991 Jul, 72 ( Pt 7), 1677 - 83
Simian hepatitis A virus (HAV) strain AGM-27: comparison of genome structure and growth in cell culture with other HAV strains; Tsarev SA et al.; Fragments of cDNA representing greater than 99% of the entire genome of wild-type hepatitis A virus (HAV) strain AGM-27, isolated from an African green monkey, were obtained by the polymerase chain reaction and sequenced . Comparison with other HAV isolates revealed differences in the predicted amino acid sequence in functionally critical parts of the genome . Comparison of the biological properties of AGM-27 with those of human wild-type and cell culture-adapted HM-175 strains revealed that AGM-27 grew in cell culture significantly better than did wild-type HM-175, but not as well as cell culture-adapted HM-175 . AGM-27 and cell culture-adapted HM-175 were distinguishable by their differential growth in CV-1, FRhK-4 and primary AGMK cells.

J Virol, 1991 Jul, 65(7), 3949 - 53
Antigenic stability of foot-and-mouth disease virus variants on serial passage in cell culture; Gonzalez MJ et al.; Two neutralizing monoclonal antibody (MAb)-resistant variants selected from an isolate of foot-and-mouth disease virus (FMDV) type A5 were repeatedly passaged in cell culture and monitored for susceptibility to neutralization by the selecting MAb . A variant isolated with a MAb to a conformational epitope (1-OG2) lost resistance in 20 passages, while a variant isolated with a MAb to a linear epitope (1-HA6) persisted for 30 passages . In both cases, the virus population emerging after passage was antigenically and genetically indistinguishable from the original wild-type parental virus (FMDV A5 Spain-86) . Coinfection assays with the wild type and each variant, and between the variants, showed rapid conversion to a homogeneous population . Wild-type virus prevailed over the variants and for coinfection between the variants, the linear epitope variant 1-HA6 . While both variants arose from a single nucleotide substitution and reversion to wild type occurred for each, it appears that the variant based on the continuous epitope (1-HA6) was more stable . We discuss the implications of these results for the antigenic diversity of FMDV and its relationship to virus evolution.

FEBS Lett, 1991 Jun 24, 284(2), 285 - 7
Nucleosomes occurring in protein-free hybridoma cell culture . Evidence for programmed cell death; Franek F et al.; In addition to monoclonal immunoglobulin, two kinds of nucleoproteins, NP1 and NP2, were isolated from the supernatants of hybridoma cultures set up in a protein-free medium . As shown by SDS-electrophoresis the two nucleoproteins shared a set of proteins (apparent Mr 11,000 to 15,000), and differed in the DNA moiety (approximately 150 bp in NP1, approximately 350 bp in NP2) . The amino acid composition of the protein moiety confirmed the nucleosomal origin of NP1 and NP2 . The findings support the view that in hybridoma cultures the cells undergo death by apoptosis, i.e . a programmed process characterized by initial fragmentation of chromatin.

Presse Med, 1991 Jun 22, 20(24), 1121 - 3
{Meningoradiculitis after injection of an antirabies vaccine . A vaccine from human diploid cell culture}; Moulignier A et al.; We report the case of a 45-year old farmer who developed meningoradiculitis after preventive anti-rabies vaccination with a vaccine obtained from human diploid cell culture . Two weeks after the second injection of vaccine, the patient complained of sensory symptoms in the right half of his body . These symptoms spontaneously regressed . The literature is reviewed and the physiopathological hypotheses are discussed.

Brain Res Dev Brain Res, 1991 Jun 21, 60(2), 123 - 32
Differentiation and survival of rat olfactory epithelial neurons in dissociated cell culture; Chuah MI et al.; Olfactory epithelial neurons in mammals are unique in that they continue to differentiate from precursor cells in the adult . These neurons extend long axons into the olfactory bulb . Previous attempts to grow these cells in dissociated cell cultures at low density, have not been entirely satisfactory . We report that when plated at very low density, rat olfactory epithelial neurons will differentiate morphologically and biochemically when cultured on astrocytes, but not on non-cellular substrata, such as polylysine, laminin or fibronectin . We demonstrate with antibodies, that these olfactory epithelial neurons require N-CAM, N-cadherin and L1 for neurite extension . Furthermore, synthetic cadherin-peptides containing the tripeptide HAV which is found in the first extracellular domain of N-cadherin, as well as the amino acids flanking this region, appear to be important in cadherin-mediated neurite growth on astrocytes . Astrocytes also appear to enhance the survival and differentiation of olfactory epithelial neurons from embryonic day 15 and 4-5 week post-natal rats, but this effect is not sustained beyond 5 days in cultures of postnatal epithelium.

Toxicol Appl Pharmacol, 1991 Jun 15, 109(2), 352 - 66
Chick embryo neural retina cell culture as a screen for developmental toxicity; Daston GP et al.; An in vitro screen for developmental toxic potential of chemicals using primary cultures of chick embryo neural retina cells is described . The neural retinas of incubation Day 6.5 White Leghorn chick embryos are dissociated into single cells, which are subsequently maintained in a rotating suspension culture . Under normal circumstances, neural retina cells form spheroidal aggregates of a consistent size over the first 24 hr of culture, an event which is dependent on competent cell-cell interactions . Over the remaining 7-day period of culture, cells continue to divide and grow, and differentiation takes place . Each of these developmentally important events--aggregation, growth, and differentiation--is objectively and quantitatively measured as aggregate size and number, aggregate protein content, and glutamine synthetase (a marker of differentiation) activity, respectively . The effects on each developmental endpoint of 22 chemicals, 14 of which have been demonstrated to be developmentally toxic in one or more mammalian species in vivo, and 8 of which are not developmentally toxic, were evaluated . Chemicals were tested up to a concentration of 40 mM, or until marked cytolethality was observed . Of the known developmental toxicants, all but one, 2-methoxyethanol, affected one or more endpoints in the assay . The teratogenic metabolite of 2-methoxyethanol, 2-methoxyacetic acid, was active in the assay . None of the 8 nondevelopmental toxicants had any effect up to a concentration of 40 mM, or at biologically achievable concentrations (e.g., in vivo systemic concentrations at the LD50) . Thus, the assay is 95% concordant with in vivo results for this set of chemicals . Quantitative comparisons were made (1) between developmentally toxic ip dosages in rats or mice in vivo and effective concentrations in the chick retina cell culture, and (2) between effective concentrations in chick retina cell culture and rodent whole embryo culture . In the first instance, 71% of the comparisons, and in the second instance, 89% of the comparisons, were within the same order of magnitude (and usually within a factor of two), indicating that the chick retina cell culture is also concordant with developmental toxic potency . Last, it was observed that test agents differentially affect developmental endpoints . Because the assay's endpoints are measured separately and objectively, it may be possible to use the assay to evaluate the effects of test agents on cellular development.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytotechnology, 1991 Jun, 6(2), 121 - 30
Selective removal of ammonia from animal cell culture broth; Nayve FR Jr et al.; Serum-free perfusion cultures of hybridoma TO-405 cells were carried out in spinner flasks coupled with zeolite A-3 packed beads . Ammonia was selectively removed from the culture broth by passing cell free permeate from ceramic cross flow filtration, through the zeolite packed bed . Ammonia concentration in the culture broth was effectively maintained between 1 to 4 mmol/l which was below the inhibitory concentration for cell growth . Maximum cell density levels of 10(7) cells/ml as well as improved percentage cell viability higher than in serum-supplemented cultures were feasible in this system . The possible effects of shear stress, generated by variation of the flow rates of the broth through the ceramic filter module, on the growth of the hybridoma cells were investigated . Backwashing, by reversing the direction of the permeate, was found necessary to prolong the life of the filter . Variation of the flow rates of the broth through the ceramic module between 0.29 m/s to 0.59 m/s did not cause immediate cell damage but growth was repressed at the higher flow rate . This study also showed that glutamine appears to be one of the factors limiting the growth of the hybridoma cells.

FEBS Lett, 1991 Jun 3, 283(2), 295 - 7
Energetic cooperation via ion-permeable junctions in mixed animal cell cultures; Aslanidi KB et al.; Low ouabain concentration (1 x 10(-6) M) is shown to decrease intracellular K+ (K+in) and to increase intracellular Na+ (Na+in) in human fibroblast cell cultures . The same ouabain concentration was without effect upon K+in ad Na+in in rodent cultures such as BHK-21, mouse fibroblasts and rat glyoma C6 cells . K+in and Na+in in the mixed cultures of human and BHK-21 fibroblasts or human and mouse fibroblasts were found to be resistant to 1 x 10(-6) M ouabain whereas that of the mixtures of human and rat glyoma C6 cells proved to be ouabain-sensitive . The gap-junction-mediated dye transfer was revealed between human and BHK-21 cells . Such an effect was very small in the human-C6 cell mixed culture . It is concluded that cells with active ion pumps can support the maintenance of K+ and Na+ gradients in cells with inactive pumps, provided that effective ion transport via gap junctions takes place.

J Immunol Methods, 1991 Jun 3, 139(2), 257 - 63
Sensitive one-step enzyme immunoassay for HIV-1 p24 antigen in human blood specimens and cell culture supernatants; Kontio S; An enzyme immunoassay (EIA) with a single incubation step has been developed to detect the HIV-1 major core polypeptide p24 in human blood specimens and in cell culture supernatants . In this assay, the solid phase is coated with monoclonal antibodies to p24, and the specimen is incubated simultaneously with peroxidase labelled polyclonal anti-HIV(Fab') . If HIV-1 p24 antigen is present in a specimen it will form a sandwich complex with the capture and tracer antibodies during the incubation step . The sensitivity of the assay for p24 antigen, 20 pg/ml, was equal to that of two commercial HIV antigen detection EIAs, when human blood specimens and virus isolation cell culture supernatants were analyzed . However, the assay described is simpler to perform than those previously reported, since only one incubation step is needed . Moreover this assay minimizes the handling of material containing potentially infectious HIV.

J Immunol Methods, 1991 Jun 3, 139(2), 191 - 5
Evaluation of a test system for measuring cytokine production in human whole blood cell cultures; Elsasser-Beile U et al.; A simple and reproducible method is described for the measurement of mitogen-induced cytokine production in cultures of both human peripheral blood mononuclear cells (PBMC) and whole blood . In the culture supernatants the cytokines interleukin-1 alpha (IL-1 alpha), interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) were determined by a rapid and sensitive immunoassay using various monoclonal and polyclonal antibodies . Comparing the PBMC cultures with the whole blood system a good correlation was obtained if the cell number was taken into account . In the post-induction supernatants the cytokine values were found to follow typical kinetic curves . The protocol was evaluated by screening 60 cancer patients with primary disease and 60 healthy controls . A markedly reduced secretion of IFN-gamma and IL-1 alpha was found in the cancer patients compared to controls, although leukocyte and lymphocyte counts were almost identical in both groups.

J Endocrinol, 1991 Jun, 129(3), 417 - 22
Immunocytochemical and morphometric studies of mammotrophs, somatotrophs and somatomammotrophs in sheep pituitary cell cultures; Thorpe JR et al.; Alterations occurring when sheep anterior pituitary cells are placed in culture for 4 days were studied using electron microscopy and immunogold labelling . The majority of cells present showed marked morphological changes during culture, with degranulation and development of extensive rough endoplasmic reticulum . The proportions of somatotrophs and mammotrophs, as identified by immunogold labelling, fell during culture . The majority (70%) of somatotrophs showed relatively little degranulation but the remainder were extensively degranulated after 4 days in culture, suggesting two subpopulations of this cell type . Conversely, most (80%) of the mammotrophs showed extensive degranulation after culture, but one-fifth remained heavily granulated, suggesting that mammotrophs too are heterogeneous . The proportion of cells labelling for both GH and prolactin (somatomammotrophs) increased during culture to about 3% of the cells present, compared with less than 0.2% of all cells before culture.

J Cell Physiol, 1991 Jun, 147(3), 427 - 33
Replacement of media in cell culture alters oxygen toxicity: possible role of lipid aldehydes and glutathione transferase in oxygen toxicity; Sullivan SJ et al.; Replacement of media in cell cultures during exposure to hyperoxia was found to alter oxygen toxicity . Following 100 hr of exposure to 95% or 80% O2, the surviving fraction (SF) of Chinese hamster fibroblasts, as assayed by clonogenicity, was less than 1 x 10(-3) when the culture media was replaced only at the onset of the O2 exposure . Media replacement every 24 hr throughout the hyperoxic exposure resulted in SFs of 1.7 x 10(-1) (95% O2) and 1.9 x 10(-1) (80% O2) at 95 hr . Cellular resistance to and metabolism of 4-hydroxy-2-nonenal (4HNE), a cytotoxic byproduct of lipid peroxidation, was examined in cells 24 hr following exposure to 80% O2 for 144 hr with media replacement . These O2-exposed cells were resistant to 4HNE, requiring 2.6 times as long in 80 microM 4HNE to reach 30% survival as compared to density-matched normoxia control . Furthermore, during 40 and 60 min of exposure to 4HNE, the O2-preexposed cells metabolized greater quantities of 4HNE (fmole/cell) relative to control . The activity of glutathione S-transferase (GST), an enzyme believed to be involved with the detoxification of 4HNE, was significantly increased in the O2-preexposed cells compared with controls . Catalase activity was significantly increased, but no change was found in total glutathione content, glutathione peroxidase, manganese superoxide dismutase, and copper-zinc superoxide dismutase activities at the time of 4HNE treatment in the O2-preexposed cells relative to density-matched control . The results demonstrate that in vitro tolerance to the cytotoxic effects of hyperoxia can be achieved through media replacement during O2 exposure . Tolerance to oxygen toxicity conferred resistance to the cytotoxic effects of 4HNE, possibly through GST-catalyzed detoxification . These results provide further support for the hypothesis that toxic aldehydic byproducts of lipid peroxidation contribute to hyperoxic injury.

J Dent Res, 1991 Jun, 70(6), 1014 - 8
The release of elements of dental casting alloys into cell-culture medium; Wataha JC et al.; Ten dental casting alloys were tested for alloy-element release into cell-culture medium, and this release was related to alloy composition, alloy microstructure, and alloy cytotoxicity (previously determined) . Cell-culture medium was analyzed for alloy elements by flame atomic absorption . Concentrations of elements in the medium were normalized by dividing them by their atomic abundance in the alloy, giving element medium-alloy ratios (EMA ratios) . Results showed that Au, In, and Pd generally did not dissolve into the medium, but that Ag, Cd, Cu, Ga, Ni, and Zn frequently dissolved . Comparison of EMA ratios for Ag, Cu, and Zn showed that each element retained a behavioral identity in diverse metallurgical environments, but that these environments influenced the release behavior to some degree . Some EMA ratios in multiphase alloys were greater than those in solid solutions, and EMA ratios showed great diversity within all the alloys . Nominal composition seemed to be of little value in the prediction of metal release unless the composition supported multiple-phase formation . In addition, release of alloy elements did not, in itself, completely predict alloy cytotoxicity measured previously . However, cytotoxicity was associated with metal release in each case . The commercial alloys used in this study exhibited more complex and less predictable release behavior than did the simpler ternary alloy systems used by previous investigators . It is believed that the use of commercial preparations is necessary for their in vivo behavior to be modeled.

J Bone Joint Surg Am, 1991 Jun, 73(5), 647 - 58
Synthesis of chondrocytic keratan sulphate-containing proteoglycans by human chondrosarcoma cells in long-term cell culture; Block JA et al.; Keratan sulphate is an integral component of the large aggregating proteoglycans of mature human articular cartilage . The keratan sulphate content of chondrocytic proteoglycans increases during maturation, and it is a useful marker of mature-type chondrocytic proteoglycans . Ordinarily, in cell culture, chondrocytes from non-neoplastic tissues dedifferentiate, diminish or cease to synthesize aggregating proteoglycans with the same amount of keratan sulphate as those formed in vivo, and do not maintain their in vivo phenotype . In tissue culture, this down-regulation of synthesis of keratan sulphate is irreversible . The study of the metabolism of mature human chondrocytes has been hampered by the absence of stable models . We report a cell-line, 105KC, derived from a human chondrosarcoma, that has maintained a stable proteoglycan phenotype during more than three years of culture . Analysis with immunofluorescence suggested that 105KC cells continued to synthesize keratan sulphate in long-term culture . Biochemical analysis demonstrated that 105KC cells maintained the production of chondrocytic large-aggregating proteoglycans and that keratan sulphate composed 13 per cent of their glycosaminoglycan content . To our knowledge, 105KC represents the first model to have maintained the post-fetal chondrocytic proteoglycan phenotype in stable culture . This study documents the feasibility of the development of mature chondrocytic cell-lines and sheds light on the biological characteristics of chondrosarcomas.

Am J Clin Pathol, 1991 Jun, 95(6), 765 - 8
Enhanced isolation of influenza virus in conventional plate cell cultures by using low-speed centrifugation from clinical specimens; Seno M et al.; To determine whether low-speed centrifugation as a means of amplifying viral adsorption to target cells can enhance the sensitivity of conventional techniques for the isolation of influenza virus from clinical specimens, the authors conducted a simultaneous comparison between two conventional plate cell cultures--one with and one without centrifugation (700 X g, 60 minutes) . Of 26 influenza virus isolates obtained from 528 clinical specimens, 17 were more efficiently isolated by the centrifugation assay compared with conventional culture methods . Centrifugation-assisted methods were found to be efficient in the recovery of 17 of 20 isolates (85%) not identified on first passage by conventional techniques . The number of isolates obtained with centrifugation was 1.5 times as high as that obtained with no centrifugation after three passages . The two assays were significantly different in the isolation of influenza virus (P less than 0.01) . Since low-speed centrifugation may increase the infectivity of influenza virus in cell culture, this technique may prove useful in the efficient and rapid isolation of this virus from clinical specimens.

Oralprophylaxe, 1991 Jun, 13(2), 55 - 60
{Comparative studies of toothpastes and toothpaste ingredients in biological systems: 1 . Can various toothpastes be differentiated by relative biological effectiveness in cell culture studies?}; Gerckens B et al.; The cytotoxicity of 15 commercial toothpastes was examined in human epithelial cell cultures . The following parameters were evaluated: protein concentration, DNA-synthesis, cell morphology and autoradiography . In comparison to the control cell culture, one toothpaste had no influence on the cell growth at all . The other pastes caused a more or less strong growth inhibition, which also depended on the concentration of the toothpaste suspension . The presented investigations showed that the cytotoxicity tests applied to toothpastes yielded a good validity.

Rev Sci Tech, 1991 Jun, 10(2), 499 - 511
Systematic morphogenesis of the viral haemorrhagic disease virus in infected rabbits and in adapted cell culture; Gong XM et al.; The morphogenesis of viral haemorrhagic disease virus (VHDV) of rabbits in infected cell cultures and in tissues and cells from infected animals has been observed systematically by electron microscopy . Viral particles have been found to assemble through a condensation process within the nucleus of infected cells, having passed partially into the cytoplasm through disrupted nuclear membranes, enlarged nuclear pores or perinuclear space . Late in infection, both mature and immature virions are observed in the cytoplasm; these are often aggregated or interspersed and remain cell-associated long after infection . Release of the virus occurs when the cell finally lyses . In studies of the localisation and development of VHDV antigens, the viral antigens have been observed to appear first in the nucleus of infected cells and often thereafter in the cytoplasm . This finding is very similar to observations made by electron microscopy.

Brain Res Mol Brain Res, 1991 Jun, 10(3), 235 - 40
Spontaneous electrical activity regulates vasoactive intestinal peptide expression in dissociated spinal cord cell cultures; Agoston DV et al.; Activity-dependent expression of vasoactive intestinal peptide (VIP) was investigated in spinal cord/dorsal root ganglia cultures derived from embryonic mice . Since all spinal cord neurons appear to exhibit spontaneous action potentials after one week in vitro, activity-dependent regulation of VIP-transcripts (mRNAVIP) could be studied with or without electrical blockade induced by tetrodotoxin (TTX) . In 10-day-old cultures, a 50% decrease in mRNAVIP was observed after 3 days of treatment with TTX . The decrease in mRNAVIP was reversed upon removal of the TTX and was dependent on the age of the cultures: no decreases from control were observed in 5-day-old cultures and much smaller decrements were produced in one month old cultures treated with TTX . A variety of neuroactive substances were tested for effects on mRNAVIP in electrically active and electrically blocked cultures . Application of 8-bromo-cAMP (cAMP), N-methyl-D-aspartate (NMDA), substance P, muscimol, A23187 and VIP to electrically active cultures resulted in a 2- to 3-fold increase in mRNAVIP, while phorbol myristate 13-acetate (PMA) and 8-bromo-cGMP (cGMP) had no effect . In contrast, electrically inactive cultures exhibited a 3 to 4-fold increase in mRNAVIP after treatment with PMA, cAMP and VIP, while NMDA, substance P, muscimol, A23187 and cGMP produced no increases . In summary, the regulation of VIP gene expression in embryonic spinal cord neurons shows a temporal sensitivity to TTX-induced electrical blockade and may be mediated by multiple neurotransmitter inputs which converge on cAMP- and calcium-related processes in an activity-dependent manner.

Curr Opin Biotechnol, 1991 Jun, 2(3), 370 - 4
Large-scale plant cell culture: methods, applications and products; Bisaria V et al.; Recent significant contributions to the design of large-scale plant cell cultures for secondary metabolite production include: the development of a strategy to control the concentration of dissolved gases at constant shear; a surface immobilization technique to retain cell mass at high mixing rates; and a modified stirred-tank reactor with a mesh cage to prevent damage of hairy root cultures.

Brain Res, 1991 May 10, 548(1-2), 322 - 5
Inhibition of Na+/Ca2+ exchange enhances delayed neuronal death elicited by glutamate in cerebellar granule cell cultures; Andreeva N et al.; Experiments have been carried out on the primary cerebellar granule cell cultures from 7- to 8-day-old Wistar rats . To study a possible contribution of Na+/Ca2+ exchange to the toxic effect of glutamate, two amiloride derivatives, 3',4'-dichlorobenzamil (DCB) and 5-(N-4-chlorobenzyl)-2',4'-dimethylbenzamil (CBDMB), known to be the potent inhibitors of this exchange system, were used . Addition of DCB or CBDMB (at 30 and 10 microM, respectively) to a 25 microM glutamate solution dramatically enhanced the delayed neuronal death observed during the 4 h after termination of glutamate treatment . Similar but insignificantly smaller effects were obtained when these agents were added to the cultures in the post-glutamate period . Removal of Na+ (by substituting for choline chloride) from the external Mg2+-free solution in the post-glutamate period also enhanced a delayed neuronal damage . The data obtained suggest that Na+/Ca2+ exchanger does not constitute the route for Ca2+ entry during the post-glutamate period but, on the contrary, attenuates glutamate neurotoxicity providing Ca2+ extrusion from the cells under the conditions of a sustained Ca2+ influx.

J Immunol Methods, 1991 May 17, 139(1), 135 - 44
Screening of various mammalian cell culture media to establish a downstream purification scheme; Graf H et al.; Various serum-free media for use in hybridoma cell cultures are reviewed and their protein composition analysed . The chromatographic behaviour of the serum-free components was analysed on ion exchange, hydrophobic and sizing columns in order to facilitate the design of a purification scheme for monoclonal antibodies produced from various cell cultures.

Brain Res, 1991 May 17, 549(1), 106 - 11
Differential expression of protein kinase C isozymes in rat glial cell cultures; Masliah E et al.; Protein kinase C (PKC) is a family of closely related enzymes implicated in molecular processes involved in growth and differentiation in a variety of cells . We studied the presence and distribution of 4 PKC isozymes in glial cell cultures of the rat hippocampus employing antisera raised against synthetic peptides predicted from the cDNA sequences corresponding to the C-terminal portion of 4 PKC isoforms, alpha, beta I, beta II, and gamma . PKC(alpha) and -(beta II), but neither PKC(beta I) nor -(gamma) isoforms were detected in glial cultures of the rat hippocampus . Anti-PKC(alpha) immunostained all glial cells, whereas anti-PKC(beta II) faintly stained about 20% of total glial cells resembling the type-2 astrocyte that were GFAP immunopositive, with few processes . Anti-PKC(beta II) did not stain about 80% of the glial fibrillary acidic protein (GFAP)-immunopositive cells with a few thick processes which resembled the type-1 astrocyte . A few cells that stained intensely with anti-PKC(beta II) were GFAP immunopositive and possessed fine, but well-developed, multiple processes . Faint PKC(beta II) immunoreactivity was also detected among anti-MBP-positive cells (possibly oligodendrocytes), RCA-1-positive cells (possibly microglia), and small, oval, anti-GFAP-positive cells . These results suggest the involvement of distinct PKC isoforms in different glial functions.

J Chromatogr, 1991 May 10, 543(2), 463 - 70
Separation and microanalysis of growth factors by Phast system gel electrophoresis and by DNA synthesis in cell culture; Kuo KW et al.; A simple, micro-scale method was established for the characterization of growth factors at picogram levels using Phast system gel electrophoresis followed by monitoring the mitogenic activity by DNA synthesis in cell culture instead of staining methods . The separations and bioassays were carried out with a procedure involving Phast polyacrylamide gel electrophoresis or isoelectric focusing, gel slicing along the template, elution of growth factors through Transwell membranes and measurement of {3H}thymidine incorporation into DNA of normal rat kidney (NRK) fibroblasts . Transwell cell culture chamber inserts separated sliced gel pieces from culture cells and also permitted the direct elution of growth factors into the culture medium . The lower limit of sensitivity for human epidermal growth factor (hEGF) and transforming growth factor type alpha (TGF-alpha) were about 50 and 200 pg, respectively . At these concentrations, they were not detectable by the current most sensitive silver staining technique . Iodinated hEGF and TGF-alpha were also used to demonstrate the feasibility of determining the isoelectric point and molecular weight of peptides at picogram levels . This method is reliable, reproducible and can improve current methods for the characterization of growth factors.

Biochim Biophys Acta, 1991 May 2, 1089(1), 83 - 7
Recombinant human interleukin-1 suppresses lipoprotein lipase activity, but not expression of lipoprotein lipase mRNA in mesenchymal rat heart cell cultures; Friedman G et al.; The effect of human recombinant interleukin-1 (IL-1) on the regulation of lipoprotein lipase (LPL) was studied in rat heart mesenchymal cell cultures . A time-dependent reduction in enzyme activity occurred with a 30% fall after 1 h . The suppression of enzyme activity was accompanied by a commensurate reduction in enzyme mass . The reduction in LPL activity was most prominent in the heparin releasable pool; IL-1 treatment resulted in a 7.2-8.3-fold decrease in the functional compartment and a 2.5-2.8-fold decrease in residual cellular activity . The effect of IL-1 could be prevented by the addition of the IL-1 inhibitor . However, in contradistinction to the effect of tumor necrosis factor (TNF), there was no change in LPL mRNA in cultures treated with IL-1 . The present results show that the regulation of LPL in mesenchymal heart cell cultures by IL-1 occurs posttranscriptionally, as has been shown in 3T3 cells . The more pronounced effect on LPL activity in the functional pool suggests that IL-1 treatment might have influenced also the processing and/or transport of the enzyme to the cell surface.

Cancer Genet Cytogenet, 1991 May, 53(1), 57 - 66
Selection of low frequency tumor cells from cell culture by growth in nude mice; Nichols WW et al.; Tissue cultures of tumor cells are frequently utilized to characterize chromosomal changes when direct cytogenetic preparations on tumors fail . The present study demonstrates that chromosomal markers found in direct tumor preparations can become undetectable in cell culture at variable rates presumably because of overgrowth of normal cell components in the culture . Injection of cultured tumor cells into nude mice followed by direct chromosomal preparations on the resulting nude mouse tumors can be used to select cells with the original tumor karyotype . This is true even when the tumor cell frequency in the culture is so low that they are not found in routine chromosomal preparations of the cultured cells . This technique can thus complement tissue culture findings and provide additional useful information about the original karyotype in cases where direct chromosomal preparations from tumors have failed.

Mol Biol (Mosk), 1991 May-Jun, 25(3), 624 - 32
{The effect of modifying terminal oligonucleotide linkages on their stability in cell cultures}; Abramova TV et al.; The effect of modification of terminal groups of deoxyribooligonucleotides on their stability in cell culture and inside mammalian cells, namely Krebs 2, ascite carcinoma (KAC) and mouse fibroblasts L929, has been investigated . Oligonucleotides and their derivatives were found to be stable in culture medium without serum during 24 h . In the medium with KAC cells or ascitic fluid, orthophosphate was rapidly eliminated from the 5'-terminus of the oligonucleotides . In KAC cells, the scission of 5'-phosphomonoester bonds was accompanied by reutilization of the phosphate and by degradation of oligonucleotides to mononucleotides . In the medium with fibroblasts L929, the oligonucleotides were degraded from the 3'-end to tetranucleotides . Modification of oligonucleotides at the 5'-terminus by amidation made the 5'-phosphate groups resistant to KAC . Modification of the oligonucleotides by coupling of cholesterol or phenazinium to the 3'-terminus sufficiently increases their stability in the medium with fibroblasts L929, in that with Krebs 2 ascite carcinoma cells and inside the cells.

Zhonghua Yan Ke Za Zhi, 1991 May, 27(3), 169 - 71
{An experimental study on the primary cell culture of human trigeminal ganglion}; Xie LX; A primary cell culture of human fetal trigeminal ganglion in vitro was first established . The growth features and morphology of the three types of cells in the trigeminal ganglion were described . The neurons in the culture were identified with the immunoperoxidase technique . A new model for studying the latent infection of herpes simplex virus type 1 in human is provided.

Anticancer Res, 1991 May-Jun, 11(3), 1093 - 5
Evaluation of cytokine levels in whole cell cultures of patients with gynaecological tumors as diagnostic parameters; Elsasser-Beile U et al.; The measurement of the cytokine production of activated lymphocytes and monocytes supposedly provides a tool for evaluating the actual status of the cellular immunity potential of a person . We measured the levels of IFN-gamma and IL-1-alpha in the 4 day post-induction supernatants of mitogen-stimulated unseparated blood cell cultures by a rapid and sensitive immunoassay using various monoclonal and polyclonal antibodies . With this reproducible and easy-to-handle system we tested 90 patients with primary gynaecological tumors and the same number of age-matched female controls . In the blood cell cultures of the tumor patients significantly lower values of IFN-gamma and IL-1-alpha were observed, although lymphocyte and monocyte counts were almost normal . There was also a difference in the IFN-gamma-levels between the four tumors, in as much as patients with ovarian and endometrial carcinomas had significantly lower values than patients with breast and cervical carcinomas.

Vopr Virusol, 1991 May-Jun, 36(3), 231 - 5
{The Mopeia virus induces pH-dependent "fusion from within" in a BHK-21 cell culture}; Glushakova SE et al.; The capacity of BHK-21 cell culture to produce Mopeia virus (Arenaviridae family) to form syncytium upon acidification of the culture medium to pH 5.5 and lower was demonstrated . The cell fusion requires their active virus production: a virus titre in the culture medium must be at least 10(5) PEU/ml . The inhibition of virus multiplication with ammonium chloride as well as treatment of the cells before the medium acidification with immune serum reduced syncytium formation markedly . No cell fusion was observed upon acidification of the medium immediately after virus adsorption to cells . Thus, the observed cell fusion under the influence of the virus is an "internal fusion" and confirms our previous data on the endocytosis mode of arenavirus penetration into the cell.

Virology, 1991 May, 182(1), 135 - 44
Analysis of spliced and unspliced Rous sarcoma virus RNAs early and late after infection of chicken embryo fibroblasts: effect of cell culture conditions; Berberich SL et al.; Quantitative determinations of spliced and unspliced viral RNA early after retrovirus infection or after transient transfection with proviral DNA have been difficult because of low intracellular viral RNA levels . In this report we describe conditions for the sensitive and quantitative assay of unspliced viral RNA and spliced mRNAs from avian sarcoma virus-infected and transiently transfected chicken embryo fibroblasts . RNase protection mapping was performed with a tandem 32P-labeled riboprobe consisting of three separate regions complementary to the major 5' splice site and the two major 3' splice sites (env and src) . The steady-state levels of viral RNA species were determined as early as 14 hr postinfection; no significant changes were observed in the relative levels of spliced mRNAs or in splice site usage as the infection progressed from 14 to 48 hr . After the cells became morphologically transformed at approximately 72 hr postinfection, the condition of the medium of the transformed cell culture affected the steady-state levels of viral RNAs . Unspliced genomic RNA predominated in the transformed cells at 4.5 to 8 hr after feeding; spliced env mRNA was the major species of RNA at 46 to 103 hr after medium change . In contrast, the steady-state levels of viral RNA in transiently transfected cells or in cells infected with a transformation-defective avian sarcoma virus were not as sensitive to the conditions of medium change . The medium-dependent changes in steady-state levels of viral RNA species may either be caused by a declining transcription rate resulting in the accumulation of the more stable env mRNA relative to the more labile unspliced RNA and src mRNA or to changes in the extent of viral RNA splicing.

J Virol, 1991 May, 65(5), 2724 - 7
An antigen encoded by the latency-associated transcript in neuronal cell cultures latently infected with herpes simplex virus type 1; Doerig C et al.; During latent infection of neurons with herpes simplex virus type 1, viral transcription is restricted to the latency-associated transcripts (LATs) . These RNAs contain open reading frames, but detection of a protein encoded by the LATs has not been reported . We used immunocytochemical techniques to demonstrate that an antiserum directed against a bacterially expressed fusion protein containing part of a LAT-encoded polypeptide recognized an antigen present in primary neurons latently infected in vitro . This antigen (called LAA, for latency-associated antigen) was not detected in mock-infected neurons, in productively infected Vero cells, or in neurons latently infected with a mutant virus carrying a deletion in the LAT gene . By Western immunoblot analysis, we demonstrated the presence of a protein with an apparent molecular mass of 80 kDa recognized by the anti-LAA antiserum in latently infected neurons.

J Virol, 1991 May, 65(5), 2699 - 701
Formation and selection of intergenogroup reassortants during cell culture adaptation of rotaviruses from dually infected subjects; Ward RL et al.; A previous study showed that intergenogroup reassortants of human rotaviruses can persist in nature (R.L . Ward, O . Nakagomi, D.R . Knowlton, M.M . McNeal, T . Nakagomi, J.D . Clemens, D.A . Sack, and G.M . Schiff, J . Virol . 64:3219-3225, 1990), but the mechanisms involved in their formation and selection had not been determined . In this study it was shown that, during cell culture adaptation of rotaviruses belonging to different genogroups from stools of dually infected subjects, intergenogroup reassortants were formed and selected, presumably mimicking the processes that occur in nature.

J Pharm Belg, 1991 May-Jun, 46(3), 197 - 200
{Cell cultures . Complementary or alternative methods to animal experimentation}; Guillot R; In vitro methods, which appeared at the beginning of the century, have had exponential development since one decade for ethical and technological reasons . They can be used for studies concerning intracellular, intercellular or environmental activities . Despite of limiting factors, these techniques offer certain advantages which have to be optimized in order to be validated . They are actually complementary, used for exploratory purposes . In the future, they could replace partially but not completely some in vivo experiments.

Mol Biol Rep, 1991 May, 15(2), 73 - 9
Plasmodium falciparum: recombinant baculoviruses direct the expression of circumsporozoite proteins in Spodoptera frugiperda cell cultures; Jacobs P et al.; The DNA coding for the circumsporozoite protein (CPS) of Plasmodium falciparum has been cloned into the baculovirus expression vector pAcYM1 and expressed in Spodoptera frugiperda (Sf9) insect cells . Three DNA constructs have been made: the first one directs the synthesis of the complete CSP (aa 1-412), the second leads to the production of a species devoid of the anchor domain (aa 1-391) and the third one to a molecule lacking both signal and membrane anchor sequences (aa 18-391) . All three recombinant CPS were produced at about 3 micrograms per 10(6) infected cells and were characterized in terms of immunoreactivity and apparent molecular weight . Analytical purification of the recombinant proteins was achieved by a combination of heat treatment, acidification, isoelectric focusing and ion exchange chromatography . The purified material, when injected into mice, generated only modest antibody responses, although antisera from immunized mice reacted with control CSP antigens carrying or not the major immunodominant repeat region.

Endocrinology, 1991 May, 128(5), 2489 - 96
Further characterization of insulin-like-growth factor binding proteins in rat osteoblast-like cell cultures: modulation by 17 beta-estradiol and human growth hormone; Chen TL et al.; Insulin-like growth factors (IGF-I and IGF-II) are endocrine and autocrine factors affecting bone growth and metabolism . Binding proteins for IGFs (IGFBPs) are synthesized by the target tissues of IGF actions . Thus, IGFBPs may act as modulators for the biological functions of IGFs . We have characterized the rat IGFBPs (rIGFBPs) and studied their regulation by 17 beta-estradiol (beta E2) and human GH (hGH) in rat osteoblast-like (ROB) cell cultures . ROB cells were prepared from 19- to 20-day fetal rat calvariae by sequential collagenase digestion and studies were performed on the serum and phenol red-free conditioned medium of confluent cultures . {125I}IGF-I ligand blots showed that the major rIGFBP in the ROB is a nonglycosylated protein of 31 kilodaltons . This protein was immunoprecipitated by a specific antibody to rIGFBP-2 and messenger RNA for rIGFBP-2 was detected by RNA hybridization indicating that the rIGFBP-2 is the major rIGFBP of ROB . A minor band at 24 kilodaltons is likely to be the rat homologue of the newly isolated inhibitory IGFBP-4 . The predominant glycosylated adult form of rIGFBPs of rat serum, rIGFBP-3, was undetectable . When cultures were treated with beta E2 for 2 days, there was a dose-dependent biphasic response which showed an inhibition of the rIGFBP-2 at low doses of 10(-11) to 10(-9) M and a stimulation at 10(-6) M . These changes in rIGFBP-2 parallel the changes in the endogenous IGF-I level . rIGFBP-2 level was not affected by 17 alpha-estradiol at the same concentration range . hGH, on the other hand stimulated the levels of rIGFBP-2 and rIGFBP-4 at doses ranging from 10(-11) to 10(-9) M without changing the IGF-I secretion . The alteration of the rIGFBPs by beta E2 and hGH suggests a role for these hormones in bone by modulating the biological functions of IGFs via their binding proteins.

J Histochem Cytochem, 1991 May, 39(5), 561 - 8
A double staining technique for simultaneous demonstration of astrocytes and microglia in brain sections and astroglial cell cultures; Castellano B et al.; We developed a double staining technique for simultaneous demonstration of astrocytes and microglial cells in histological brain sections and cell cultures . The procedure included a histochemical stain specific for microglial cells and an immunocytochemical stain specific for astroglial cells, with postponement of the final visualization of the staining products until both reactions had been performed . First, microglial cells were specifically but invisibly labeled by histochemical reaction for nucleoside diphosphatase (NDPase) . Then the astroglial cells were labeled by performing the first parts of the immunocytochemical reaction for glial fibrillary acidic protein (GFAP) . Finally, in a series of intervening steps, the NDPase reaction product was visualized and stabilized by treatment with ammonium sulfide and silver nitrate, while the 1-naphthol basic dye method was used to visualize the GFAP immunoreactive product . As an end product, the NDPase-positive microglial cells were brown and the GFAP-reactive astroglial cells blue . The two types of glial cells were clearly distinguishable in vibratome sections of rat brain tissue and in primary astroglial cell cultures, and we never observed cells that stained for both NDPase and GFAP . When the GFAP antibody was replaced by the OX-42 antibody, which recognizes microglial cells and macrophages, double staining of microglial cells was observed . The staining protocol has wide applications in studies of the functional interactions between microglial and astroglial cells in the normal brain and in different pathological states with neuronal or axonal degeneration, just as it can be used for experimental studies in cell cultures.

J Neurosci, 1991 May, 11(5), 1325 - 33
Dopamine content and metabolism in mesencephalic and diencephalic cell cultures: sex differences and effects of sex steroids; Beyer C et al.; Sexual differentiation of dopaminergic neurons was studied in gender-specific cultures . Dissociated cell cultures were prepared from di- or mesencephalon of gestational day 14 rat embryos and raised in the absence or presence of 17 beta-estradiol or testosterone for up to 13 days in vitro (DIV) . Developmental profiles of levels of dopamine (DA) and metabolites as well as capacity for vesicular storage of the transmitter were determined by HPLC . Tyrosine hydroxylase-immunoreactive (TH-IR) neurons were counted . Higher levels of DA were measured in female than in male cultures of both brain regions . In mesencephalic cultures, the differences in DA levels were fully accounted for by sex differences in numbers of TH-IR cells, whereas no sex differences in cell numbers were found in diencephalic cultures . Dihydroxyphenylacetic acid (DOPAC) levels and vesicular storage capacity matured faster in mesencephalic than in diencephalic cultures, but no sex differences were observed . Homovanillic acid (HVA) could not be detected except in 13-DIV mesencephalic cultures . Hormonal treatment did not erase sexual differentiation of dopaminergic neurons . Irrespective of the gender, however, both steroids decreased DA and DOPAC contents in diencephalic cultures but not in mesencephalic cultures . It is proposed that sexual differentiation of dopaminergic systems proceeds in a region-specific fashion and that neurogenesis and development of various parameters of dopaminergic activity may be differentially affected . Sexual differentiation of dopaminergic neurons may be initiated independently of the action of gonadal steroid hormones and may subsequently be modified by differences in hormonal environment.

Vopr Virusol, 1991 May-Jun, 36(3), 209 - 12
{The reproduction of the hepatitis A virus in cell cultures}; Farashian VR et al.; A comparative analysis of reproduction of 9 strains of hepatitis A viruses (HSA-15, CF-979, MI, MBB 11/5, H-141, KMW-1, GBM, IH-26, WR-61) from different regions of the world in cell cultures (PLC/PRF/5, HEL-240, FRhK-4, MK) revealed the differences in the capacity of the viruses for reproduction in these cell lines . The factors influencing HAV reproduction in cell cultures such as temperature, medium, and sera were studied . In one-cycle infection, accumulation of vRNA and formation of virus particles were analysed.

Brain Res Mol Brain Res, 1991 May, 10(2), 115 - 21
Regulation of proopiomelanocortin messenger RNA concentrations by opioid peptides in primary cell cultures of rat hypothalamus; l'Hereault S et al.; The effects of opioid peptides on a 1.1-kb long proopiomelanocortin messenger RNA (POMC mRNA) have been investigated in rat hypothalamic cells maintained in culture . Most opioid peptides exerted an inhibitory control on POMC mRNA steady-state concentrations . beta-Endorphin caused a 65% maximal inhibitory effect (IC50 = 6.1 x 10(-9) M) while slightly less inhibition was caused by Met- and Leu-enkephalin, dynorphin A and DADLE ({D-Ala2,D-Leu5} enkephalin) . The effects of beta-endorphin and of Met-enkephalin were completely reversed by the delta opioid antagonist ICI 174,864 while the kappa-receptor specific antagonist binaltorphimine or the sigma-receptor specific antagonist DTG (1,3-di(2-tolyl) guanidine) respectively blocked the inhibitory actions of dynorphin A and of DADLE . The mu-receptor specific agonist DAGO ({D-Ala2,N-Me-Phe4,Gly5-OL}enkephalin) did not affect POMC mRNA levels . The failure of the dopaminergic D2 antagonist haloperidol to modify the inhibitory effects of opioid peptides argues for a direct inhibitory opioid peptide modulation of hypothalamic POMC mRNA levels mediated by the delta-, kappa- and sigma- (but not mu-) receptors in vivo.

Hum Genet, 1991 May, 87(1), 6 - 10
The size of small polydisperse circular DNA (spcDNA) in angiofibroma-derived cell cultures from patients with tuberous sclerosis (TSC) differs from that in fibroblasts; Motejlek K et al.; Cell cultures were derived from angiofibromas of three patients with tuberous sclerosis (TSC), from the unaffected skin of these patients, and from the skin of five healthy donors . The length distributions of the small polydisperse circular DNA (spcDNA) fraction of these cell cultures were then analyzed . Nearly half the spcDNA molecules from the angiofibroma cultures were longer than 0.4 micron, whereas only about 7% exceeded this threshold in the spcDNA preparations from the skin fibroblast cultures . The percentage of the larger size class of spcDNA showed an increase at higher numbers of in vitro passages in all three types of cultures, but this effect was much more conspicuous in the angiofibroma-derived cultures than in those from the skin fibroblasts . An age-dependent increase in the overall amount of spcDNA was only seen in the angiofibroma-derived cultures . Our earlier finding of elevated amounts of spcDNA in angiofibroma cultures was confirmed in cultures from an additional TSC patient.

Cytotechnology, 1991 May, 6(1), 49 - 54
Primary cell culture from embryos of the Japanese scallop Mizuchopecten yessoensis (Bivalvia); Odintsova NA et al.; Primary cell cultures obtained from embryos of Mizuchopecten yessoensis (Bivalvia) survived for four months . Although the number of cells progressively decreased during the cultivation, mitotic cells were observed both at the first stages and at the end . A possibility of growing marine invertebrates cells in long term primary culture is discussed.

Neurosci Lett, 1991 Apr 29, 125(2), 101 - 6
Primary dissociated cell culture of embryonic rat metencephalon: presence of GABA in serotonergic neurons; Belin MF et al.; This study was performed to determine whether neurons, where gamma-aminobutyric acid (GABA) and serotonin (5-HT) coexist, represent neuronal entities which can survive in vitro . In dissociated cultures from 18-day-old embryonic rat metencephalon, it was possible to develop glial and neuronal cells . Among the neurons, some of them, which contain glutamate decarboxylase or are capable of accumulating {3H}GABA are GABAergic; others, containing tryptophan hydroxylase or 5-HT are serotoninergic . By combining radioautography and immunocytochemistry, it was possible to observe neurons where 5-HT and GABA coexist . Cultures might be a suitable model to study the functioning (release or synthesis of both neurotransmitters) of neurons where two classical neurotransmitters coexist.

Biochim Biophys Acta, 1991 Apr 26, 1064(1), 162 - 4
Mycoplasma contamination greatly enhances the apparent transport and concentrative accumulation of formycin B by mammalian cell culture; Plagemann PG; S49 mouse leukemia cells exhibit both equilibrative and Na(+)-dependent, concentrative formycin B transport . The latter represents only a minor nucleoside transport component and is detectable only when equilibrative nucleoside transport is inhibited by dipyridamole or another transport inhibitor . Thus in uncontaminated S49 cells formycin B accumulated only to slightly above the intracellular-extracellular equilibrium level . In contrast, in suspensions of S49 cells contaminated with mycoplasma, formycin B accumulated in the intracellular water space in unmodified form to 40-50-times the extracellular concentration in a dipyridamole-independent manner during 90 min of incubation at 37 degrees C . The mycoplasma active formycin B transport system was inhibited by all nucleosides tested, including thymidine and deoxycytidine, which are not substrates for the concentrative nucleoside transporter of S49 cells . Mycoplasma contamination was detected by the presence of cell-associated adenosine phosphorylase activity.

J Immunol Methods, 1991 Apr 25, 138(2), 273 - 83
Preparation of clinical grade monoclonal antibodies from serum-containing cell culture supernatants; Jiskoot W et al.; Three mouse monoclonal antibodies (Mab), RIV6, MN12, and WT31, were purified from cell culture supernatants containing foetal bovine serum (FBS) by two-step purification protocols, involving protein A affinity and ion exchange chromatography . Provided that the purification conditions were adapted to the physico-chemical properties of the individual Mab, clinical grade products could be obtained . The residual levels of bovine IgG originating from FBS were below 1% on a protein basis . Endotoxin levels were below 1 ng/ml . The contents of other serum proteins, DNA, and protein A were below or near the detection limits . The final products met the requirements for therapeutic Mab . Special attention was paid to the behaviour of foetal bovine IgG in the different purification steps . Large variations in the IgG contents of different batches of FBS were observed . However, the properties of the IgG fractions of the batches were very similar . A major IgG fraction with a low affinity for protein A and with components with relatively acidic isoelectric points (pIs) was distinguished from a minor fraction exhibiting a high affinity for protein A and a more diverse pI pattern . The impact of these findings on the purification strategy used for the Mab is discussed.

Biologicals, 1991 Apr, 19(2), 87 - 92
Quality control testing of surfaces for mammalian cell culture using cells propagated in a protein-free medium; Cinatl J Jr et al.; Mouse NCTC clone 929 L (L-929) cells were propagated continuously for 3 years as monolayers in a protein-free chemically-defined medium . These cells, designated L-929-WS, were used for quality control testing of the surfaces of commercially available cell culture plastic flasks . Differences in attachment and saturation density of L-929-WS cells in a protein-free culture medium were taken to define various levels of quality of the culture vessels tested . The rate of attachment and growth of L-929-WS cells on a surface of a given quality correlated directly with that of human embryonal fibroblasts and embryonal epithelial cells grown in a serum-free medium supplemented with growth factors and hormones . L-929-WS cells propagated continuously in a protein-free medium provide a simple and sensitive assay system for more general quality control testing of surfaces used for the culture of monolayer cells.

Can J Physiol Pharmacol, 1991 Apr, 69(4), 526 - 30
Porphyrinogenic effects in chick embryo liver cell culture of chloramphenicol analogues that are mechanism-based inactivators of cytochrome P-450; Green-Thompson RP et al.; Structural analogues of chloramphenicol (CAP) cause mechanism-based inactivation of rat liver cytochrome P-450 (P450) either via protein acylation or destruction of the heme prosthetic group . The goal of the present work was to determine whether CAP analogues that cause loss of the P450 heme moiety also cause porphyrin accumulation in chick embryo liver cell culture . The porphyrin profiles produced by exposure of cells to CAP analogues (160 microM) were determined by high-performance liquid chromatography with fluorescence detection . Of three CAP analogues that do not cause loss of the heme moiety of rat liver P450IIB1, two dichloroacetamides were not porphyrinogenic . The third compound, a chlorofluoroacetamide, caused porphyrin accumulation . This result may be due to the presence of P450 isozymes in chick embryo hepatocytes, distinct from rat liver P450IIB1, that are susceptible to destruction by this analogue . Of four CAP analogues that inactivate rat liver P450IIB1 with concomitant heme loss, a dichloroacetamide and two chlorofluoroacetamides caused porphyrin accumulation . The remaining compound, a monochloroacetamide, was not porphyrinogenic, perhaps because the P450 apoprotein cannot be reconstituted with fresh heme drawn from the regulatory "free heme pool" following inactivation by this analogue . Alternatively, there may be no P450 isozyme in chick embryo liver cell culture that is susceptible to inactivation by this compound.

Teratology, 1991 Apr, 43(4), 319 - 24
Comparative studies of embryotoxic action of ethylenethiourea in rat whole embryo and embryonic cell culture; Tsuchiya T et al.; The effects of ethylenethiourea (ETU) were investigated using rat (Wistar-imamichi) embryos cultured from days 11 to 13 of gestation or cultured rat embryonic cells extracted on day 11 . Malformations in cultured embryos at the concentration of 30 micrograms/ml of ETU were found in the head and tail, which were severely affected, as well as the limb and face . All embryos exposed to 150 and 300 micrograms/ml of ETU had malformed heads, tails, limbs, and facial configurations . Protein contents of the cultured embryos were decreased dose-dependently at the concentrations ranging from 30 to 300 micrograms/ml . In the histological studies of the cultured embryos with ETU, thinner neuroepithelium in head was observed . In the embryonic cells extracted on day 11 of gestation, ETU dose-dependently inhibited the differentiation of midbrain (MB) cells into neurons and that of limb bud (LB) cells into chondrocytes at the concentrations ranging from 30 to 600 micrograms/ml of ETU . The concentrations of ETU that inhibited the production of differentiated foci by 50% (IC50) were 170 micrograms/ml in LB cells of day 11, and greater than 600 micrograms/ml in LB cells on day 12 of development . Therefore, differentiation of MB cells was more sensitive to ETU than the differentiation of LB cells . These results indicated that there was a reasonable correlation of ETU induced changes in cultured whole embryos and embryonic cells.

Exp Mol Pathol, 1991 Apr, 54(2), 159 - 71
Lysosomal hydrolysis of lipids in a cell culture model of smooth muscle foam cells; Minor LK et al.; Rabbit aortic smooth muscle cells take up lipid droplets when they are presented using an inverted culture technique . These droplets were localized in secondary lysosomes as demonstrated by staining for acid phosphatase . Initially, 69% of the cell volume was occupied by lipid, and 94% of the lipid was in lysosomes . After a 24-hr clearance period, the cell volume occupied by lipid decreased to 53%, although there was no change in the fraction of cell lipid that was in lysosomes . To confirm that hydrolysis of droplet lipid was occurring in lysosomes, cultures were exposed to medium containing Sandoz 58-035, an inhibitor of acyl CoA:cholesterol acyl transferase, for 24 hr in the presence and absence of chloroquine, ammonium chloride, or methylamine . Although the hydrolysis of cholesteryl oleate was sensitive to these lysosomotropic agents, the hydrolysis of triolein was not . Using reconstituted LDL containing cholesteryl oleate and triolein, we demonstrated that the hydrolyses of cholesteryl oleate and triolein were equally sensitive to the lysosomotropic agents when the cells were not loaded with droplet lipid . However, in cells loaded with lipid, hydrolysis of LDL cholesteryl ester was sensitive to the lysosomotropic agents but hydrolysis of triolein was not . We therefore conclude that both droplet lipids were hydrolyzed in lysosomes, and we attribute the failure of the lysosomotropic agents to inhibit fully the hydrolysis of droplet triolein to the presence of a large mass of free fatty acids in the lysosome that maintains a sufficiently low pH to sustain the triglyceridase activity, but not the cholesteryl esterase activity, of the lysosomal acid lipase.

Am J Clin Pathol, 1991 Apr, 95(4), 578 - 82
Comparison of the Gen-Probe PACE 2 system, direct fluorescent-antibody, and cell culture for detecting Chlamydia trachomatis in cervical specimens; Iwen PC et al.; A chemiluminescent-labeled DNA probe (Gen-Probe PACE 2 System) for detection of Chlamydia trachomatis in endocervical specimens was evaluated . For each specimen, tissue cell culture, direct immunofluorescent staining (DFA), and DNA probe assay were performed . Thirty of 318 (9.4%) specimens were positive for C . trachomatis . The sensitivities, specificities, and positive and negative predictive values of the DNA probe compared with cell culture were 93%, 98%, 85%, and 99%, respectively, and for DFA these same values were 81%, 99%, 83%, and 99%, respectively . The Gen-Probe PACE 2 System is a reliable method for the rapid detection of endocervical chlamydial infection.

Proc Soc Exp Biol Med, 1991 Apr, 196(4), 415 - 20
Effects of amiodarone on elastin biosynthesis in primary hamster lung cell cultures; Costa KA et al.; Amiodarone is a Class III antiarrhythmic agent that has been implicated as a cause of human pulmonary fibrosis . Pulmonary fibrosis is associated with increased levels of connective tissue proteins such as collagen and elastin . The purpose of this investigation was to determine whether elastin synthesis would be altered by in vitro amiodarone administration . Primary hamster lung cell cultures were utilized . Cultures were treated with 2, 10, and 20 micrograms/ml amiodarone . Following treatment, elastin synthesis was monitored by a biochemical tracer assay based on the presence of the cross-linking amino acids: desmosine/isodesmosine . These cross-links are found only in elastin . Addition of {14C} lysine to cultures results in uptake of the radiolabel into the cross-links . Cross-links were isolated and identified using chromatography and electrophoresis . At all doses of amiodarone, elastin synthesis was seen to increase above control levels . Light and electron microscopy confirmed the presence of an extracellular matrix . The morphologic studies also revealed the presence of cytoplasmic inclusion bodies and vacuoles that are often associated with cationic, amphiphilic drugs such as amiodarone.

Endocrinology, 1991 Apr, 128(4), 1907 - 17
Steroid action on estrogen and progestin receptors in monkey pituitary cell cultures; Sprangers SA et al.; Estradiol (E) treatment of spayed macaques induces progestin receptors (PR) in pituitary gonadotropes, but not lactotropes . In contrast, levels of pituitary estrogen receptors (ER) remain constant in spayed or E-treated monkeys . In monkey pituitary cultures, the number of PR-positive cells varies depending on the donor status, whereas the percentage of ER-positive cells in similar . We sought to determine whether E can directly induce PR in monkey pituitary gonadotropes in culture and to provide further evidence that monkey pituitary ER are constitutively expressed . Dispersed pituitary cells from intact or gonadectomized male and female macaques were cultured on extracellular matrix with and without E, phenol red, high or low insulin, insulin-like growth factor-I, or 5% ovariectomized monkey serum, ER- and PR-positive parenchymal cells were immunohistochemically detected with monoclonal antibodies H222 and D75 (against human ER) and B39 (against human PR) . ER levels were also measured with a gradient shift assay incorporating H222 . Neither the percentage of ER-positive cells nor the levels of nuclear ER were altered by donor status or by 8 days of culture in phenol red or E . In contrast, cultures from gonad-intact donors exhibited the highest average percentage of PR-positive cells . The lower number of PR-positive cells in cultures from spayed donors did not vary for 8 days on extracellular matrix without phenol red, E, insulin, or serum . E directly increased the percentage of PR-expressing cells in serum-free cultures . Addition of serum also increased the percentage of PR-positive cells . Addition of E to serum-containing cultures further increased the percentage of PR-positive cells . Neither insulin nor insulin-like growth factor-I directly affected the number of PR-positive cells, but a high level of insulin blunted the action of E on PR induction in serum-free culture . We conclude that E treatment has no obvious effect on ER expression in macaque pituitary . However, the induction of pituitary PR by E treatment of spayed monkeys can be accounted for by a direct action of E on PR gene expression in gonadotropes.

Toxicol Appl Pharmacol, 1991 Apr, 108(2), 307 - 20
Pig Leydig cell culture: a useful in vitro test for evaluating the testicular toxicity of compounds; Brun HP et al.; In vivo studies have shown that the testis is a target organ for drugs and chemicals . In order to evaluate the testicular toxicity of compounds and to identify the mechanisms of their toxicity, we have developed a miniaturized primary culture of immature pig Leydig cells . Five well-known drugs with differing mechanisms of toxicity on testicular functions were tested to validate the model . Testosterone and progesterone secretion were measured to evaluate testicular function . Cell viability was assessed quantitatively using a colorimetric assay based on the reduction of a tetrazolium salt (3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide) which stains viable cells only, thus allowing discrimination between specific inhibitors of Leydig cell function and nonspecific cytotoxic drugs . Ketoconazole and aminoglutethimide inhibited both testosterone and progesterone secretion, but without modifying cell viability . Spironolactone specifically blocked testosterone secretion and increased progesterone concentration without inducing cell mortality . Cycloheximide altered testicular steroid secretion by another mechanism of action . Chlorpromazine, which interferes with the secretion of gonadotropins in vivo, produced a significant inhibition of progesterone and testosterone secretion as a result of the cytotoxic effects of the drug . In conclusion, this in vitro test enables one to discriminate accurately between specific and nonspecific inhibitors of steroidogenesis and could reduce the number of false positives when screening for potential testicular toxins.

Am J Med Genet, 1991 Apr 1, 39(1), 81 - 3
"Pseudomosaicism" for 4p- in amniotic fluid cell culture proven to be true mosaicism after birth; Vockley J et al.; Pseudomosaicism is noted in approximately 1% of amniotic fluid cell studies . Some represent numerical abnormalities, but pseudomosaicism for structural chromosomal abnormalities is also seen . Pseudomosaicism is not usually considered clinically significant . Recently, we evaluated a 13-month-old girl with developmental delay and minor anomalies suggestive of 4p- syndrome . In 5 of 100 peripheral lymphocytes, she had a deletion 46,XX,del(4)(p15) . Review of a prenatal amniocentesis study performed on the mother of our patient disclosed that one colony of 18 examined from 2 in situ cultures had the same abnormality, whereas none of the 27 cells from a flask culture showed the abnormality . Results of this study had originally been reported as showing pseudomosaicism . To our knowledge, amniotic fluid pseudomosaicism of a structural abnormality has not previously been shown to reflect true mosaicism in fetal tissue or liveborn children . The actual incidence of this phenomenon is unknown, but it may be present in unexamined children with minimal clinical findings . Apparently only one previous case of mosaic 4p- in a liveborn individual has been reported.

J Pharmacol Methods, 1991 Apr, 25(2), 133 - 45
A cell culture assay for the detection of cardiotoxicity; Low-Friedrich I et al.; An important step in minimizing the number of animal experiments in medical research is the study of in vitro model systems . We propose the use of "shock protein" formation, which is a cellular response to cell-damaging stress as an assay to monitor cardiotoxicity . Isolated and cultured cardiac myocytes were prepared by a trypsin digestion method from 18-day-old fetal mice . These cells respond to typical substances inducing "shock protein" formation in other cellular systems as well as to known cardiotoxins with the de novo synthesis of "shock proteins." Pharmaceuticals relevant in transplant medicine were tested for possible cardiotoxic effects: Cyclosporine A evokes "shock protein" formation at subtherapeutic concentrations . Azathioprine and methyl-prednisolone exert the same effect but at concentration ranges highly above the therapeutic level . The ability to induce "shock protein" synthesis obviously seems to be restricted to toxic drugs . The data presented demonstrate that the proposed in vitro model system for cardiotoxicity is animal saving and sensitive.

Acta Virol, 1991 Apr, 35(2), 187 - 9
Detection of nuclear cytomegalovirus antigen in cell culture as compared to classical virus isolation; Horacek J et al.; Rapid detection of human cytomegalovirus early nuclear protein in human diploid cells by immunofluorescence (IF) using commercial (Du Pont) monoclonal antibody was compared with the classical virus isolation in these cells . From 30 specimens tested early nuclear protein was detected in 16 cases but the virus was isolated out of 4 samples only.

Neurochem Res, 1991 Apr, 16(4), 423 - 8
Hypoxia induced metabolism dysfunction of rat astrocytes in primary cell cultures; Tholey G et al.; In order to study the astroglial contribution to hypoxic injury on brain tissue metabolism, modifications of glutamine synthetase (GS) lactate dehydrogenase (LDH) enolase and malate dehydrogenase activity produced by reduced oxygen supply have been determined in primary cultures of astrocytes prepared from newborn rat cerebral cortex . Enzymatic activities were measured immediately after the hypoxic treatment (9 h) and during post injury recovery . GS level is significantly decreased in response to low oxygen pressure and increased above control value during the post hypoxic recovery period . The magnitude of GS reduction by hypoxia depends on the age of the cells in culture . Lactate dehydrogenase and enolase levels were significantly enhanced during the two periods considered . No modification of the MDH level was observed . The synthesis of LDH isoenzymes containing mainly M subunits is specifically induced by hypoxia . Our results suggest that astroglial cells may represent a particularly sensitive target toward hypoxia injury in brain tissue . Low oxygen pressure available may modify some fundamental metabolical functions of these cells such as glutamate turnover and lactic acid accumulation.

J Hosp Infect, 1991 Apr, 17(4), 287 - 96
Assay of antiseptic agents in cell culture: conditions affecting cytotoxicity; Tatnall FM et al.; In-vivo studies suggest that chlorine-releasing antiseptic agents inhibit wound healing . Studies which have used cell culture systems to evaluate cytotoxicity have generated conflicting results for the toxicity of free-chlorine agents relative to other antiseptics . Here we examine the following three factors which may influence the toxicity of individual agents within a cell culture assay: (1) cell number; (2) duration of exposure; and (3) the nature of the antiseptic diluent . Three agents (sodium hypochlorite, chlorhexidine and hydrogen peroxide) were tested on transformed human keratinocytes (SVK 14 cells) . It was found that increasing cell number, and using serum or medium as a diluent, reduced the toxicity of all agents but had the greatest effect on sodium hypochlorite . In contrast, increasing the duration of exposure increased the toxicity of all agents but had the greatest effect on hydrogen peroxide . These observations may explain the high toxicity of hydrogen peroxide and relatively low toxicity of sodium hypochlorite which have been observed in vitro and are the reverse of in-vivo findings . Culture systems in which high cell numbers are coupled with an agent diluted in serum or medium, and a long exposure time, seem likely to decrease the toxicity of chlorine-releasing agents relative to hydrogen peroxide.

J Biotechnol, 1991 Apr, 18(1-2), 161 - 72
On-line determination of glucose concentration throughout animal cell cultures based on chemiluminescent detection of hydrogen peroxide coupled with flow-injection analysis; Huang YL et al.; A flow-injection analysis (FIA) system for the on-line determination of glucose in animal cell cultures is described . The system is based on immobilized glucose oxidase (GOD) . The hydrogen peroxide generated in the enzyme reaction is determined via a highly sensitive chemiluminescent reaction with luminol . Based on the measurement of the maximum emitted light intensity, the system was able to analyse hydrogen peroxide over the concentration range of 10(-7) to 10(-2) M . For glucose determination, the system has a linear range of 10(-5) to 5 x 10(-2) M glucose, with an r.s.d . of 3% at the 1 mM level (5 measurements) . The influence of luminol and buffer concentrations, pH and temperature on the chemiluminescent reaction were investigated . The enzyme reactor used was stable for more than 4 weeks in continuous operation, and it was possible to analyse up to 20 samples per h . The system has been successfully applied to on-line monitoring of glucose concentration during an animal cell culture, designed for the production of human antithrombin III factor . Results obtained with the FIA system were compared with off-line results, obtained with a Yellow Springs Instrument Company Model 27 (YSI).

Biochim Biophys Acta, 1991 Mar 19, 1092(1), 101 - 9
Effect of luteinizing hormone releasing hormone pulse characteristics on comparative luteinizing hormone and follicle stimulating hormone secretion from superfused rat anterior pituitary cell cultures; Kellom TA et al.; We have shown that 4 ng luteinizing hormone releasing hormone (LHRH) pulses induced significantly greater luteinizing hormone (LH) release from proestrous rat superfused anterior pituitary cells with no cycle related differences in follicle stimulating hormone (FSH) . Current studies gave 8 ng LHRH in various pulse regimens to study amplitude, duration and frequency effects on LH and FSH secretion from estrous 0800, proestrous 1500 and proestrous 1900 cells . Regimen 1 gave 8 ng LHRH as a single bolus once/h; regimen 2 divided the 8 ng into 3 equal 'minipulses' given at 4 min intervals to extend duration; regimen 3 gave the 3 'minipulses' at 10 min intervals, thereby further extending duration: regimen 4 was the same as regimen 2, except that the 3 'minipulses' were given at a pulse frequency of 2 h rather than 1 h . In experiment 1, all four regimens were employed at proestrus 1900 . FSH was significantly elevated by all 8 ng regimens as compared to 4 ng pulses; further, 8 ng divided into 3 equal 'minipulses' separated by 4 min at 1 and 3 h frequencies (regimens 2 and 4) resulted in FSH secretion that was significantly greater than with either a single 8 ng bolus (regimen 1) or when the 'minipulses' were separated by 10 min (regimen 3) . In experiment 2, at proestrus 1500, FSH response to the second pulse of regimen 4 was significantly greater than in regimen 2; LH release was significantly suppressed at pulse 2 compared to regimen 2 accentuating divergent FSH secretion . At estrus 0800, FSH response to the second pulse of regimen 4 was significantly stimulated FSH at proestrus 1900, 1500 and estrus 0800, FSH divergence was most marked at proestrus 1500 . These data indicate a potential role for hypothalamic LHRH secretory pattern in inducing divergent gonadotropin secretion in the rat.

J Biol Chem, 1991 Mar 5, 266(7), 4251 - 6
Thrombin attenuates the stimulatory effect of histamine on Ca2+ entry in confluent human umbilical vein endothelial cell cultures; Neylon CB et al.; Human umbilical vein endothelial cells were grown to confluence, as first passage cells, on coverslips . Treatment with ionomycin or histamine caused a sustained rise in intracellular Ca2+ (measured by Fura-2 fluorescence), but after treatment with thrombin, only a transient rise in Ca2+ was observed . Furthermore, the addition of thrombin after ionomycin or histamine lowered the raised Ca2+ back to near control levels . This effect of thrombin was concentration dependent, with increasing concentrations producing increases in both the rate and extent of the lowering of Ca2+ . A similar effect of thrombin was seen on video imaging of Fura-2-loaded cell monolayers . The Ca2(+)-lowering effect of thrombin was not mimicked by phorbol 12-myristate 13-acetate nor blocked by staurosporine, indicating a lack of involvement of protein kinase C; intracellular pH also does not appear to be involved . The mechanism by which thrombin lowers cytoplasmic Ca2+ is due mainly to inhibition of Ca2+ entry since thrombin prevented the stimulated influx of Mn2+ caused by histamine or ionomycin . It may therefore be that in vivo under certain physiological or pathological conditions, thrombin's effects on intracellular Ca2+ are more transient than those of histamine, and thrombin also may induce transience in histamine's actions.

Brain Res Bull, 1991 Mar, 26(3), 333 - 8
Electrophysiological comparison of pyramidal and stellate nonpyramidal neurons in dissociated cell culture of rat hippocampus; Buchhalter JR et al.; Differences in electrophysiological properties between pyramidal and nonpyramidal neurons have been previously demonstrated in the hippocampal slice preparation . However, it has also been shown that nonpyramidal neurons from several hippocampal regions have different morphologies as well as different active membrane characteristics . In this study, active and passive electrophysiological characteristics of pyramidal and a single morphological type of nonpyramidal neuron in rat dissociated hippocampal culture were examined with intracellular recording at room temperature and at 33-35 degrees C . No significant differences were noted between the two groups of neurons, at either temperature, with regard to action potential amplitude, rate of rise and fall, duration at half maximal amplitude, adaptation to steady state depolarization or resting membrane potential and input resistance . We conclude that these electrophysiological properties cannot be used to distinguish these two neuron types in dissociated hippocampal cell culture.

J Cell Physiol, 1991 Mar, 146(3), 370 - 8
Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures; Leboy PS et al.; We have examined the ability of dexamethasone, retinoic acid, and vitamin D3 to induce osteogenic differentiation in rat marrow stromal cell cultures by measuring the expression of mRNAs associated with the differentiated osteoblast phenotype as well as analyzing collagen secretion and alkaline phosphatase activity . Marrow cells were cultured for 8 days in primary culture and 8 days in secondary culture, with and without 10 nM dexamethasone or 1 microM retinoic acid . Under all conditions, cultures produced high levels of osteonectin mRNA . Cells grown with dexamethasone in both primary and secondary culture contained elevated alkaline phosphatase mRNA and significant amounts of type I collagen and osteopontin mRNA . Addition of 1,25-dihydroxyvitamin D3 to these dexamethasone-treated cultures induced expression of osteocalcin mRNA and increased osteopontin mRNA . The levels of alkaline phosphatase, osteopontin, and osteocalcin mRNAs in Dex/Dex/VitD3 cultures were comparable to those of 1,25-dihydroxyvitamin D3-treated ROS 17/2.8 osteosarcoma cells . Omitting dexamethasone from either primary or secondary culture resulted in significantly less alkaline phosphatase mRNA, little osteopontin mRNA, and no osteocalcin mRNA . Retinoic acid increased alkaline phosphatase activity to a greater extent than did dexamethasone but did not have a parallel effect on the expression of alkaline phosphatase mRNA and induced neither osteopontin or osteocalcin mRNAs . In all conditions, marrow stromal cells synthesized and secreted a mixture of type I and III collagens . However, dexamethasone-treated cells also synthesized an additional collagen type, provisionally identified as type V . The synthesis and secretion of collagens type I and III was decreased by both dexamethasone and retinoic acid . Neither dexamethasone nor retinoic acid induced mRNAs associated with the chondrogenic phenotype . We conclude that dexamethasone, but not retinoic acid, promotes the expression of markers of the osteoblast phenotype in cultures of rat marrow stromal fibroblasts.

Mol Pharmacol, 1991 Mar, 39(3), 324 - 31
Thymopoietin, a potent antagonist at nicotinic receptors in C2 muscle cell cultures; Quik M et al.; Recent work has shown that thymopoietin, a polypeptide with actions in the immune and nervous systems, potently binds to the alpha-bungarotoxin (alpha-BGT) receptor . The present study was done to characterize the interaction of thymopoietin at the nicotinic alpha-BGT binding site in cultured muscle cells and to correlate these findings with the effects of the polypeptide on nicotinic receptor-mediated function . Inhibition studies showed that thymopoietin potently inhibited 125I-alpha-BGT binding in C2 muscle cells in culture, with an IC50 of 1.1 nM, a value similar to that for alpha-BGT . Thymopoietin bound to the alpha-BGT receptor in the cells in culture relatively slowly; at 10(-8) M thymopoietin, maximal inhibition occurred after 45 to 75 min of exposure to the polypeptide . Dissociation of thymopoietin from the receptor exhibited a much longer time course; recovery of alpha-BGT binding to control values after exposure to 10(-8) M thymopoietin occurred approximately 16 hr after removal of the polypeptide . The effects of thymopoietin on 125I-alpha-BGT binding correlated well with those on nicotinic function . Thymopoietin potently inhibited nicotinic receptor-mediated 22Na uptake in muscle cells in culture, with an IC50 of 2 nM . This effect was dependent on the length of the preincubation period with thymopoietin, with maximal inhibition occurring after 60 min of exposure to the polypeptide . Recovery of the functional response after thymopoietin (10(-8) M) exposure required about 16 hr . The mode of inhibition of receptor-mediated ion flux by thymopoietin was similar to that observed with alpha-BGT but distinct from that obtained with d-tubocurarine and gallamine . To conclude, thymopoietin, a thymic polypeptide associated with the immune system, potently inhibited both 125I-alpha-BGT binding and nicotinic receptor-mediated function in C2 muscle cells . These findings may have implications for myasthenia gravis and/or other neuromuscular disorders.

Br J Cancer, 1991 Mar, 63(3), 358 - 62
SR 4233 cytotoxicity and metabolism in DNA repair-competent and repair-deficient cell cultures; Biedermann KA et al.; In order to understand in more detail the mechanism underlying the preferential hypoxic cytotoxicity of the benzotriazine N-oxide SR 4233, we have compared the hypoxic cytotoxicity of this drug to the rates of hypoxic metabolism in both DNA double strand break repair-competent and repair-deficient cell cultures . Rodent SCCVII cells and repair deficient, radiation sensitive cells (rodent XR-1, V-3, and human AT5BI) were most sensitive to SR 4233 under hypoxia with a lethal dose needed to kill 50% of cells (LD50) of less than 5 microM . SR 4233 was less cytotoxic to human AG 1522 (LD50 = 18 microM), CHO 4364 (LD50 = 25 microM) and human HT 1080 cells (LD50 = 33 microM) . The sensitivities to SR 4233 were found to be inversely proportional to the rates of SR 4233 metabolism in repair-competent cells (R2 = 0.9) . However, XR-1 and V-3 cells were more sensitive to SR 4233 than predicted by the metabolism rate . Thus, the toxicity by SR 4233 towards hypoxic cells appears to result from two mechanisms; the rate of drug metabolism and the ability to repair DNA double strand breaks.

Mutat Res, 1991 Mar, 254(2), 153 - 60
Effect of passaging on Mer phenotype of human fetal cell cultures; Day RS 3rd et al.; With increasing passage in culture, the human fibroblast cell strain GM11 lost the Mer+ phenotype (the ability to support the growth of adenovirus 5 damaged prior to infection by MNNG) . All of 46 embryonic strains prepared either from various organs of 20 fetuses from 6-7 to 13-14 weeks of gestational age or from two hydatidiform moles showed normal repair of MNNG-treated virus . We conclude that human fetal strains are not usually deficient in such repair, and that the behavior of GM11 is atypical.

FASEB J, 1991 Mar 1, 5(3), 287 - 94
Cell culture models of differentiation; Watt FM; It is now possible to culture cells from most organs of the body under conditions in which they continue to express at least some of their differentiated traits, and to model some of the differentiation processes that occur during embryonic and adult life . How much can these cultures tell us about the acquisition and maintenance of the differentiated state? To answer this question I shall outline the features of several cell culture models, dividing them into categories according to whether they mimic differentiation during development, differentiation of adult stem cell progeny, or the transition from one differentiated phenotype to another . In spite of the diversity of cell types under consideration, it is possible to detect some common themes: the stability of the differentiated state; the relationship between proliferation and differentiation; the relative importance of intrinsic cellular programming and environmental regulation; and possible mechanisms for transcriptional control of the genes that are activated during differentiation . In recent years cell culture models have yielded a great deal of information about differentiation and the way is now clear for even more exciting discoveries.

Proc Natl Acad Sci U S A, 1991 Mar 1, 88(5), 1756 - 9
Heme inhibits human immunodeficiency virus 1 replication in cell cultures and enhances the antiviral effect of zidovudine; Levere RD et al.; The effects of heme alone and heme administered together with 3'-azido-3'-deoxythymidine (AZT) on human immunodeficiency virus replication in human peripheral blood lymphocytes and in the H9 cell line were studied . Heme enhanced the antiviral action of AZT against both drug-resistant and drug-sensitive viral strains; the heme effect was more pronounced against the latter . Moreover, heme alone displayed a significant ability to inhibit viral replication in concentrations markedly smaller than those required to inhibit the reverse transcriptase of Rauscher murine leukemia virus . The results of this study extend the range of pharmacological actions that metalloporphyrins exert in biological systems and suggest that further study of the interactions of the natural compound heme and human immunodeficiency virus chemotherapeutic agents such as AZT may be useful.

J Orthop Res, 1991 Mar, 9(2), 289 - 96
Cell association of fretting corrosion products generated in a cell culture; Merritt K et al.; The nature and distribution of corrosion products released into the body from orthopaedic implants remains an important issue . Various approaches to study this problem have been taken, such as the injection of metal salts, the injection of corrosion products, analysis of retrieved implants and adjacent tissue, and stimulated corrosion in vivo, with collection of body fluids and tissues for analysis . Tissue culture techniques have also been used to study the cellular response to metal salts or to corrosion or wear products that were generated in a separate environment . In this study, fretting corrosion of stainless steel plates and screws and of cobalt-chromium alloy plates with stainless steel screws was undertaken within a cell culture . The results showed that the cell cultures remained viable despite considerable metal ion release . Nickel was released in all cultures with fretting corrosion and was found mainly in the tissue culture medium (supernatant of the harvested cultures) . Cobalt was detected only in those cultures with fretting corrosion of the cobalt-chromium alloy, and it was present mainly in the tissue culture medium . Chromium was released in all cultures with fretting corrosion, and it was found to be associated mainly with the cells with little in the culture medium . This compartmentalization of cell-associated chromium and fluid-associated cobalt and nickel supports in vivo studies showing chromium accumulation in red blood cells or tissue sites and comparatively low levels of nickel and cobalt.

Basic Res Cardiol, 1991 Mar-Apr, 86(2), 79 - 86
Cell culture systems to study progression and inhibition of intimal proliferations; Betz E; Vascular smooth muscle cells, endothelial cells, and fibroblasts are the main cellular constituents of artery walls . Mass cultures and clone cultures of these cell types have meanwhile become valuable tools in the research of the genesis, pathophysiology, and therapy of vessel-wall diseases . With transfilter co-culture systems the three-layered construction of the artery wall can be imitated in vitro, and it has become possible to induce smooth muscle cell proliferates in these in vitro system which resemble, in many respects, intimal proliferates as they often occur after angioplasty, stent- or bypass operations in the form of secondary stenoses . With this technique the interaction of the three cell species of artery walls can be easily studied . The time-course of the development of smooth muscle cell proliferates in vitro resembles the in vivo scenario . Addition of oxidized lipoproteins and monocytes to the culture medium of transfilter cultures leads to atheroma-like proliferates . Culturing whole artery segments is another in vitro technique for induction of intimal proliferates, and enables the production of intimal proliferates in a way similar to transfilter culture systems . Because of the striking similarities of the cellular responses of transfilter- and organ-culture systems with in vivo processes in atherogenesis, and in the development of secondary stenoses after angioplasty, the described co-culture systems are suitable for studying the genesis, pathophysiology, and therapy of stenosing artery processes, as well as to obtain further insight into basic problems of cell interaction in vessel walls.

Diagn Microbiol Infect Dis, 1991 Mar-Apr, 14(2), 131 - 4
A comparison of direct immunofluorescence, shell vial culture, and conventional cell culture for the rapid detection of influenza A and B; Johnston SL et al.; Direct immunofluorescence (FA) and shell vial contrifugation cultures (SVCs) were compared with conventional tube cultures for the rapid detection of influenza A and B by using a commercial antibody . Of the 439 specimens tested, 82 were positive by conventional culture (CC) . The direct smear prepared from pelleted cells or direct swab material exhibited positive fluorescence in only seven (8.5%) of these cases, whereas the SVC was positive in 30 (37%) . The SVC method detected 12 additional positive isolates that were not recovered in CC . The mean time to isolation in CC was 3.6 days for influenza A and 4.3 days for influenza B . The use of SVC provided more rapid results (36-48 hr) . The FA method, although more rapid, may be of limited sensitivity and difficult to interpret depending on the quality of the specimen . The results indicate that SVC complements conventional culture in the rapid detection of influenza and can detect infections that may be missed in conventional tubes, but should not be used to the exclusion of conventional culture.

Mikrobiol Zh, 1991 Mar-Apr, 53(2), 95 - 8
{The characteristics of glucose consumption by an L-929 cell culture infected with Chlamydia}; Mavrov II et al.; The effect of the intracellular parasite Chlamydia trachomatis on the host cell energy metabolism has been studied . Glucose consumption by L-929 cell cultures infected or uninfected by C . trachomatis was studied in comparison during a 3-days cultivation . The content of glucose in the cultural medium was determined every 5, 24, 48, 72 hrs according to the developmental cycle of the parasite . It was shown that cell infection by C . trachomatis induced the alteration of energy metabolism via an increase in the glucose consumption rate.

What Is Fermentation?, What Is Environmental Microbiology?, What Is Genetic Engineering?, What Is Activated Sludge?, What Is Biotechnology?, a, Microorganisms, i, Bacterium, i, Microbe, e, Microbes, i, Microorganism, r, Gram positive, c, Microorganism, i, Escherichia coli, s, Saccharomyces yeast, e, Erythromycin, r, Candida albicans, c, Antibiotics, e, Microbial, e, Yeasts, e, Salmonella, o, Citrobacter, o, Streptococci, c, Yeasts, i, Streptococcal, n, Antimicrobials, s, S. cerevisiae, e, Escherichia coli, n, Gram positive, e, Candida albicans, n, Bacteriological, r, Escherichia coli

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005