Cell cultures

Eur J Pharmacol, 1994 Nov 3, 264(3), 361 - 9
GABAA receptor-mediated inhibition of N-methyl-D-aspartate-evoked {3H}dopamine release from mesencephalic cell cultures; Chaudieu I et al.; Direct activations of both GABAA and GABAB receptors are known to hyperpolarize dopaminergic neurons . However systemic or intra-ventral tegmental administration of a GABAA receptor agonist produces paradoxical depolarization of mesencephalic dopaminergic neurons and increases dopamine release . Thus indirect excitation appears to preclude observation of inhibitory GABAA effects on dopamine release in intact tissue . The present study used cultures of isolated cells from rat ventral mesencephalon to characterize effects of GABAA and GABAB receptor activation on evoked dopamine release . The GABAA receptor agonist, muscimol, produced a potent and complete inhibition of N-methyl-D-aspartate (NMDA)-evoked {3H}dopamine release . This effect was blocked by the GABAA receptor antagonist, picrotoxin, and enhanced by flunitrazepam . Omission of Mg2+ greatly reduced the inhibitory effect of muscimol on NMDA-evoked {3H}dopamine release . Muscimol had little or no effect on {3H}dopamine release evoked by the non-NMDA receptor agonists, quisqualate and kainate . The GABAB receptor agonist, baclofen, slightly inhibited NMDA-evoked {3H}dopamine release and had no effect on release evoked by quisqualate or kainate . Endogenous GABA released by the mesencephalic cells also appeared to inhibit NMDA-evoked {3H}dopamine release mainly via a GABAA receptor-mediated mechanism . This is suggested by the observations that NMDA-evoked {3H}dopamine release was potentiated by picrotoxin but not by the GABAB receptor antagonist, phaclofen, and that blockade of extracellular GABA removal, with amino-oxyacetic acid and beta-alanine, inhibited NMDA-evoked {3H}dopamine release in a picrotoxin-sensitive manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1994 Nov, 60(11), 4203 - 6
Cell culture and PCR determination of poliovirus inactivation by disinfectants; Ma JF et al.; Inactivation of poliovirus type 1 by 1 N HCl, 1 N NaOH, 0.5 and 1.0 mg of free chlorine per liter, and UV light was compared by using cell culture and seminested PCR (30 cycles of reverse transcriptase-PCR plus 30 cycles of seminested PCR) . A minimum contact time of 45 min with HCl, 3 min with NaOH, 3 and 6 min with 1.0 and 0.5 mg of free chlorine per liter, respectively, was required to render 1.64 x 10(2) PFU of poliovirus type 1 per ml undetectable by seminested PCR . In cell culture, a minimum contact time of 5 min to HCl, 30 s to NaOH, and 1 min to either chlorine concentration was required to render the viruses undetectable by the plaque assay method . No correlation was observed between results by PCR and cell culture when viruses were exposed to UV light . These data suggest that inactivated virus with intact nucleic acid sequences can be detected by PCR.

Cancer Immunol Immunother, 1994 Nov, 39(5), 305 - 12
Development of four donor-specific phenotypes in human long-term lymphokine-activated killer cell cultures; Vollenweider I et al.; A series of 62 lymphokine-activated killer cell (LAK) cultures from 44 different donors was investigated for the distribution of various CD markers during a cultivation period of 3 weeks . Great differences in the phenotypic pattern were found between different donors, but similar changes of the subset pattern of various donors allowed a classification of the LAK cultures into four distinct LAK types . LAK type 1 was characterised by low numbers of CD3+ cells and high values for CD56+ cells . In LAK type 2 cultures gamma/delta TCR+ cells extensively proliferated, whereas in LAK type 3 cultures the CD57 and CD8 values increased considerably . LAK type 4 cultures did not show any of these characteristics . The resulting phenotype of a LAK culture was donor-specific, as LAK cultures established from the same peripheral blood mononuclear cells (PBMC), fresh or after cryopreservation, or from PBMC obtained from the same donor at different venous punctures, always developed the same phenotype . A clear correlation between phenotype and killing activity could only be found for LAK type 1 cultures, which always developed high lytic activity . Long-term IL-2 stimulation induced high levels of perforin-positive cells in LAK cultures but the perforin content did not correlate with the cytotoxicity . The transcription pattern for various cytokines only varied slightly between the cultures . Messenger RNA for granulocyte/macrophage- colony-stimulating factor, interferon gamma, tumour necrosis factor alpha, interleukin-4 (IL-4) and IL-5 were found in almost all cultures during the entire cultivation period, whereas mRNA for IL-2 was never detected . Most variations in the transcription pattern were observed for IL-6 and IL-7 . However, no correlation could be found between the endogenous cytokine production and the phenotype or lytic activity of the LAK cultures . Further studies are required to determine the factors that cause lymphocyte subsets from a specific donor to proliferate preferentially under long-term IL-2 stimulation.

J Neurosci, 1994 Nov, 14(11 Pt 1), 6402 - 11
Hippocampal synaptogenesis in cell culture: developmental time course of synapse formation, calcium influx, and synaptic protein distribution; Basarsky TA et al.; The formation of chemical synapses between hippocampal neurons in primary cell culture was studied using electrophysiology, calcium imaging, and immunocytochemical approaches . Inhibitory and excitatory synapses formed within 12 d in cell culture (DIC) that were sensitive to the N-type calcium channel blocker omega-conotoxin GVIA (omega-CgTx) . At 4 DIC, immature connections were present in which spontaneous, but rarely evoked, synaptic currents were detected . At both 4 and 12 DIC, the synaptic proteins rab3a, synapsin I, and synaptotagmin were present in hippocampal neurons, but the subcellular distribution changed from one in which immunoreactivity was initially distributed within soma and neurites to a punctate varicose appearance . Correlated with the transformation from immature to mature synaptic states was the onset of omega-CgTx-sensitive calcium influx . Taken together, these data suggest that the expression of functional omega-CgTx-sensitive calcium influx is temporally coincident with synapse formation, and that during the maturation of the synapse there is a redistribution of synaptic proteins.

Comp Biochem Physiol C Pharmacol Toxicol Endocrinol, 1994 Nov, 109(3), 269 - 76
Effects of thyroid hormones on chick embryo muscle cell culture; Vyboh P et al.; The importance of thyroid hormones (TH) in the normal development of muscles has been repeatedly postulated . The effects of physiological TH doses on ornithine decarboxylase (ODC) and protein synthesis in muscle cells have been studied using cell cultures prepared from 11-day-old chick embryos . Triiodothyronine nuclear receptors in primary muscle cell culture were characterized on the basis of the specific binding analysis as a single receptor class with the equilibrium dissociation constant Kd = 1.2 +/- 0.4 x 10(-10) mol/l and binding capacity Bmax = 0.21 +/- 0.09 fmol/micrograms DNA . While the physiological amounts of both triiodothyronine (T3) and thyroxine (T4) stimulated ODC activity after 2 hr of treatment, only T3 had the same stimulatory effect after 4 hr of treatment . Twenty-four hour exposure of muscle cell culture to TH did not change ODC activity . The incorporation of {3H}leucine into proteins was elevated only after 120 hr incubation in the presence of T4 . Application of T4 caused also an increase in the protein content after 24 hr.

Differentiation, 1994 Nov, 58(1), 37 - 46
Effects of chronic electrical stimulation on myosin heavy chain expression in satellite cell cultures derived from rat muscles of different fiber-type composition; Wehrle U et al.; Myotube cultures were established from satellite cells of three rat muscles of different fiber-type composition, slow-twitch soleus, diaphragm, and fast-twitch tibialis anterior (TA) . Effects of chronic electrical stimulation were studied by exposing these cultures for up to 13 days to a stimulus pattern consisting of 250 ms impulse trains of 40 Hz, repeated every 4 s . Changes in myosin expression were assessed at the mRNA level by Northern blotting and in situ hybridization . Expression of slow myosin at the protein level was analysed by immunoblotting and immunohistochemistry with two antibodies, one specific to adult slow myosin, the other reacting with developmental and adult slow myosin heavy chain (MHCI) isoforms . In all three myotube cultures stimulation enhanced the mRNA and protein expression of a developmental isoform of slow myosin (MHCI) . However, the three myotube cultures differed in the extent of the increase in MHCI . It was greatest in soleus-derived myotubes, least in TA-derived myotubes, and intermediate in diaphragm-derived myotubes . In addition to the increase in slow myosin, long-term stimulation led to an isoform switch, as indicated by an increase in myotubes reacting with the antibody specific for the adult MHCI . Our results suggest that enhanced contractile activity promotes the expression of the slow phenotype predetermined in satellite cells of slow-twitch, type I fibers . The different extents of increased slow myosin expression may thus be explained as reflecting different percentages of type I fibers and consequently of slow-type satellite cells in the corresponding donor muscles.

J Neurol Sci, 1994 Nov, 126(2), 133 - 7
Prevention of HIV coat protein (gp120) toxicity in cortical cell cultures by riluzole; Sindou P et al.; Neurological complications observed in HIV-infected patients are very frequent . Neocortical lesions include reduced neuronal density due to neuronal degeneration . The HIV envelope protein gp120 has potent neurotoxic properties in cell cultures blocked either by NMDA antagonists or calcium channel antagonists . Moreover, human monocytoid cell lines infected by HIV release endogenous toxic factors with comparable cellular actions . We have analysed the effects of riluzole, a compound reducing the excitatory amino acid release on gp120-induced neurotoxicity in primary neuronal cultures . Riluzole, which blocks the release of glutamate and aspartate from nerve terminals, prevents (10(-7) M) the neuronal degeneration produced by 20 pM of gp120 in cortical cell cultures . This result could suggest that toxic factors produced by activated macrophages might increase glutamate release, and that this may be prevented by riluzole.

J Clin Microbiol, 1994 Nov, 32(11), 2655 - 9
Diagnosis of cytomegalovirus infections by shell vial assay and conventional cell culture during antiviral prophylaxis; Manez R et al.; A total of 3,552 specimens for conventional cytomegalovirus (CMV) culture and shell vial assay for CMV immediate-early antigen were obtained during a prospective randomized trial for prophylaxis of CMV disease after liver transplantation . Prophylaxis with ganciclovir for 2 weeks and then high-dose acyclovir for 2.5 months was compared with high-dose acyclovir alone for 3 months . During the first 12 weeks after transplantation, when the patients were on prophylaxis, there were significantly more clinical samples positive by the shell vial assay and negative by standard culture in comparison with the number of samples obtained from weeks 13 to 24, after prophylaxis was discontinued, that were positive by the shell vial assay and negative by standard culture . In contrast, significantly fewer samples were positive by both the shell vial assay and standard culture during the first 12 weeks compared with the number obtained 13 to 24 weeks after transplantation that were positive by both methods . Samples positive by the shell vial assay only were obtained significantly more frequently from patients with asymptomatic than symptomatic CMV infections, while samples positive by both methods were obtained significantly more often from patients with symptomatic CMV infection . It was concluded that antiviral prophylaxis with high-dose acyclovir or ganciclovir and then high-dose acyclovir and asymptomatic CMV infection are associated with a decrease in the level of CMV isolation by standard cell culture in comparison with that by the shell vial assay.

Am J Hypertens, 1994 Nov, 7(11), 953 - 9
Altered phospholipid fatty acid content and metabolism in heart cell cultures from newborn spontaneously hypertensive rats; Millanvoye-Van Brussel E et al.; Genetic hypertension has been proposed to be associated with impaired lipid metabolism . To investigate whether lipid metabolism is altered in young rats of the spontaneously hypertensive Okamoto strain (SHR), we have compared the phospholipid fatty acid content and metabolism in cultured heart myocytes and fibroblasts from SHR and normotensive Wistar-Kyoto (WKY) newborn rats . The phospholipid-bound fatty acid profile and metabolism were altered in SHR cardiomyocytes and unchanged in SHR fibroblasts . In SHR myocytes, the fatty acid composition of the phospholipid fraction was modified, with a lowered proportion of linoleic (P < .05) and eicosapentaenoic acid (P < .001), resulting in a decreased polyunsaturated to saturated fatty acid ratio (1.16 +/- 0.08 in SHR v 1.44 +/- 0.08 in WKY, P < .02) . The metabolism of radioactive arachidonate (C20:4) and linoleate (C18:2) also differed between SHR and WKY myocytes . Their release was increased (P < .004 and .05 for C20:4 and C18:2, respectively) . The labeled phospholipid species also differed between the two strains, suggesting an altered phospholipid turnover in SHR . This study demonstrates modifications of phospholipid fatty acid profile and metabolism in spontaneously contractile cardiac cells from newborn prehypertensive SHR, in the absence of neural, hormonal, and hemodynamic influences.

J Immunoassay, 1994 Nov, 15(4), 411 - 28
Development of an ELISA for quantification of human protein S in cell culture fluids using commercial polyclonal antisera; Phillips DJ et al.; An enzyme-linked immunosorbent assay (ELISA) was developed to measure protein S antigen released into cell culture fluids . We used readily available commercial polyclonal antisera to develop the assay . This assay was sensitive with a detection limit of about 0.086 ng/ml . Between-assay precision (coefficient of variation) at levels of 0.2, 1.1, and 13.9 ng/ml was 14%, 15%, and 11% respectively . Specificity and accuracy were demonstrated from the use of: 1) culture fluids from 3-primary endothelial cell cultures and 7-cell lines known to constitutively produce protein S; 2) 2-cell lines not synthesizing protein S; and 3) from selected samples of normal and protein S deficient plasma . The ELISA described here was about 12-fold more sensitive and 40-fold more cost effective when compared to a commercial ELISA kit . Thus the assay provided a sensitive, specific, precise and economical method useful for the measurement of the nanogram amounts of protein S commonly encountered in cell culture fluids.

Enzyme Microb Technol, 1994 Nov, 16(11), 957 - 63
Principles of an efficient new method for the removal of ammonia from animal cell cultures using hydrophobic membranes; Schneider M et al.; To overcome ammonia inhibition in animal cell culture, a method utilizing porous polytetrafluoroethylene membranes with an associated pH gradient was investigated . Model experiments without cells were carried out in three different reactor configurations suitable for animal cell culture processes to characterize mass transfer properties of the process . The actual fluxes of ammonia across the membrane are of the same order of magnitude as the typical ammonia production rate in an animal cell process, thus indicating the potential of the method for preventing the accumulation of toxic ammonia in cell culture.

J Androl, 1994 Nov-Dec, 15(6), 543 - 50
Testosterone regulation of proto-oncogene c-myc expression in primary Sertoli cell cultures from prepubertal rats; Lim K et al.; The expression of c-myc has been associated with cell proliferation through changes of nuclear function . To evaluate the possibility that the proto-oncogene c-myc plays a role in testosterone-dependent gene regulation, the effects of testosterone on the expression of c-myc have been investigated in primary Sertoli cell cultures . Testosterone increased c-myc mRNA levels, with maximal stimulation reached in 16 hours . The induction of c-myc mRNA was dependent on the concentration of testosterone . Testosterone-induced c-myc mRNA levels were also increased in cells after addition of cycloheximide but reduced by actinomycin-D pretreatment . Even in the absence of hormone in culture medium, c-myc mRNA was clearly detectable in Sertoli cells from 8-day-old rats but hardly detectable in cells from 14 and 28 days of age . Testosterone stimulated c-myc mRNA expression in the Sertoli cells from only 8-/and 14-day-old rats . These results suggest that testosterone induces c-myc mRNA levels in the primary Sertoli cells from prepubertal rats, and then transient expression of c-myc may be responsible for some of the regulatory roles of testosterone-dependent genes in the Sertoli cells . The biological significance of testosterone-dependent c-myc induction is not known.

Eur J Clin Microbiol Infect Dis, 1994 Nov, 13(11), 994 - 9
Animal and cell-culture models for the study of mycobacterial infections and treatment; Orme IM et al.; Emerging problems with the treatment of infections caused by Mycobacterium avium and Mycobacterium tuberculosis require the development of new models, both in vitro and in vivo, in which new chemotherapeutic and immunotherapeutic approaches can be tested . In this brief review, the use of cell culture models, in which drugs can be tested for their capacity to inhibit mycobacterial growth within the infected host macrophage, and new models in vivo in which drugs and/or cytokines can be tested in infected mice are discussed . In this latter case, new emerging mouse models include animals with engineered gene disruptions, in which severely disseminated infections can be produced, thus mimicking events in severely immunocompromised human patients.

Regul Pept, 1994 Oct 21, 53(3), 203 - 9
Immunoregulatory properties of hexapeptide isolated from porcine bone marrow cell culture; Mikhailova A et al.; Myelopeptide 1 (MP-1) is hexapeptide originally isolated from porcine bone marrow cell culture . It was synthesized and its immunoregulatory properties were studied . MP-1 caused a 1.5-2-fold dose-dependent increase of antibody production in the culture of mouse immune lymph node cells . It abolished Con A induction of T suppressors in the suspension of mouse spleen cells and counteracted the inhibitory effect of T suppressors on antibody production . The inoculation of MP-1 (1 x 10(-9) g/mouse) to mice two weeks after their gamma-irradiation (2 Gy) resulted in an increase of antibody production up to 80.2 +/- 15.5% as compared to that in the irradiated control 37.6 +/- 12.0% . Immunofluorescent analysis revealed the specific binding of MP-1 with receptors on the target cells in the suspension of mouse spleen cells . It is supposed that MP-1 participates in the immunoregulatory processes in the living organism.

Neuroreport, 1994 Oct 3, 5(15), 1946 - 8
Cell density and exogenous CNTF affect CNTF mRNA levels in glial cell cultures; Meyer V et al.; Regulation of CNTF mRNA was investigated in primary cortical astrocytes cultured from newborn rats and in C6 glioma cells . Northern blot analysis indicated that semi-confluent astrocyte cell cultures . CNTF added to confluent astrocyte cultures down-regulated CNTF message in a dose-dependent fashion . In contrast, when CNTF was given to semi-confluent astrocyte cultures the level of CNTF mRNA was up-regulated . C6 glioma cells also showed a cell density-dependent expression of CNTF: cells from confluent cultures expressed detectable amounts of CNTF mRNA whereas those from semi-confluent cultures did not . Thus, CNTF mRNA expression by astroglial and glioma cells is regulated by cell contact and exogenous CNTF in vitro.

Virology, 1994 Oct, 204(1), 60 - 8
Mutational events in consecutive passages of hepatitis A virus strain GBM during cell culture adaptation; Graff J et al.; In order to study the adaptation of hepatitis A virus (HAV) in cell culture, we examined the mutational events of the genome in early passages of HAV strain GBM propagated either in FRhK-4 cells (fetal rhesus monkey kidney-derived) or in human embryonic kidney (HEK) and human embryonic fibroblast cells (HFS) in relation to their growth characteristics . Sequence analysis of the nucleotide region encoding 2B, 2C, and the beginning of 3A as well as the nucleotide region encompassing the 5' noncoding region (5'NCR) of the genome was performed on consecutive virus passages after amplification of the viral RNA from the cell culture supernatant by antigen-capture PCR . By the 2nd passage of the GBM variants cultured in FRhK-4 or in HEK cells we found a mutation at nucleotide position 3889 (2B coding region) which results in an amino acid substitution from alanine to valine . Further mutations present in the 2B/2C region of the cell culture-adapted GBM variants differ from each other and occur after the 10th or even the 40th virus passage . Another early change, an in frame deletion of nine nucleotides in the 3A region, appeared in the 5th virus passage only in GBM cultured on FRhK-4 cells . This genome region showed different mutations in the virus passages on HEK and HFS cells . The 5'NCR of the cell culture-adapted GBM variants, in contrast, did not show any mutations before the 8th virus passage . The faster and more efficient growth of the HAV strain GBM during successive propagation on cell cultures seems to correlate with the appearance of mutations in the investigated genome regions.

Stroke, 1994 Oct, 25(10), 2085 - 9; discussion 2089-90
Dipyridamole increases oxygen-glucose deprivation-induced injury in cortical cell culture; Lobner D et al.; BACKGROUND AND PURPOSE: Adenosine transport inhibitors attenuate ischemic central neuronal damage in vivo, but the locus of this protective action is presently unknown . To help address the question of whether adenosine transport inhibitors have a protective effect directly on brain parenchyma, we tested the effect of the adenosine transport inhibitor dipyridamole on neuronal loss induced by oxygen-glucose deprivation in vitro . METHODS: Murine cortical cultures were exposed to combined oxygen and glucose deprivation, N-methyl-D-aspartate, or kainate . The extracellular concentrations of glutamate and adenosine were measured by high-performance liquid chromatography; neuronal cell death was assessed by morphological examination and measurement of lactate dehydrogenase release . RESULTS: Cultures exposed to oxygen-glucose deprivation for 30 to 75 minutes exhibited an insult-dependent increase in extracellular adenosine, followed shortly by an increase in extracellular glutamate and 24 hours later by neuronal death . Addition of the A1 receptor antagonist 8-cyclopentyltheophylline during oxygen-glucose deprivation enhanced both glutamate release and neuronal damage . Addition of 10 mumol/L dipyridamole decreased extracellular adenosine and also enhanced extracellular glutamate and neuronal death . In contrast, dipyridamole increased the levels of extracellular adenosine stimulated by N-methyl-D-aspartate or kainate . CONCLUSIONS: These results are consistent with the idea that endogenous adenosine has a neuroprotective effect directly on cortical cells exposed to oxygen-glucose deprivation . However, inhibition of adenosine transport with dipyridamole was surprisingly not an effective strategy for enhancing this protective effect . The beneficial effects of adenosine transport inhibitors observed in vivo may be mediated indirectly--for example, by effects on the vasculature.

Cardiovasc Res, 1994 Oct, 28(10), 1500 - 6
Effects of 21-aminosteroids in neonatal rat cardiac myocyte cell cultures exposed to free radicals; Burton KP; OBJECTIVE: The aim was to test a group of 21-aminosteroids, U74006F, U75412E, and U74500E, known as lazaroids, for their ability to prevent alterations in neonatal rat cardiac myocytes exposed to solutions containing a xanthine oxidase mediated free radical generating system . METHODS: Myocytes were either left untreated (non-treated cultures) or pretreated for 15 min with the drug vehicle or one of the lazaroids . Myocytes were either examined as non-exposed control cultures or exposed to the free radical generating system for 60 min . Measurement of {3H}arachidonate label in a lipid extract of the culture medium and release of lactate dehydrogenase (LDH) into the medium were analysed . RESULTS: Myocytes not treated with lazaroids and vehicle treated myocytes exposed to free radicals showed a significant release of {3H}arachidonate and lactate dehydrogenase (LDH) . At a dose 1 x 10(-5) M, all lazaroid treated myocytes showed significantly lower release of {3H}arachidonate measured in the total lipid extract compared to the non-treated or vehicle treated cultures . Release of {3H}arachidonate was significantly lower for the myocytes treated with U74006F and U74500E at 1 x 10(-6) M concentration . Only the U74006F treated myocytes showed protection at 1 x 10(7) M . LDH release was significantly attenuated at a dose of 1 x 10(-5) M for the U75412E treated myocytes and at 1 x 10(-5) and 1 x 10(-6) M for the U74500E treated myocytes compared to the myocytes not pretreated with a lazaroid and exposed to free radicals . CONCLUSIONS: Lazaroids provide protection against the release of {3H}arachidonate and LDH from myocardial cells exposed to free radical mediated injury . U74006F appeared to have the higher efficacy, at equal molar concentrations, in protecting against the release of {3H}arachidonate, whereas U74500E was observed to have the higher potency in inhibiting LDH release.

Biotechnol Appl Biochem, 1994 Oct, 20 ( Pt 2), 291 - 307
Multichannel flow-injection-analysis biosensor system for on-line monitoring of glucose, lactate, glutamine, glutamate and ammonia in animal cell culture; Blankenstein G et al.; The application of a chemiluminometric method for the on-line monitoring of a hybridoma cell culture is described . Enzyme sensors for glucose, lactate, glutamine, glutamate and ammonia, based on oxidase-catalysed reactions, were developed and connected to a flow-injection-analysis (f.i.a.) biosensor . H2O2 produced by the oxidase-catalysed enzyme reaction was detected by luminol chemiluminescence with a fibre-optic H2O2 biosensor . The system has been used to monitor animal cell cultures . A continuous hybridoma cell cultivation for the production of monoclonal antibodies is presented as an example . It was possible to monitor the bioprocess over a period of 15 days . A complete analysis of all five components could be performed within 42 min . The enzyme sensors were stable during the whole cultivation time without significant loss of activity . The computer-controlled biosensor f.i.a . works with good reliability . The precision for all five components ranged between 2.2 and 4.5% . It was possible to determine glutamine in one step using an anti-interference enzyme reactor . Endogenous glutamate was completely removed up to a level of 0.5 mM.

Biochem J, 1994 Oct 1, 303 ( Pt 1), 89 - 96
Effects of the S-adenosylmethionine decarboxylase inhibitor, 5'-({(Z)-4-amino-2-butenyl}methylamino)-5'-deoxyadenosine, on cell growth and polyamine metabolism and transport in Chinese hamster ovary cell cultures; Byers TL et al.; The regulation of polyamine transport and the roles of polyamine transport and synthesis in cell growth were investigated using cultured Chinese hamster ovary (CHO) cells and CHOMG cells which are mutants lacking polyamine-transport activity . Metabolically stable methylated polyamine analogues were used to measure polyamine accumulation, and the irreversible S-adenosyl-L-methionine decarboxylase inhibitor, 5'-({(Z)-4-amino-2-butenyl}methylamino)-5'-deoxyadenosine (AbeAdo), was used to inhibit synthesis . Exposure to AbeAdo lead to a dose-dependent decrease in growth for both cell lines, although CHOMG cells were more sensitive . Intracellular putrescine levels were greatly increased in AbeAdo-treated CHO cells and to a lesser extent in CHOMG cells, whereas intracellular spermidine and spermine levels were substantially reduced in both . Treatment with AbeAdo increased putrescine content in the culture medium to a much greater extent in CHOMG cultures indicating that a portion of the excess putrescine synthesized in response to AbeAdo treatment is excreted, but that CHO cells salvage this putrescine whereas it is lost to CHOMG cells which cannot take up polyamines . AbeAdo treatment increased polyamine transport into CHO cells despite high intracellular putrescine, suggesting that spermidine and/or spermine, and not putrescine, are the major factors regulating transport activity . The accumulation of either 1-methylspermidine or 1,12-dimethylspermine was significantly increased by AbeAdo treatment . Accumulation was increased even further when protein synthesis was blocked by cycloheximide, indicating that a short-lived protein is involved in the regulation of polyamine uptake . In the presence of cycloheximide and AbeAdo or alpha-difluoromethylornithine, methylated polyamine derivatives accumulated to very high levels leading to cell death . These results show that the polyamine-transport system plays an important role in retaining intracellular polyamines and that down-regulation of the transport system in response to increased intracellular polyamine content is necessary to prevent accumulation of toxic levels of polyamines.

J Nutr, 1994 Oct, 124(10), 1942 - 9
Uptake, transport and metabolism of exogenous nucleosides in intestinal epithelial cell cultures; He Y et al.; The uptake and transport of nucleosides (adenosine, cytidine, guanosine, thymidine, uridine and inosine) were examined in intestinal cell lines (Caco-2 and IEC-6) . Well-differentiated Caco-2 cells took up significantly more uridine and thymidine than less-differentiated cells over 24 h . These differences were reflected in the resulting trichloroacetic acid-soluble (nucleotide, nucleoside and nucleobase) pool sizes rather than in differences in nucleic acid incorporation . IEC-6 cells have smaller soluble pools than Caco-2 cells, and the uptake of nucleosides over 60 min reflected this difference . Caco-2 cells transported significantly more thymidine, cytidine, guanosine, adenosine and inosine into acid-soluble pools than IEC-6 cells . Caco-2 cells were grown on filters to examine the transcellular transport of nucleosides and to examine the chemical form in which nucleosides appeared at the basolateral aspect of the cell monolayer . The pattern of transport varied for individual nucleosides . The transport of cytidine and guanosine was significantly greater in the apical-to-basolateral direction than in the opposite direction over 2 h . No purine nucleoside was transported intact across the Caco-2 cell monolayer in either direction, with the majority of the transported material appearing as nucleobases . Nucleobases also represented the main metabolites of pyrimidines transported . However, some cytidine and uridine were detectable, but each constituted < 20% of transported material . Nucleosides are therefore transported by enterocytes but metabolized during transport.

Exp Cell Res, 1994 Oct, 214(2), 543 - 50
Establishment of adult rat Schwann cell cultures: effect of b-FGF, alpha-MSH, NGF, PDGF, and TGF-beta on cell cycle; Peulve P et al.; Mitotically active Schwann cells isolated from adult rat sciatic nerve segments were cultivated, using a bivariate BrdU/DNA flow cytometry analysis, to test the effect of b-FGF (10 ng/ml), alpha-MSH (10 ng/ml), NGF (10 ng/ml), PDGF-AB (10 ng/ml), and TGF-beta 1 (10 ng/ml) on the cell cycle . Compared to control or cholera toxin-treated cultures, no significant differences (P < 0.05; Newmann-Keuls test) were observed in the proportion of G0G1 cells, S cells, G2M cells, and in the LI when alpha-MSH, NGF, PDGF-AB, or TGF-beta 1 were present . The S phase duration varied from 6.16 +/- 0.24 to 7.86 +/- 2.6 h, and the deduced potential doubling time was estimated at between 46.70 +/- 7.09 and 56.05 +/- 7.55 h . In contrast, when b-FGF was added to the culture, the cell cycle was significantly modified, and the proportion of G0G1 cells decreased from 68-77% to 59-64%, while the proportion of S cells increased from 14-16% to 24.0-26.4% . Although S phase duration was not significantly changed (6.02 +/- 0.36 h), the 1.7- to 2.8-fold LI increase reduced the potential doubling time to 25.99 +/- 6.13 h . We conclude from these results that only b-FGF-induced adult rat Schwann cells dramatically reenter in cell cycle and that this growth factor may be an axonally derived signal-promoting mitogenesis after nerve injury.

Clin Exp Immunol, 1994 Oct, 98(1), 158 - 62
The development of transplant coronary artery disease after cardiac transplantation is correlated with a predominance of CD8+ T lymphocytes in endomyocardial biopsy derived T cell cultures; Jutte NH et al.; Long-term survival of heart transplant recipients is limited by the development of transplant coronary artery disease (TCAD) . We analysed whether the development of TCAD is correlated with the incidence of acute rejection episodes, with the formation of anti-HLA antibodies or with the composition and function of T lymphocyte cultures derived from endomyocardial biopsies . TCAD was assessed by visual analysis of annually performed coronary angiograms and defined as the presence of all vascular changes, including minor wall irregularities . One year after transplantation, 31 of the 77 patients studied had TCAD (40%) . The median age and mean number of HLA mismatches in patients with or without TCAD were highly comparable . The patient groups did not differ in incidence of acute rejection episodes, nor in percentage of endomyocardial biopsies yielding T cell cultures . At 1 year after transplantation, lymphocyte cultures from 18/31 TCAD+ patients (58%) and 27/46 TCAD- patients (57%) were analysed . The TCAD+ patients had, compared with the TCAD- patients, a higher median percentage of CD8+ T cells (71% versus 25%, P = 0.06) and a lower median percentage of CD4+ T cells (4% versus 40%, P = 0.04) . Similar differences were found in a longitudinal analysis of the culture results of endomyocardial biopsies (EMBs) obtained during the first year . The cytotoxic reactivity of the cultures against donor HLA class I or class II antigens was comparable in the two groups, although a difference in recognition of heart specific antigens remains possible . The fact that EMB-derived cultures from TCAD+ and TCAD- patients differed in T cell phenotype populations gives some support to the hypothesis that cellular immunological processes are involved in the development of TCAD . However, while the median values differed, the overlap of the percentages of CD8+ cells in cultures from TCAD- and TCAD+ patients shows that other factors besides CD8+ T cells also play a role.

Glycobiology, 1994 Oct, 4(5), 611 - 6
Purification and characterization of alpha-L-fucosidase from Chinese hamster ovary cell culture supernatant; Gramer MJ et al.; In this study, alpha-L-fucosidase from Chinese hamster ovary (CHO) cell culture supernatant was purified 11 200-fold to apparent homogeneity to assess the rate of fucose hydrolysis from oligosaccharide and glycoprotein substrates . The fucosidase migrated as a single band of 51 kDa on SDS-PAGE and is a glycoprotein, as determined by retention on concanavalin A-Sepharose, and by lectin blotting with concanavalin A . Hydrolysis of the artificial substrate 4-methyl-umbelliferyl-alpha-L-fucoside (4MU-Fuc) followed simple Michaelis-Menten kinetics, and was competitively inhibited by free fucose and by two known fucosidase inhibitors, fucosylamine and deoxyfuconojirimycin . Hydrolysis of fucose from oligosaccharides including 2'-fucosyllactose, 3-fucosyllactose, Fuc alpha(1,6)GlcNAc and pooled gp120 oligosaccharides with the Fuc alpha(1,6)GlcNAc linkage also followed simple Michaelis-Menten kinetics . However, activity toward 4MU-Fuc was optimal near pH 7, while activities toward the oligosaccharide substrates were optimal near pH 5 . No fucose was released from the recombinant CHO cell-produced glycoproteins gp120 or soluble CD4 with the Fuc alpha(1,6)GlcNAc linkage, or from human serum alpha 1-acid glycoprotein with the Fuc alpha(1,3)GlcNAc linkage . Enzymatic removal of sialic acid and galactose from gp120 oligosaccharides did not alter the susceptibility of gp120 to fucosidase attack . These data suggest that released CHO cell fucosidase does not contribute to the heterogeneity of fucosylation that has been observed in CHO cell culture-produced glycoproteins.

Biochem Mol Biol Int, 1994 Oct, 34(4), 809 - 16
Tentative evidence of a rosmarinic acid peroxidase in cell cultures from lavandin (Lavandula x intermedia) flowers; Lopez-Arnaldos T et al.; The oxidative stability of rosmarinic acid (alpha-O-caffeoyl-3,4-dihydroxyphenyllactic acid) in lavandin (Lavandula x intermedia) cell cultures was studied in an attempt to explain the decrease in the rosmarinic acid content of aging cell cultures, a process which is associated with the appearance of brown pigments . The oxidation of rosmarinic acid by a partially purified protein fraction was followed spectrophotometrically and by HPLC . The results showed that rosmarinic acid oxidation was almost totally dependent on the presence of H2O2 and protein, and that brownish products were the results of this oxidation, resembling those shown by aging cell cultures . Since this protein fraction contains peroxidase activities and shows the total absence of tropolone-sensitive polyphenoloxidase (catecholase) and laccase activities, rosmarinic acid oxidation is tentatively proposed to be caused by a peroxidase-like activity . These results support the existence of a rosmarinic acid peroxidase in cell cultures of lavandin flowers, which may be involved in the oxidative destruction of rosmarinic acid, and which may also be responsible for the formation of brown pigments during aging, lowering the yields of rosmarinic acid.

New Microbiol, 1994 Oct, 17(4), 341 - 4
The propagation of a porcine hemagglutinating encephalomyelitis virus in swine kidney cell cultures; Kadoi K et al.; An established cell line, KSEK6, derived from swine embryo kidney proved to be a suitable host for the propagation of a hemagglutinating encephalomyelitis virus, strain 67N . Infected cells showed clear cytopathic effect as early as 24 hours incubation at 37 degrees C . Plaques easily visible to the naked eye were also produced under agar overlay medium . The infective titer of 3rd passage level in the cells was in the order of 10(8) PFU per ml . Detecting antibodies against this virus strain in swine sera was considered to be more accurate by neutralization test than by hemagglutination inhibition.

Antiviral Res, 1994 Oct, 25(2), 123 - 31
Anti-influenza virus activity of the neuraminidase inhibitor 4-guanidino-Neu5Ac2en in cell culture and in human respiratory epithelium; Hayden FG et al.; The anti-influenza activity of the neuraminidase inhibitor 4-guanidino-Neu5Ac2en (4-G-NAc) was determined in Madin-Darby canine kidney (MDCK) cells by yield reduction and ELISA and in explants of human respiratory epithelium by yield reduction . In MDCK cells, 50% inhibitory concentrations (EC50) averaged 0.5 microgram/ml for influenza A/Virginia/88(H3N2) and 0.04 microgram/ml for A/Texas/36/91(H1N1) by ELISA, and < 0.01 microgram/ml for influenza A/Virginia by yield reduction . In human adenoid explants, concentrations causing at least 1.0 log10 TCID50/ml decrease in yield (EC90) at 48 h were < 0.01 microgram/ml for A(H1N1) and A(H3N2) viruses and 0.25 microgram/ml for B/Hong Kong/5/72 . 100 micrograms/ml 4-G-NAc did not inhibit outgrowth of human adenoid epithelium at 6 days, whereas ribavirin 10 micrograms/ml reduced outgrowth by > 50% . 4-G-NAc is a potent and selective inhibitor of clinical isolates of influenza A and B viruses in human respiratory epithelium.

Toxicon, 1994 Oct, 32(10), 1287 - 91
Comparison of the hepatotoxicity of toxin T-514 of Karwinskia humboldtiana and its diastereoisomer in primary liver cell cultures; Garza-Ocanas L et al.; Toxin T-514 of Karwinskia humboldtiana has been demonstrated to be hepatotoxic in vivo and in vitro . Recently a diastereoisomer of T-514 has been isolated . In the present study we have evaluated and compared the in vitro hepatoxicity of the diastereoisomer of T-514 using primary cultures of rat hepatocytes . Cytotoxicity was evaluated by release of cytoplasmic enzyme lactate dehydrogenase (LDH), and mitochondrial metabolic function (MTT reduction) . The diastereoisomer was shown to be almost as hepatoxic in vitro as toxin T-514.

Prostaglandins Leukot Essent Fatty Acids, 1994 Oct, 51(4), 241 - 4
The effect of zearalenone and 17 beta-estradiol on prostacyclin and thromboxane production in cell cultures from human umbilical cord veins; Neuer A et al.; The effect of zearalenone, a nonsteroidal mycotoxin with estrogenic activity, and of 17 beta-estradiol on prostacyclin and thromboxane production in human endothelial cells was investigated . Zearalenone stimulated prostacyclin production in low concentrations (10(-7) and 10(-8)M) and inhibited it at a higher concentration (10(-5)M) . Estradiol alone in the concentration range 10(-5)-10(-8)M had no clear-cut effect on the prostacyclin production . The combination of both substances effected changes in prostacyclin production similar to that from zearalenone alone; with the exception of estradiol at a concentration of 10(-6)M which enhanced the effect of zearalenone . No distinct changes in the thromboxane production from the two substances could be found, either alone or in combination.

Antimicrob Agents Chemother, 1994 Oct, 38(10), 2404 - 8
Activity of (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine against human cytomegalovirus when administered as single-bolus dose and continuous infusion in in vitro cell culture perfusion system; Moore MR et al.; HPMPC {(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine} is a potent inhibitor of human cytomegalovirus (HCMV) replication as determined by conventional tissue culture methods in which the drug concentration remains constant over time . Previous studies have shown HPMPC to have a long intracellular half-life . Despite its relatively short extracellular half-life, HPMPC might provide significant anti-HCMV activity long after the elimination of the drug by first-order kinetics . We addressed this hypothesis by measuring the activity of HPMPC in a novel cell culture perfusion system . This system allows us to compare the activity of HPMPC when given as a continuous infusion with its activity when given as a single-bolus dose followed by elimination that simulates the drug's in vivo pharmacokinetics . We show that continuous infusions maintaining maximum concentrations (Cmaxs) of 0.05, 0.10, 0.31, and 1.0 micrograms/ml and achieving areas under the drug concentration-time curves (AUCs) of 8.4, 17, 50, and 162 micrograms.h/ml, respectively, result in 27, 56, 63, and 88% inhibition of viral DNA accumulation, respectively, compared with an untreated control . Single-bolus doses achieving Cmaxs of 0.10, 1.25, 3.0, and 7.7 micrograms/ml with an elimination half-life of 20 h achieved AUCs of 2.4, 32, 78, and 138 micrograms.h/ml and resulted in 0, 48, 69, and 87% inhibition of HCMV DNA accumulation . Single-bolus doses achieving Cmaxs of 3.9 and 12 micrograms/ml with an elimination half-life of 6.5 h achieved AUCs of 34 and 105 micrograms.h/ml, respectively, resulting in 15 and 76% inhibition of viral DNA accumulation . Comparison of Cmax-versus-effect curves for these three regimens suggests that maximum concentration is not the only important pharmacokinetic determinant of HPMPC's antiviral activity . Similar comparisons of AUC-versus-effect curves for continuous and bolus dosing suggest that the AUC is an important determinant of antiviral activity for AUCs greater than 100 micrograms . h/ml . We conclude that single-bolus doses of HPMPC potently inhibit HCMV DNA accumulation but that this activity is more heavily influenced by the AUC than the Cmax at the upper end of the AUC range tested . At lower AUCs, some other parameter may be the primary determinant of antiviral activity . Our cell culture perfusion system provides a novel, efficient, and convenient method for addressing questions relating the effects of constantly changing drug concentrations to antiviral effects.

Pflugers Arch, 1994 Oct, 428(3-4), 296 - 9
A novel two-compartment culture dish allows microscopic evaluation of two different treatments in one cell culture simultaneously . Influence of external pH on Na+/Ca2+ exchanger activity in cultured rat cardiomyocytes; Atsma DE et al.; A new type of culture dish containing two separate compartments is described, that can be used in high-magnification microscopy . Using the dish, two halves of a single-cell culture, grown on a standard coverslip, can be exposed to different treatments simultaneously, allowing the effect of one treatment to be compared with that of the other treatment in the same culture . This way, the natural variability that might exist between different individual cultures is circumvented . In addition, by simultaneously conducting two experiments per dish, the number of experiments needed can be decreased . This both reduces the time to complete a series of experiments and allows the optimal use of specimens that are difficult to obtain, such as human material . We found there is an excellent barrier between the two compartments for lipophilic and hydrophilic compounds, and for low-molecular-mass cations . To illustrate the use of the dish we describe the influence of external pH on the activity of the Na+/Ca2+ exchanger in intact cultured neonatal rat ventricular cardiomyocytes . The intracellular free calcium concentration ({Ca2+}i) in the cardiomyocytes, measured using fura-2 and imaging fluorescence microscopy, was studied during sodium-free incubation . The resulting rise in {Ca2+}i at pH 7.4 in one compartment was compared with that in the other compartment in which the pH was either 6.0, 7.0, 7.4 or 8.0 . It was found that below pH 7.4, Na+/Ca2+ exchanger activity was diminished, whereas at pH higher than 7.4 the Na+/Ca2+ exchanger activity was increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Curr Opin Biotechnol, 1994 Oct, 5(5), 546 - 9
The effect of cell-culture conditions on the oligosaccharide structures of secreted glycoproteins; Andersen DC et al.; Glycoprotein oligosaccharide structure influences numerous important protein properties . In recent years, a number of studies have demonstrated that cell-culture methodology can significantly affect the oligosaccharide structures of recombinant proteins and antibodies, and, in the past year in particular, several of the specific environmental variables responsible for these effects have been identified.

Rev Latinoam Microbiol, 1994 Oct-Dec, 36(4), 231 - 41
{Identification of enterotoxins and cytotoxins of Escherichia coli by Vero cell culture and solid-phase hybridization (colony blot)}; Giono-Cerezo S et al.; We studied 40 E . coli strains from meat and hamburger and 64 strains from stools of people: 14/64 from traveler's diarrhea and 50/64 from infants with and without diarrhea . We want to know if they could be producing of cytotoxin and enterotoxin in Vero culture cells as well as phenotypic and genotypic relationships . The serotypes that we isolated were different than O:157H:7 . We found 3 cytotoxic strains, 77 heat labile enterotoxic strains on Vero culture cells, and 19 E . coli strains with cytotoxin and LT enterotoxin . One strain isolated from infant with diarrhea was negative sorbitol but cytotoxic effect and it was O:55 serotype . The colony blot and cytotonic were found in 90 (86.5%) E . coli strains . The sensitivity of colony blot was 93.75%.

Cell Mol Neurobiol, 1994 Oct, 14(5), 557 - 68
Design and application of antisense oligonucleotides in cell culture, in vivo, and as therapeutic agents; Brysch W et al.; 1 . Synthetic oligonucleotides can inhibit the expression of a gene in a sequence specific manner on the transcriptional and translational level . These molecules are usually referred to as antisense oligonucleotides . 2 . Antisense mediated inhibition of gene expression is a valuable tool to analyze the function of a gene in vivo and can also be used for therapeutic gene suppression . 3 . A number of factors such as the mode of action, specificity, chemistry, and pharmacology must be carefully considered for the design and successful application of antisense oligonucleotides . 4 . Assay systems and controls must be chosen as to assure that the observed biological effects of antisense oligonucleotides do in fact reflect the result of a specific gene inhibition . 5 . This article critically discusses these factors in view of the literature and our own experience with a wide range of cell types and animal models, targeting different genes . The emphasis is on the use of phosphorothioate oligodeoxynucleotides in cell cultures, in vivo, and as potential drugs.

J Antimicrob Chemother, 1994 Oct, 34(4), 589 - 94
Susceptibility in cell culture of feline immunodeficiency virus to eighteen antiviral agents; Smyth NR et al.; The in-vitro susceptibilities of two strains of feline immunodeficiency virus to 18 antiviral agents were determined in two cell lines . In terms of inhibiting p24 antigen production, the nucleoside-analogue reverse transcriptase inhibitors were the most effective compounds . Inhibition was also observed with aurintricarboxylic acid, phosphonoformate and butyldeoxynorjirimycin, but not with the other agents tested.

Antimicrob Agents Chemother, 1994 Oct, 38(10), 2465 - 8
Anti-human immunodeficiency virus type 1 activities of U-90152 and U-75875 in human brain cell cultures; Peterson PK et al.; Antiviral activities of the reverse transcriptase inhibitors U-90152 and 3'-azido-2',3'-dideoxythymidine and the protease inhibitor U-75875 were compared in two culture models of human immunodeficiency virus type 1 brain infection . In a model involving acutely infected microglial cells, U-90152 was the most active, whereas in a model using chronically infected promonocytes, U-75875 was the most active.

Neurochem Int, 1994 Oct, 25(4), 377 - 84
Excitotoxicity of glutamate and four analogs in primary spinal cord cell cultures; Wells K et al.; Continuous glutamate exposure produced widespread neuronal damage in mixed whole dissociated murine spinal cord cell cultures . Ethidium bromide and acridine orange staining revealed that a 24 h glutamate exposure produced nearly 98% neuronal cell death but the underlying glia were spared . Continuous exposure to glutamate, N-methyl-D-aspartate (NMDA), kainate and quisqualate produced time-dependent and dose-dependent cell death as measured by the assay of lactate dehydrogenase activity in the cell culture media . Glutamate (500 microM), NMDA (100 microM) and kainate (500 microM) were equally neurotoxic . In contrast, quisqualate (100 microM) was only partially neurotoxic compared to the other glutamate analogs . The neurotoxicity of glutamate was blocked by the NMDA antagonist, MK-801 . The neurotoxicity of kainate and quisqualate was blocked with the non-NMDA antagonist CNQX . Continuous exposure to (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) was not neurotoxic, even at concentrations up to 1 mM.

J Immunol, 1994 Sep 15, 153(6), 2579 - 91
Characterization and purification of a macrophage-triggering factor produced in Mycoplasma arginini-infected L5178Y cell cultures; Yang G et al.; The supernatant of Mycoplasma arginini-infected murine L5178Y T lymphoma cell cultures (SN-L51) synergizes with small concentrations of IFN-gamma to activate murine peritoneal, thioglycollate-elicited macrophages (M phi) to exhibit cytostatic activity against tumor cells . Treatment of M phi with IFN-gamma and SN-L51 sequentially, but not in the reverse order, activates M phi, which indicates that SN-L51 contains a M phi-triggering factor (MTF) . MTF activity could be inhibited by small concentrations of prostaglandin E2, but not by polymyxin B . M phi activated by IFN-gamma plus MTF produce cytostatic effects on tumor cells through a nitric oxide-dependent pathway . MTF activity in SN-L51 is associated with infection of L5178Y cells by M . arginini . Mycoplasma-free L5178Y cells do not produce MTF activity, infection of these L5178Y cells with M . arginini generates the activity, and supernatants of pure M . arginini cultures contain MTF activity . MTF activity is thermostable and resistant to acid, dilute alkali, proteases, and nucleases . MTF was partially purified by ammonium sulfate precipitation, chromatography, electrophoresis, and electroelution . On 12.5% SDS-urea gels, MTF activity migrated with a molecular mass of 2.5 to 4 kDa . MTF activity and the silver staining of this band was resistant to proteinase K; however, Coomassie staining of this band was abolished by proteinase K . The combined data suggest that MTF is either a stable peptide or a peptide linked to lipid or carbohydrate.

Biochem Pharmacol, 1994 Sep 15, 48(6), 1145 - 54
Antiproliferative activity of the topoisomerase I inhibitors topotecan and camptothecin, on sub- and postconfluent tumor cell cultures; Pizao PE et al.; We have assessed the antiproliferative effects of a 24-hr exposure to the topoisomerase I inhibitors, topotecan and camptothecin, on two colon and one ovarian human tumor cell lines, cultured as subconfluent and as multilayered postconfluent cultures . Chemosensitivity was measured by the sulforhodamine B assay . In general, postconfluent cultures were less sensitive to these agents, yielding GI50S (drug concentrations inhibiting growth by 50%) from 1.2 to more than 6000 times higher than those of subconfluent cultures . Both compounds displayed similar effects on subconfluent cells, inducing complete growth inhibition at concentrations ranging from 0.03 to 0.5 microM . Topotecan, however, was more potent than camptothecin in two out of the three cell lines tested as multilayered postconfluent cultures . Topoisomerase I mRNA expression on postconfluent cultures was 50% lower than on subconfluent cultures in the three cell lines studied . However, we did not detect any reproducible differences in topoisomerase I protein expression and in relaxation activity of supercoiled DNA between the two types of cultures . From accumulation experiments it appeared that the peak concentration of the lactone form of topotecan as well as the area under the concentration-time curve (AUC) were 2-fold higher in the monolayer than in the multilayer cultures . Therefore, the differences in the activity of topoisomerase I inhibitors under our experimental conditions were likely due to a decreased rate of proliferation of postconfluent cells, associated with a reduction in drug uptake.

J Immunol Methods, 1994 Sep 14, 174(1-2), 281 - 96
In situ hybridization for localization of mRNAs in mononuclear phagocytes in cell culture and tissue sections; Vignaud JM et al.; We report an in situ hybridization procedure to detect in cell preparations and tissue sections messenger RNAs coding for mononuclear phagocyte proteins . The multistep procedure is described for use in conjunction with isotopic and non-isotopic probes.

Exp Cell Res, 1994 Sep, 214(1), 177 - 88
Fate of a headless vimentin protein in stable cell cultures: soluble and cytoskeletal forms; Andreoli JM et al.; The non-alpha-helical head domains of cytoskeletal intermediate filaments (IFs) are considered to play an important role in IF assembly and stability . We have investigated the fate of a "headless" mutant vimentin protein in cell types that either lack cytoplasmic IFs or contain preexisting IF networks (keratin and vimentin) . Stable clones expressing a transfected headless vimentin cDNA were individually analyzed in order to avoid variabilities introduced by transient transfection and to compare the levels and effects of the mutant vimentin protein more accurately . In cells lacking IFs, the mutant protein existed in a diffuse, soluble form as determined by immunofluorescence and biochemical protein fractionation . In cells possessing vimentin IFs, the headless vimentin was highly dispersed throughout the cytoplasm, including lamellopodia . Expression levels in individual clones were as high as sevenfold greater than the endogenous vimentin component . Although the majority of the headless vimentin was highly soluble, a residual portion of the mutant vimentin colocalized with vimentin filaments and consistently comprised about 25% of the cytoskeletal vimentin network . These results demonstrate that the mutant protein can be stably expressed at relatively high levels without deleterious cellular effects or disruption of endogenous vimentin filaments . The observed specific ratio of mutant to wild-type vimentin (1:3) in the cytoskeleton supports IF in vivo assembly via specific hybrid tetramer formation and, further, that at least three intact head domains are required for competent tetramer formation and IF assembly.

Cornea, 1994 Sep, 13(5), 435 - 9
Activity of ganciclovir against human adenovirus type-5 infection in cell culture and cotton rat eyes; Trousdale MD et al.; The most common causes of acute viral infections of the eye for which there are no effective antiviral drugs are the adenoviruses . Until recently, pathogenesis studies and antiviral drug testing for adenovirus-induced ocular disease were not practical because no animal model was available . However, new animal models for human adenovirus-induced ocular and respiratory infections have now made such studies possible . We assessed the in vitro and in vivo activity of ganciclovir against a genetically defined adenovirus (Ad5 wt 300) known to cause severe ocular disease . The 50% inhibitory dose (ID50) values were determined by plaque reduction assays in human cells . The ID50 values of 47 and 604 microM were determined for ganciclovir and acyclovir, respectively, against Ad5, and 26 and 152 microM, respectively against Ad8 . Cotton rats were inoculated bilaterally with 10(5) plaque-forming units per eye and treated topically with ganciclovir (3%, 1%, or 0.3%) or placebo for 21 days . All inoculated eyes were culture positive on days 1-3 with increased infectivity titers, regardless of treatment . However, the incidence, duration, and titer of virus shed in eyes treated with 3% ganciclovir was reduced, and the antiadenovirus enzyme-linked immunosorbent assay titers in serum were lower in these animals . Although these differences were not statistically significant, the observed trend suggested that the highest ganciclovir dose had a suppressive effect on some disease parameters.

J Orthop Res, 1994 Sep, 12(5), 709 - 19
Device for the application of a dynamic biaxially uniform and isotropic strain to a flexible cell culture membrane; Schaffer JL et al.; A large number of studies have demonstrated that mechanical perturbation modulates cellular metabolism; however, the systematic characterization of the molecular and cellular transduction mechanisms underlying mechanically induced metabolic modulation has been impeded, in part, by the limitations of the mechanical device . The objective of this investigation was to develop an in vitro experimental system that would provide independent control of the spatial and temporal biaxial strain distribution imposed on a flexible transparent tissue culture membrane that permits attachment, proliferation, and maintenance of the phenotypic expression of cultured embryonic osteoblasts . Such a device would permit a systematic investigation of the cellular response to specific, independently controlled parameters of mechanical deformation . Using a prototype device designed to impose a dynamic sinusoidal spatially isotropic biaxial strain profile, we confirmed experimentally that the strain was biaxially uniform and isotropic (radial = circumferential strain over the entire culture membrane) to within 14% (SD/mean) for the range of the peak strains tested (2.3-9.4%) . Additionally, the uniformity was maintained at 1 Hz for at least 5 days of continuous operation . This experimental verification of the theoretically predicted isotropic strain profile suggests that the design principle is sound . Embryonic osteoblasts cultured on the flexible substrate proliferated and exhibited a temporal pattern of phenotypic expression (extracellular matrix accumulation and mineralization) comparable with that observed on polystyrene of tissue culture grade.

J Biomech, 1994 Sep, 27(9), 1169 - 77
Strain profiles for circular cell culture plates containing flexible surfaces employed to mechanically deform cells in vitro; Gilbert JA et al.; Cells in the body are constantly subjected to cyclic mechanical deformation involving tension, compression, or shear strain or all three . A mechanical loading system which deforms cultured cells in vitro was analyzed in order to quantify the deformation or strain to which the cells are subjected . The dynamic system utilizes vacuum pressure to deform a circular silicone rubber substrate on which cells are cultured . These thick circular growth surfaces or plates are formed in the bottoms of the wells of 6-well culture plates . An axisymmetric model was formulated and analyzed using rectangular hyperelastic elements in a finite element analysis (FEA) software package . The thick circular plate has some disadvantages such as difficulty in observing cells and a nonhomogeneous strain profile which is maximum at the periphery and minimal at the center . A thinner circular surface (a thin plate) was also investigated in order to provide a more homogeneous strain profile . The radial strain on the thick circular plate, as determined by FEA, was nonlinear with a peak strain value of 0.30 (vacuum pressure of 22 kPa) about three-quarters of the distance from the center to the edge . In contrast, the radial strain of the thin circular plate was moderately constant across the surface . The circumferential strain for both of these models was less than the radial strain except for the center where they are equal . Avian tendon cells were cultured on the surface of a thick plate and exposed to cyclic strains for 24 h at a rate of 0.17 Hz and observed for cellular alignment.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Biochem, 1994 Sep 1, 224(2), 353 - 64
Characterization of the functional role of E-box elements for the transcriptional activity of rat acetylcholine receptor epsilon-subunit and gamma-subunit gene promoters in primary muscle cell cultures; Durr I et al.; The expression of gamma and epsilon subunits of the acetylcholine receptor from mammalian skeletal muscle is regulated independently during myogenic differentiation and innervation . Genomic DNA fragments containing 5'-flanking sequences of the epsilon-subunit and gamma-subunit genes were characterised by a series of 5' deletions fused to the chloramphenicol-acetyltransferase gene and transiently expressed by transfection of primary cultures of rat muscle cells and non-muscle cells . A 6.3-kb epsilon-subunit fragment can be reduced to yield a 270-bp fragment that confers 5-10-times higher expression levels in muscle cells compared to in non-muscle cells . The region composed of nucleotides -185 to -128 increases the transcriptional activity moderately while the 14-bp palindrome containing a single E box at nucleotides -88 to -83 may interact with the promoter but has no enhancer properties in muscle cells . From a 1.1-kb genomic fragment of the gamma-subunit gene, 167 bp were sufficient for muscle-specific expression . Two promoter-proximal E-box elements enhance promoter activity in muscle and mediate transactivation by myogenic factors . Myogenin and myf5 were much more efficient than MRF4 or MyoD1 which exerted only little transactivation . Cotransfection experiments show that increased expression of Id in primary muscle cells inhibits chloramphenicol-acetyltransferase expression mediated by the gamma-subunit gene promoter and support the view that myogenic factors play an important role in the transcriptional regulation of the gamma-subunit gene.

Metabolism, 1994 Sep, 43(9), 1108 - 13
Ethanol and glucose-deprivation neurotoxicity in cortical cell cultures; Singh SP et al.; The toxic effects of ethanol on rat cortical cell cultures were compared with neuronal damage induced by glucose deprivation . Exposure to decreased glucose concentrations produced dose-dependent neuronal injury, as indicated by the release of lactate dehydrogenase (LDH) into the culture medium . Complete glucose deprivation resulted in mean LDH release that was more than 60% greater than that from sister cultures incubated in the presence of 5.5 mmol/L glucose . Exposure to ethanol (25, 50, or 100 mmol/L) similarly resulted in dose-related LDH release . The degree of injury resulting from complete glucose deprivation or 100 mmol/L ethanol approximated that produced by exposure to 100 mmol/L glutamic acid . Ethanol did not significantly alter LDH release from cultures consisting of only astrocytes . Both effects were inhibited by the N-methyl-D-aspartate (NMDA) receptor antagonist, D,L-2-amino-5-phosphonovaleric acid (APV) . Glutamate levels were increased in the culture medium to 191% +/- 8% of the control value after glucose deprivation (P < .001) and to 186% +/- 16% after exposure to 100 mmol/L ethanol (P < .01) . 3H-glutamate uptake by cultured astrocytes was reduced by glucose deprivation and by ethanol . This range of ethanol concentrations has previously been shown to inhibit hexose uptake by cultured astrocytes . The present results suggest that decreased glucose uptake by astrocytes in the presence of ethanol impairs their uptake of glutamate, which contributes to excitotoxic neuronal injury.

Infect Immun, 1994 Sep, 62(9), 4059 - 62
Immunoelectron-microscopic quantitation of differential levels of chlamydial proteins in a cell culture model of persistent Chlamydia trachomatis infection; Beatty WL et al.; Electron immunolabeling techniques were used for quantitative evaluation of alterations in the steady-state levels of chlamydial antigens in persistent Chlamydia trachomatis cultures . Gamma interferon-mediated persistent chlamydial development correlated with an increase in the levels of the chlamydial heat shock protein (hsp60) and with a significant reduction in the levels of the major outer membrane protein.

Mol Chem Neuropathol, 1994 Sep, 23(1), 63 - 76
Prenatal ethanol exposure reduces phosphoinositide hydrolysis stimulated by quisqualate in rat cerebellar granule cell cultures; Rhodes PG et al.; Prenatal ethanol exposure-induced alteration in poly-phosphoinositide (PPI) hydrolysis stimulated by excitatory amino acids (EAA) was studied in rat cerebellar granule cells previously labeled with {3H}myoinositol . The prenatal exposure to ethanol was achieved via maternal consumption of a Sustacal (chocolate flavored) liquid diet containing either 5% ethanol (w/v, 35% of calories) or isocaloric sucrose (pair-fed) substituted for ethanol from gestation d 11 until the day of parturition . The ionotropic glutamate receptor agonists, N-methyl-D-aspartate, kainate or (+/-)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) (100 microM each) induced a two- to four-fold increase in PPI hydrolysis over the basal level, regardless of the liquid dietary treatment . Stimulation with quisqualate (QA), an agonist activating both metabotropic and ionotropic glutamate receptors, resulted in a much stronger and dose-dependent response in PPI hydrolysis and exposure in utero to ethanol significantly reduced this response . Tetrodotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), or (+/-)-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP) had no effect on QA-stimulated PPI hydrolysis nor on the suppression of this hydrolysis by ethanol . Exposure in utero to ethanol did not affect PPI hydrolysis stimulated by a selective metabotropic glutamate receptor agonist, trans-(+/-)-l-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD) . Although the PPI hydrolysis stimulated by t-ACPD could be blocked by (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG), an antagonist of the metabotropic glutamate receptor, MCPG was incapable of affecting QA-induced PPI hydrolysis and the suppressive effects of prenatal ethanol exposure on this hydrolysis . Taken together, the data suggest that the long-lasting suppressive effects of prenatal ethanol exposure on QA-stimulated PPI hydrolysis in cerebellar granule cell cultures is through a metabotropic QA receptor pathway that may be different from the one activated by t-ACPD.

Vet Microbiol, 1994 Sep, 42(1), 65 - 77
Detection of feline coronaviruses in cell cultures and in fresh and fixed feline tissues using polymerase chain reaction; Li X et al.; Feline coronavirus infections in cell cultures and in fresh and fixed feline tissues were detected using a polymerase chain reaction (PCR) test . Cell cultures were inoculated with feline infectious peritonitis virus (FIPV), feline enteric coronavirus (FECV) or sham inoculum . The tissue samples of liver, kidney and spleen were taken from specific-pathogen-free (SPF) cats that were inoculated intranasally with 10(3) TCID50 of FIPV 79-1146 (n = 10), FIPV UCD1 (n = 3) or sham inoculum (n = 3), from clinical cats (n = 43), and from formalin-fixed archived feline tissues (n = 49), respectively . Additional tissue samples were taken from the FIPV-inoculated cats (n = 6) and were kept at 4 degrees C, room temperatures (20-24 degrees C) and 37 degrees C respectively for 0, 6, 12, 24, 48, 72, and 96 hours before frozen (-70 degrees C) for PCR to evaluate the effects of the ambient temperatures and post-mortem intervals on the test . The samples were also fixed in 10% neutrally buffered formalin, 95% ethanol, and Bouin's solution respectively to evaluate the effects of the fixatives on the test . Positive PCR results were obtained from the cell cultures that were inoculated with FIPV and FECV and from the FIPV-inoculated cats (13/13) . Negative PCR results were obtained from the sham-inoculated cell cultures and cats (3/3) . Of the 92 clinical cats, 7 of the 8 FIP-suspected cats (87.5%) and 51 of the 84 non-FIP-suspected cats (60.7%) were shown to be virus-positive in at least one of the tissue samples . There was no significant difference in the PCR results between the fresh and the formalin-fixed tissues of the clinical cats (P > 0.05) . Of the FIPV inoculated cats, the virus was detectable equally well in fresh and formalin-, Bouin's solution- or ethanol-fixed tissues . However, the amounts of total RNA extracted from the fixed tissues were significantly less than those from fresh tissues (P < 0.01) . In tissues that were kept at 4 degrees C, the virus was detectable up to 96 h; at room temperatures, up to 48 h; and at 37 degrees C, up to 24 h, respectively.

J Virol Methods, 1994 Sep, 49(2), 235 - 44
Enhancement of infectivity of hantavirus in cell culture by centrifugation; Kariwa H et al.; Centrifugation was introduced during virus adsorption to Vero E6 cells to improve the infectivity of hantavirus . Centrifugal adsorption of a stock solution of Hantaan virus strain 76-118 to a monolayer of Vero E6 cells enhanced virus infectivity depending on the centrifugation time and the centrifugal force . The maximum level of infectivity (3.1 x 10(6) FFU/ml) was enhanced after a 2 h centrifugation at 671 x g, which was almost 9-times higher than that of conventional adsorption of the virus at 37 degrees C for 1 h . Vero E6 cells were inoculated with a new hantavirus strain, KI-91-40, isolated with a low infectious titer (400 FFU/ml) from an urban rat and adsorbed by centrifugation . A higher virus titer was detected sooner compared to when using conventional adsorption . To analyze the mechanism of the enhancement, the centrifugation was carried out before and after virus adsorption . The infectivity was reduced when Vero E6 monolayers were centrifuged before virus inoculation . When the centrifugation proceeded after inoculation, the infectivity was almost equal to that without centrifugation . The infectivity was only enhanced when centrifugation was carried out during inoculation . These results indicate that centrifugation promotes a very early event of infection, probably attachment of the virus to cells.

J Virol Methods, 1994 Sep, 49(2), 153 - 6
An efficient and easy method of infection of mosquito larvae from virus-contaminated cell cultures; Barreau C et al.; A new method for efficient infection of Aedes aegypti larvae by the Aedes albopictus densovirus, AaPV is described . It consists of placing first or third instar larvae in culture flasks containing a chronically infected mosquito cell line . After 24 or 48 h of exposure to the contaminated culture, the larvae acquired the virus by feeding on infected cells . Using this technique, up to 95% of first instar Ae . aegypti larvae were found infected by the AaPV.

Exp Eye Res, 1994 Sep, 59(3), 257 - 69
Characterization and novel activation of 72-kDa metalloproteinase in retinal interphotoreceptor matrix and Y-79 cell culture medium; Jones BE et al.; Analysis of bovine interphotoreceptor matrix and conditioned medium from human Y-79 retinoblastoma cells by gelatin SDS-PAGE zymography reveals abundant activity of a 72-kDa M(r) gelatinase . The 72-kDa gelatinase from either source is inhibited by EDTA but not aprotinin or NEM, indicating that it is a metalloproteinase (MMP) . The 72-kDa MMP is converted to a 62-kDa species with APMA treatment after gelatin sepharose affinity purification, typical of previously described gelatinase MMP-2 . The latent 72-kDa gelatinase from either bovine IPM or Y-79 media autoactivates without APMA in the presence of calcium and zinc after 72 hr at 37 degrees C, producing a fully active mixture of proteinase species, 50 (48 in Y-79 medium), 38 and 35 kDa in size . The presence of inhibitory activity was examined in both whole bovine IPM and IPM fractions separated by SDS-PAGE . Whole IPM inhibited gelatinolytic activity of autoactivated Y-79-derived MMP in a dose-dependent manner . Inhibitory activities are observed in two protein fractions of 27-42 and 20-25 kDa . Western blots using antibodies to human tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1 and -2) reveal the presence of two TIMP-1-like proteins at 21 and 29 kDa in inhibitory fractions of the bovine IPM . TIMP-2 was not detected in the inhibitory IPM fractions, consistent with the observed autoactivation of bovine IPM 72-kDa gelatinase . Potential roles for this IPM MMP-TIMP system include physiologic remodelling of the neural retina-RPE cell interface and digestion of shed rod outer segment as well as pathological processes such as retinal detachment, PE cell migration, neovascularization and tumor progression . Cultured Y-79 cells appear to be a good model for studying the production and regulation of this proteinase system.

Biophys J, 1994 Sep, 67(3), 1301 - 15
Multiple site optical recording of transmembrane voltage (MSORTV) in patterned growth heart cell cultures: assessing electrical behavior, with microsecond resolution, on a cellular and subcellular scale; Rohr S et al.; We have applied multiple site optical recording of transmembrane voltage (MSORTV) to patterned growth cultures of heart cells to analyze the effect of geometry per se on impulse propagation in excitable tissue, with cellular and subcellular resolution . Extensive dye screening led to the choice of di-8-ANEPPS as the most suitable voltage-sensitive dye for this application; it is internalized slowly and permits optical recording with signal-to-noise ratios as high as 40:1 (measured peak-to-peak) and average fractional fluorescence changes of 15% per 100 mV . Using a x 100 objective and a fast data acquisition system, we could resolve impulse propagation on a microscopic scale (15 microns) with high temporal resolution (uncertainty of +/- 5 microseconds) . We could observe the decrease in conduction velocity of an impulse propagating along a narrow cell strand as it enters a region of abrupt expansion, and we could explain this phenomenon in terms of the micro-architecture of the tissue . In contrast with the elongated and aligned cells forming the narrow strands, the cells forming the expansions were aligned at random and presented 2.5 times as many cell-to-cell appositions per unit length . If the decrease in conduction velocity results entirely from this increased number of cell-to-cell boundaries per unit length, the mean activation delay introduced by each boundary can be estimated to be 70 microseconds . Using this novel experimental system, we could also demonstrate the electrical coupling of fibroblasts and endotheloid cells to myocytes in culture.

Neurosurgery, 1994 Sep, 35(3), 434 - 8; discussion 438
Human acoustic neuromas secrete interleukin-6 in cell culture: possible autocrine regulation of cell proliferation; Adams EF et al.; Interleukin-6 (IL-6) secretion by cell cultures of human acoustic neuromas was examined . Secretory rates varied from 0.02 to 5.4 ng/10(5) cells per 4 days, depending on the tumor . The IL-6 immunoreactivity eluted from a Sephadex G-100 column in a major peak corresponding to an M(r) of 30,000 and a lesser peak corresponding to an M(r) of 50,000 . Western blot analysis revealed three IL-6 immunoreactive bands with M(r)s corresponding to 53,000, 29,000, and 24,000 . Tumor necrosis factor-alpha, interleukin-1-beta, and cholera toxin all stimulated IL-6 secretion . An antisense phosphorothioate oligonucleotide against IL-6 messenger RNA inhibited both {3H}thymidine uptake and IL-6 secretion by acoustic neuroma cells in culture . In addition, {3H}thymidine uptake was inhibited by a specific polyclonal antibody against IL-6 . We conclude that human acoustic neuroma cells produce and secrete IL-6, which may act in an autocrine manner to stimulate cellular proliferation.

J Nat Prod, 1994 Sep, 57(9), 1320 - 4
Yunnanxane and its homologous esters from cell cultures of Taxus chinensis var . mairei; Ma W et al.; From cell cultures of Taxus chinensis var . mairei, yunnanxane {2 alpha, 5 alpha, 10-beta triacetoxy-14 beta-(2'-methyl-3'-hydroxyl)-butyryloxy-4(20),11-taxadiene, {1}, and four new homologous esters, 2 alpha, 5 alpha, 10 beta, 14 beta- tetra-acetoxy-4(20),11-taxadiene {2}, 2 alpha, 5 alpha, 10 beta- triacetoxy-14 beta-propionyloxy-4(20),11-taxadiene {3}, 2 alpha, 5 alpha, 10 beta- triacetoxy-14 beta-isobutyryloxy-4(20),11- taxadiene {4}, and 2 alpha, 5 alpha, 10 beta- triacetoxy-14 beta-(2'-methyl)-butyryloxy-4(20),11- taxadiene {5} have been isolated . Their structures were determined by spectroscopic methods.

In Vitro Cell Dev Biol Anim, 1994 Sep, 30A(9), 622 - 35
Mouse pancreatic acinar/ductular tissue gives rise to epithelial cultures that are morphologically, biochemically, and functionally indistinguishable from interlobular duct cell cultures; Githens S et al.; Most of the pancreatic exocrine epithelium consists of acinar and intralobular duct (ductular) cells, with the balance consisting of interlobular and main duct cells . Fragments of mouse acinar/ductular epithelium can be isolated by partial digestion with collagenase and purified by Ficoll density gradient centrifugation . We investigated whether previously developed culture conditions used for duct epithelium would result in the selective survival and proliferation of ductular cells from the acinar/ductular fragments . The fragments were cultured on nitrocellulose filters coated with extracellular matrix . After 2 to 4 wk the filters were covered with proliferating cells resembling parallel cultures of duct epithelium by the following criteria: protein/DNA ratio, light and electron microscopic appearance, the presence of duct markers (carbonic anhydrase {CA} activity, CA II mRNA, the cystic fibrosis transmembrane conductance regulator), the near absence of acinar cell markers (amylase and chymotrypsin), a similar polypeptide profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the presence of spontaneous and secretin-stimulated electrogenic ion transport . Both duct and ductular epithelia formed fluid-filled cysts in collagen gels and both could be subcultured . We conclude that acinar/ductular tissue gives rise to ductular cells in culture by some combination of acinar cell death and/or transdifferentiation to a ductular phenotype, accompanied by proliferation of these cells and preexisting ductular cells . These cultures may be used to investigate the properties of this part of the pancreatic duct system, from which most of the pancreatic juice water and electrolytes probably originates.

Carcinogenesis, 1994 Sep, 15(9), 1847 - 52
Growth characteristics and Ha-ras mutations of cell cultures isolated from chemically induced mouse liver tumours; Pedrick MS et al.; Cells have been isolated from liver tumours that have arisen in control C3H/He mice, in mice given 10 micrograms diethylnitrosamine (DEN) during the neonatal period or in mice given a diet containing phenobarbitone (PB) to allow a daily intake of 85 mg/kg/day . The cells were grown to the 8 degrees subculture when their growth characteristics were investigated in monolayer culture and following suspension in soft agar and on transplantation into nude mice . In addition, DNA was isolated from the cultures and from tumours that grew in nude mice and analysed for mutations at codon 61 of the Ha-ras oncogene . All cells derived from DEN-induced hepatocellular carcinomas (HCC) demonstrated a lack of density inhibition of growth in monolayer culture, grew in soft agar and formed tumours in nude mice with an average mean latency of 29 days . Three of the seven lines showed mutations in Ha-ras: two were CAA-->AAA transversions and one showed a CAA-->CTA transversion . In contrast, cells isolated from eosinophilic nodules in mice given PB showed inhibition of growth at confluence, did not grow in soft agar and only four of eight formed tumours in nude mice with a mean average latent period of 181 days . Cells grown from HCC in mice given PB showed a lack of density inhibition of growth, however, they did not grow in soft agar nor did they form tumours in nude mice . A single spontaneous HCC from a control mouse showed a similar growth pattern to HCC cells isolated from mice given PB . Cells from a basophilic nodule, taken from a control untreated mouse grew vigorously in culture and in soft agar and formed tumours in nude mice with a latency of 6 days . None of the cells isolated from control mice or from mice given PB showed evidence of mutations at codon 61 of Ha-ras . These data confirm that there are fundamental differences in the biology of cells grown from tumours that develop in mice under different treatment regimes . These studies also demonstrate the utility of cell culture and molecular biology in addressing the fundamental mechanism of mouse hepatic neoplasia.

Infect Immun, 1994 Sep, 62(9), 3844 - 9
Effect of lipopolysaccharide on mouse mast cell induction by a splenic cell culture system; Hu ZQ et al.; We have previously reported a method of mast cell induction by long-term culture of mouse spleen cells without using exogenous mast cell growth factor (Z.-Q . Hu, T . Yoshida, and T . Shimamura, J . Immunol . Methods 149:173, 1992) . Supernatants recovered from the long-term cultures contain endogenous interleukin 3 and soluble stem cell factor . These were assessed by the capacity of the recovered supernatants to foster the growth of a mast cell growth factor-dependent cell line and by neutralizing antibodies . Besides the soluble factors, cell-to-cell contacts mediated by membrane stem cell factor on splenic stromal cells and c-Kit receptors on mast cells also affect mast cell induction . Different lots of fetal calf serum (FCS) were examined to determine a possible trigger for cytokine production . FCS can be divided into mast cell-inducible and noninducible sera by this process . However, not all FCS lots contain mast cell growth factor . The mast cell-inducible lots contain lipopolysaccharide (LPS) confirmed by a Limulus assay . Polymyxin B can neutralize the mast cell induction activity . Non-mast cell-inducible FCS can be converted to inducible FCS by adding exogenous LPS . The results indicate that LPS as a trigger of cytokine production is responsible for mast cell induction.

Biochem Pharmacol, 1994 Aug 17, 48(4), 801 - 7
Relationship between cytotoxicity and conversion of thiosangivamycin analogs to toyocamycin analogs in cell culture medium; Renau TE et al.; Non-nucleoside analogs of the pyrrolopyrimidine nucleosides toyocamycin, sangivamycin and thiosangivamycin have been synthesized and their cytotoxicity in mammalian cells determined . While studying the effects of 5-thioamide-substituted analogs on cell growth, we observed an interesting phenomenon in which cells recovered spontaneously from growth inhibition during extended incubations . HPLC studies demonstrated that the 5-thioamide moiety of several structurally dissimilar 7-substituted 4-aminopyrrolo{2,3-d}pyrimidines, including thiosangivamycin, is unstable in cell culture medium and is converted to the corresponding 5-nitrile with a half-life of approximately 48 h . In contrast, different substituents at the 4-position of the heterocycle significantly affected the stability of the 5-thioamide moiety . Conversion of the thioamide to the nitrile was caused by components in the cell culture medium, not components of serum . The above observations demonstrate that caution should be exercised in interpreting biological data obtained in vitro for 5-thioamide pyrrolo{2,3-d}pyrimidines.

Arch Biochem Biophys, 1994 Aug 15, 313(1), 120 - 5
Iron ion induces mitochondrial DNA damage in HTC rat hepatoma cell culture--role of antioxidants in mitochondrial DNA protection from oxidative stresses; Itoh H et al.; The mitochondrial DNA (mtDNA) is exposed to the reactive oxygen species generated in the mitochondrial electron transfer system . While mitochondrial superoxide dismutase (Mn-SOD) scavenges superoxide anions to prevent damage of mtDNA, excess amount of reactive oxygen generated by quinone compounds impairs the mtDNA, in which involvement of iron ion-catalyzed reactions is suggested . In this communication, we report that the mtDNA in HTC rat hepatoma cells was markedly damaged in the presence of either ferrous (Fe2+) or ferric (Fe3+) iron in the culture medium . The mtDNA of HTC cells was damaged in the presence of 100 microM of iron ions in the culture medium within 3 h . There was no significant difference in the potency of Fe2+ and Fe3+ . Deferoxamine, a Fe(3+)-specific chelating reagent, blocked the iron ion-induced mtDNA damage completely . The mtDNA in rat hepatocyte primary culture was, on the other hand, not damaged by either Fe2+ or Fe3+ . To understand the difference between iron ion-induced damage of mtDNA in HTC cells and in the primary hepatocytes, the Mn-SOD activity and lipid-soluble antioxidant contents were compared . The Mn-SOD activity in HTC cells was markedly lower (< 0.02 U/mg cell protein) than that in rat hepatocyte primary cell cultures (1.5 U/mg cell protein) . Immunoreactive Mn-SOD content in HTC cells was also lower (21 ng/mg cell protein) than in the primary cultures (99 ng/mg cell protein) . HTC cells contain much smaller amounts (3-11% of that of normal rat hepatocytes) of alpha-tocopherol and coenzyme Q homologs (CoQn) . These results suggest that reactive oxygen species produced by iron ion impaired mtDNA of HTC cells, in which antioxidant activity was markedly decreased.

Brain Res, 1994 Aug 15, 654(1), 118 - 28
Alpha-sialyl cholesterol increases laminin in Schwann cell cultures and attenuates cytostatic drug-induced reduction of laminin; Konings PN et al.; Schwann cells play an important role in peripheral nerve regeneration . Here, we report the effect of alpha-sialyl cholesterol (alpha-SC), a derivative of the sialic acid-containing natural gangliosides, and the cytostatic agents, cisplatin, taxol and vincristine on the laminin production in Schwann cell cultures isolated from rat sciatic nerves . Laminin, one of several extracellular matrix components produced by Schwann cells, is known to potentiate axonal outgrowth . Laminin content was increased by alpha-SC, starting at 7.0 micrograms/ml with a maximal effect at 22.4 micrograms/ml (30%, P < 0.001) . The three cytostatic drugs, dose-dependently reduced laminin content in Schwann cell cultures: (1) cisplatin at a threshold dose of 2 micrograms/ml (-26.4%, P < 0.001); (2) taxol, starting at a dose of 1 ng/ml (-8.0%, P < 0.05); and (3) vincristine, starting at 0.5 ng/ml (-5.9%, P < 0.05) . Cultured Schwann cells were incubated with cytostatic drugs in combination with increasing amounts of alpha-SC and it was found that, depending on the cytostatic drug concentration used, alpha-SC could reduce or completely prevent the cytostatic drug-induced reduction of laminin in Schwann cell cultures . Co-treatment with alpha-SC also reduced part of the morphological changes caused by the cytostatic drugs . alpha-SC did not counteract the anti-proliferative effect of the cytostatic drugs on K-562 human erythroleukemia cells . In conclusion, alpha-SC increased laminin content in Schwann cell cultures and protected Schwann cell cultures against the decrease of laminin by cytostatic drugs without interfering with the anti-proliferative potential, suggesting that alpha-SC may have clinical use in protecting cancer patients against the neurotoxic effects of cytostatic drugs.

Microsc Res Tech, 1994 Aug 15, 28(6), 492 - 504
Adenosine 5'-triphosphate promotes mineralization in differentiating chick limb-bud mesenchymal cell cultures; Boskey AL et al.; When chick limb-bud mesenchymal cells are plated in micromass culture, they differentiate to form a mineralizable cartilage matrix . Previous studies have demonstrated that, when the total inorganic phosphate concentration of the medium is adjusted to 3-4 mM by adding inorganic phosphate to the basal medium, the mineralized matrix formed resembles that of chick calcified cartilage in ovo . When the high-energy phosphates adenosine 5'-triphosphate (ATP) or creatine phosphate are used as supplements in place of inorganic phosphate, the mineralized matrix as analyzed by electron microscopy and Fourier transform infrared microscopy is also similar to that in ovo . This is in marked contrast to the mineralized matrix formed in the presence of 2.5-5 mM beta-glycerophosphate, where mineral deposition is random and mineral crystal sizes in general are larger . This is also in contrast to the known ability of ATP to inhibit mineral deposition in solution in the absence of cells . In the differentiating mesenchymal cell culture system, ATP does not alter the rate of cell proliferation (DNA content), the rate of matrix synthesis (3H-leucine uptake), the mean crystallite length, or the rate of mineral deposition (45Ca uptake) when contrasted with cultures supplemented with inorganic phosphate . However, ATP does increase the mineral to matrix ratio, especially around the edge of the culture, where a type I collagen matrix is presented . It is suggested that ATP promotes mineral deposition by providing a high-energy phosphate source, which may be used to phosphorylate extracellular matrix proteins and to regulate calcium flux through cell membranes.

Toxicology, 1994 Aug 12, 91(3), 221 - 34
Methylene chloride: an inhalation study to investigate toxicity in the mouse lung using morphological, biochemical and Clara cell culture techniques; Foster JR et al.; Single exposures of mice to methylene chloride (MC) cause vacuolation and necrosis of the bronchiolar Clara cells which subsequently recover normal morphology on continued exposure . Both cytochrome P-450 (CYP)- and glutathione S-transferase (GST)-dependent metabolism of MC are known to occur . The current studies have investigated the metabolism of MC in mouse lung using inhibitors of both GST and CYP-dependent routes of metabolism, the consequences of metabolic inhibition on the Clara cell vacuolation, and any changes in cell proliferation, assessed in vitro, in Clara cells cultured from exposed individuals . Vacuolated bronchiolar cells were seen in mice exposed to 2000 and 4000 ppm MC but were not seen at lower concentrations, while addition of the CYP inhibitor, piperonyl butoxide, significantly reduced the bronchiolar cell vacuolation seen following exposure to 2000 ppm MC . Treatment of mice with the glutathione depletor, buthionine sulphoximine, had no effect on the number of vacuolated bronchiolar cells following MC . Exposure of mice to 1000 ppm MC and above for 6 h caused a burst of DNA synthesis in bronchiolar Clara cells cultured in vitro from the lungs of exposed animals . The results suggest that the Clara cell vacuolation following MC exposure is mediated via CYP metabolism, that depression of the CYP metabolic pathway occurs following exposure, and that Clara cell vacuolation may have a priming role in stimulating cell proliferation in the unaffected cell population.

J Biol Chem, 1994 Aug 5, 269(31), 19671 - 4
Activated Gq-alpha potentiates platelet-derived growth factor-stimulated mitogenesis in confluent cell cultures; De Vivo M et al.; We studied the effects of activation of the Gq-alpha signaling pathway on mitogenesis by expressing a mutant (Q209L), activated alpha-subunit of Gq (alpha q*) in NIH-3T3 cells . A clonal NIH-3T3 cell line expressing alpha q* in an inducible manner was isolated . Expression of alpha q* is induced with dexamethasone, allowing the use of non-induced cells as controls for the effects of alpha q* expression . We found that, by itself, expression of alpha q* did not increase either DNA synthesis or mitogen-activated protein (MAP) kinase activity in serum-starved cells . Because alpha q* transforms cells grown in the presence of serum (De Vivo M., Chen, J., Codina, J., and Iyengar, R . (1992) J . Biol . Chem . 267, 18263-18266), we tested whether growth factor-stimulated signaling and mitogenesis were affected by expression of alpha q* . Platelet-derived growth factor (PDGF) stimulated thymidine incorporation modestly (50%) in contact-inhibited, confluent cell cultures . In cells expressing alpha q*, PDGF stimulated DNA synthesis up to 3-fold over basal . Concomitant with the potentiation of PDGF-stimulated DNA synthesis, expression of alpha q* potentiated PDGF-stimulated p44 MAP kinase activity . PDGF was much more effective in stimulating both DNA synthesis and p44 MAP kinase activity in subconfluent cell cultures and expression of alpha q* exerted little or no effect on PDGF-stimulated effects in subconfluent cells . These data show that cooperation between signaling pathways may occur in a cell state-specific fashion . Such cooperation in part may be responsible for the triggering of complex cellular responses such as cell transformation.

J Pharmacol Exp Ther, 1994 Aug, 270(2), 822 - 30
1-Methyl-4-phenylpyridinium-like neurotoxicity of a pyridinium metabolite derived from haloperidol: cell culture and neurotransmitter uptake studies; Bloomquist J et al.; It is now generally accepted that the nigrostriatal degenerative properties of the parkinsonian-inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine are mediated by the brain monoamine oxidase B generated 1-methyl-4-phenylpyridinium metabolite (MPP+) . In this article, the results are described of ongoing efforts to evaluate the MPP(+)-type neurotoxic potential of the haloperidol (HP)-derived pyridinium metabolite HPP+, a 1,4-disubstituted structural analog of MPP+, which is formed in humans and rats treated with HP . Previous studies in the rat have shown that intrastriatal perfusion of HPP+ leads to the irreversible depletion of striatal dopamine and serotonin . Furthermore, HPP+ was a potent inhibitor of NADH-supported mitochondrial respiration . This article reports that HPP+ also is toxic to dopaminergic and serotonergic neurons in cultures of embryonic mesencephalic cells, as measured by loss of the ability of exposed cells to accumulate tritium-labeled dopamine and serotonin and by immunochemical staining techniques . HPP+ also inhibited the uptake of these labeled neurotransmitters by synaptosomes prepared from mouse neostriata (dopamine) and cortical tissues (serotonin) . Because HP is unlikely to be a substrate for brain monoamine oxidase B, the production and accumulation of HPP+ in the brain is probably not comparable to that of MPP+ . On the other hand, chronic exposure to HP could result in brain levels of this lipophilic quaternary pyridinium species that might coincide with the late-appearing tardive dyskinesias that are observed in some HP-treated patients months and, more often, years after the initiation of HP therapy.

J Cell Physiol, 1994 Aug, 160(2), 336 - 44
Hormonal regulation of some steps of thyroglobulin synthesis and secretion in bicameral cell culture; Desruisseau S et al.; Porcine thyroid cells were cultured for 15 days on porous bottom chambers with or without different mixtures of hormones added to serum-free basal medium . Assays with 10% serum were also performed for comparison with previously published results . The effects of the hormones, particularly insulin, TSH and hydrocortisone, were studied on total RNA content, thyroglobulin mRNA level, the amount of thyroglobulin secreted into the apical medium and on glycosylation . Insulin and TSH similarly increased the total RNA content, and their effects were additive . Thyroglobulin mRNA content was increased twofold by insulin and threefold by TSH . When they were added simultaneously, the maximal level of thyroglobulin mRNA was reached, showing that TSH and insulin effects on thyroglobulin gene expression were additive . Hydrocortisone alone did not modify total RNA or thyroglobulin mRNA content but the hormone amplified total RNA when insulin and TSH were present together . The basal level of thyroglobulin secreted into the apical medium was increased threefold by insulin and fourfold by TSH . The effects of these two hormones added together appeared to be additive . Hydrocortisone had no effect alone or even when combined with insulin or TSH . However, when the three hormones were added together, the hormonal response was amplified . TSH effect and insulin effect on the incorporation of 3H-mannose into thyroglobulin as well as on the anionic residue content of the molecule were additive.

Biomaterials, 1994 Aug, 15(10), 859 - 64
Antibody response in rats against non-toxic glucose sensor membranes tested in cell culture; Ziegler M et al.; Several glucose sensors have been developed, but none are commercially available . The most urgent in vivo problem is the drift of glucose sensor output with time, which may be caused by leakage or denaturation of glucose oxidase, and events at the body-sensor interface such as protein coating, encapsulation with cells, toxicity of the device and inflammation . In the present study, a specific immune response against the outer cellulose acetate membrane of a glucose sensor implanted into rats has also been proved . The immune response against polymeric membranes can be confirmed by detection of specific immunoglobulin G antibodies to cellulose acetate, polyurethane and regenerated cellulose after implantation of the respective membrane . The individually different antibody formation against polymers in rats was amplified by one application of complete Freund's adjuvant in combination with the first implantation . The cell culture results using the fibroblast cell line L-929 showed only a minor toxicity of regenerated cellulose, whereas the other polymers had no effect on cell growth and viability . From the results of this study, it is proposed to integrate immunogenicity as a further parameter for evaluation of the biocompatibility and biosafety of materials or medical devices which are provided for implantation.

Vet Microbiol, 1994 Aug 1, 41(3), 267 - 79
Infection of leucocyte cell cultures derived from different species with pig circovirus; Allan GM et al.; Cultures of leucocyte cells were prepared from pig bone marrow, peripheral blood, lung washings, thymus and lymph nodes . Cell cultures were also prepared from peripheral blood from sheep, cattle and a human . Immunofluorescent (IF) staining of all these cultures, following inoculation with pig circovirus (PCV), detected virus replication in all the cell cultures derived from pigs and in the cell cultures derived from cattle . Virus replication in pig leucocyte cell cultures was confirmed by demonstrating the production of infectious virus . Double immunostaining of PCV infected cells using monoclonal antibodies specific for cell membrane markers indicated infection was confined to monocyte/macrophage cell types . No PCV antigen was detected in T or B cells in infected cell cultures.

Mol Neurobiol, 1994 Aug-Dec, 9(1-3), 41 - 54
Alzheimer's disease cerebrospinal fluid antibodies display selectivity for microglia . Investigations with cell cultures and human cortical biopsies; Dahlstrom A et al.; Previous investigations demonstrated that the cerebrospinal fluid (CSF) from Alzheimer's disease (AD) patients contains antibodies that recognize specific neuronal populations in the adult rat central nervous system (CNS) . These findings suggest a pathogenic role for immunological aberrations in this disorder . To determine if antibodies may provide a means to differentially diagnose the dementias, CSF from a diversified dementia population was screened against the developing rat CNS and a cell culture system . Markings produced by AD CSF were distinctly different from those of vascular dementias (VAD) against the developing rat CNS . More importantly, some AD CSF recognized amoeboid microglia . The recognition of amoeboid microglia by antibodies in AD CSF is particularly interesting since these cells proliferate in response to nervous system disease and also engulf debris . A cell culture technique was developed to allow the rapid screening of CSF antibodies . Patient CSF produced five different types of markings in the cell culture: microglia, glioblasts, fibers, nonspecific, or negative . Correlations with these structures and the diagnosis of four different dementia populations revealed that, in comparison to the other groups, AD CSF displayed remarkable selectivity toward microglial cells . Cortical biopsies from patients suspected to have AD were incubated with the patient's own CSF and that of confirmed AD patients . Both CSF samples recognized microglial cells in the patient's cortical biopsy . The same CSF samples incubated against normal human cortical autopsy or a biopsy from a 3-mo-old child displayed negative immunoreactivity . These three approaches suggest that the presence of CSF microglial antibodies may be a means to distinguish AD patients from other dementias . The results add further support to the widely growing concept that inflammation and similar immune mechanisms may contribute to AD pathogenesis.

J Gen Physiol, 1994 Aug, 104(2), 287 - 309
Characterization of impulse propagation at the microscopic level across geometrically defined expansions of excitable tissue: multiple site optical recording of transmembrane voltage (MSORTV) in patterned growth heart cell cultures; Rohr S et al.; Impulse propagation across sudden expansions of excitable tissue has been shown to exhibit various forms of conduction disturbance on a macroscopic scale, ranging from small delays to unidirectional or complete conduction block . With the present study, we attempted to characterize systematically the dependence of impulse propagation on the geometry of the underlying excitable tissue on a microscopic scale by investigating the spatio-temporal pattern of transmembrane voltage changes associated with impulse propagation from a narrow cell strand to a large cell area using multiple site optical recording of transmembrane voltage (MSORTV) in conjunction with patterned growth of neonatal rat heart cells in culture . While action potential propagation was smooth in the case of funneled expansions, delays of variable size occurred during propagation into rectangular or incised expansions . Close to the abrupt expansion, which functionally represented an increased electrical load to the narrow cell strand, the delays were accompanied by marked distortions of the action potential upstroke, exhibiting, in extreme cases, an initial depolarization to 50% followed by a delayed secondary depolarization to 100% of the full-signal amplitude . These distortions, which were based on bidirectional electrotonic interactions across the transition, were maximal immediately downstream from the expansion . The maximal slowing of impulse conduction across abrupt expansions was, in agreement with recently published results obtained from two-dimensional computer simulations, always situated in the expanded region . At high stimulation rates, the delays sometimes turned into intermittent unidirectional blocks, as revealed by reverse stimulation . These blocks were always characterized by a marked abbreviation of the action potentials upstream from the region causing the block which might, in an appropriate network, facilitate reentry because of the associated shortening of the refractory period . Because the patterns were composed of cells having identical membrane properties, the results show that the local action potential shape can be modulated profoundly by the two-dimensional architecture of the underlying cell ensemble alone.

Wei Sheng Wu Xue Bao, 1994 Aug, 34(4), 328 - 31
{Propagation of the HTV in primary human embryonic kidney and lung cell culture}; Liu B et al.; 2 strains of Hantaan virus (HTV, 76-118, Hubei-114) have been propagated successfully in cultured primary human embryonic kidney (HEK) and lung (HEL) cells . Cytopathic effect (CPE) was observed in the two kind of cells on day 5 to 7 postinoculation which showed the cell became round and clustered, then detached . The replicating peak of the Hubei-114 in two kinds of cell cultures appeared on the 11th day and another strain on the 14th or 17th day after infection . The ultrastructure changes were observed with EM and IEM, which stained by ICGT before embedding . It was discovered that the mitochondia atrophied and decreased, and inclusion bodies in the cytoplasma of HEK and KEL cells . A large amount of gold granulae were found in the inclusion bodies and the virions were seen occasionally . Contamination with other agents have been ruled out . Our data suggest that the replicating characters of HTV in these cell systems might be possible for the pathogenicity of HFRS for human.

J Cell Sci, 1994 Aug, 107 ( Pt 8), 2343 - 51
Development of a spontaneous permanent cell line of rabbit corneal epithelial cells that undergoes sequential stages of differentiation in cell culture; Castro-Munozledo F; Established epithelial cell lines that retain their differentiation potential and growth regulatory characteristics can provide valuable tools for studying gene regulation, extracellular matrix synthesis or growth factor response . They are also useful for drug development and toxicity testing . Experiments were therefore carried out to optimize culture conditions for the long-term, serial transfer of corneal epithelial cells in the presence of 3T3 feeder layers; and to establish a permanent cell line . In such experiments, rabbit corneal epithelial cells were seeded at low inoculation densities, and transferred every 5 days . After 80 population doublings, an epithelial cell line, RCE1, emerged . The cell line is heteroploid, with an average population doubling time of 15.5 hours (vs 18 hours for primary cultures) . When RCE1 cells reached confluence, they stratified to form a three- to five-layered epithelium and expressed the differentiation-related keratin pair K3/K12 as shown by immunoblot and immunostaining . Biosynthetic labeling of proliferating, confluent and stratified cultures further showed that RCE1 cells expressed keratin pairs K5/K14, K6/K16 and K3/K12, thus mimicking faithfully the stage-dependent differentiation of primary cultures of rabbit corneal keratinocytes . The results demonstrated that RCE1 cells provide a useful model for studying corneal cell growth and differentiation.

J Nutr, 1994 Aug, 124(8 Suppl), 1540S - 1545S
Cell culture as a tool for identifying nutritional disease therapies; Ballard FJ; Cell culture methodologies can be used to help develop therapies for the treatment of polytrauma . An overview is presented of research with the L6 myoblast cell line that led to the discovery of the truncated insulin-like growth factor (IGF) variant, des(1-3)IGF-I and that gave an explanation of the enhanced potency of this growth factor . Subsequent efforts to develop an even more potent variant also utilized cultured cells as indicator bioassays . Such experiments not only provided mechanistic information on IGF action but also directed attention towards IGF variants that were later shown to be more potent in vivo at reversing catabolic conditions.

Cancer Res, 1994 Jul 15, 54(14), 3766 - 71
Increased activity and expression of NAD(P)H:quinone acceptor oxidoreductase in confluent cell cultures and within multicellular spheroids; Phillips RM et al.; NAD(P)H:quinone acceptor oxidoreductase (NQO1, EC 1.6.99.2) is an enzyme that is believed to play a central role in the bioreductive activation of several compounds, particularly quinones . The results of this study demonstrate that the activity of NQO1 is significantly elevated (2.5-fold) in HT-29 human colon cells that are in the plateau phase of the growth curve as opposed to cells in the exponential phase . Analysis of gene expression using semiquantitative reverse transcription-polymerase chain reaction and Northern blot analysis demonstrates that the increased enzyme activity is associated with increased NQO1 mRNA levels . Sequential trypsinization of layers of cells from HT-29 multicellular spheroids and analysis of gene expression by reverse transcription-polymerase chain reaction demonstrate that NQO1 expression is elevated in cells close to the necrotic center . Maximum expression occurs at a depth of 90-110 microns, with reduced expression as the distance toward both the surface and the necrotic center decreases . HT-29 spheroids were significantly more responsive than monolayers (concentration producing 50% inhibition, 124.6 and 364 nM, respectively) to the experimental drug, 2,5-dimethyl-3,6 diaziridinyl-1,4 benzoquinone . While the environmental stimulus responsible for causing elevated NQO1 expression has not been identified, the fact that NQO1 expression is influenced by microenvironmental conditions will have important implications for those drugs that are activated by NQO1.

ASAIO J, 1994 Jul-Sep, 40(3), M594 - 7
Microfabricated surface designs for cell culture and diagnosis; Matsuda T et al.; Grooved and holed surfaces with a well fabricated design may serve as microsubstrates for cell culture and microreactors for diagnosis . In this study, the authors prepared chemically treated, micrometer scale grooved and holed glass surfaces by combined surface modification and ultraviolet (UV) excimer laser ablation techniques, as follows . 1) Microcell-culture substrate: Amino group attached glass surfaces, prepared by the treatment with an aminopropylsilane, were condensed with a carboxylated radical initiator . Subsequently, polyacrylamide was grafted by surface initiated radical polymerization to create a very hydrophilic surface layer . Ultraviolet excimer laser beams (KrF: 248 nm) were irradiated through a microscope onto surfaces to create grooves or holes that were 10 and 50 microns in width or diameter, respectively . The depth, depending on the irradiation light strength, ranged from a few to several tenths of a micrometer . On endothelial cell (EC) seeding, ECs adhered and grew on the bottoms of the grooved or holed surface where glass was exposed on ablation . Little cell adhesion was observed on non ablated, grafted surfaces . Endothelial cells aligned along the groove, resulting in very narrow tube like tissue formation, whereas ECs tended to form a multilayered spherical aggregate in a hole . A single cell resided in a 10 microns square hole . 2) Microreactor for diagnosis: The glass surface, treated with a fluorinated silane, was ablated to create round holes . On addition of a few microliters of water, water could be quantitatively transferred into a hole because of the water repellent characteristics of non ablated, fluorinated glass . As a model of a microreactor, enzyme reactions to affect different levels of glucose were carried out in tiny holed surfaces.

Eur J Haematol, 1994 Jul, 53(1), 6 - 10
Serum immunoreactive erythropoietin in hyper- and hypothyroidism: clinical observations related to cell culture studies; Brenner B et al.; Laboratory experiments have demonstrated that tetra- and triiodothyronine (T4, T3) enhance hypoxia-induced erythropoietin (Epo) production . In the present study serum immunoreactive Epo was measured in 29 patients with hyperthyroidism and in 10 patients with hypothyroidism . Epo levels were inversely correlated to the blood haemoglobin concentration {Hb} in both groups of patients . However, Epo levels at given {Hb} were significantly higher in the hyperthyroid state . In vitro studies confirmed that T4 and T3 stimulate Epo synthesis in the human liver cell line HepG2 . This stimulating effect persisted for at least 1 day after the removal of T4 and T3 from the cultures . Thus, while thyroidal disorders affect steady-state levels of circulating Epo, it seems unlikely that thyroid hormones play a major role in abrupt adjustments of Epo production, such as the diurnal changes.

Metabolism, 1994 Jul, 43(7), 878 - 82
Insulin increases the release of endothelin in endothelial cell cultures in vitro but not in vivo; Metsarinne K et al.; Endothelin-1 (ET-1), a potent vasoconstrictor and mitogenic peptide for vascular smooth muscle cells, may be a marker for development of vascular disorders in diabetic patients . The aim of this study was to elucidate the possible role of insulin in the regulation of ET-1 production . The effect of hyperinsulinemia (with and without concomitant hyperglycemia) on the release of ET-1 was studied in 23 healthy men in vivo, as well as in human umbilical cord vein endothelial cell (HUVEC) cultures in vitro . Plasma glucose and insulin were maintained at four desired levels (from 5 to 22 mmol/L and 0.065 to 12.9 nmol/L, respectively) during the in vivo studies . The mean (SEM) plasma ET-1 during normoglycemia and a fasting insulin concentration in healthy men was 3.8 (0.4) pg/mL, and ET-1 levels did not change in response to changes in the concentration of glucose (from 5.0 to 22 mmol/L) or insulin (from 0.065 to 12.9 nmol/L) . The ET-1 concentration in HUVEC culture medium increased linearly during 24 hours, and insulin further enhanced the release of ET-1 dose-dependently . ET-1 release was stimulated by angiotensin II, thrombin, and transforming growth factor-beta (TGF-beta), whereas treatment with glucose and insulin-like growth factor-1 (IGF-1) was not associated with changed ET-1 levels in culture medium . Our results show that although high insulin concentrations stimulate ET-1 release in vitro, hyperinsulinemia is not associated with increased plasma ET-1 levels in healthy men in vivo . The role of insulin in the regulation of ET-1 production in vivo, if any, remains unsettled.

Hepatology, 1994 Jul, 20(1 Pt 1), 142 - 8
Prostaglandin F2 alpha and D2 release from primary Ito cell cultures after stimulation with noradrenaline and ATP but not adenosine; Athari A et al.; Rat liver Ito cells were cultured for 24 hr with 20% newborn calf serum . Stimulation with the sympathetic neurotransmitter noradrenaline (0.1 mumol/L to 1 mmol/L) led to a dose-dependent increase in prostaglandin F2 alpha release and a slightly smaller enhancement of prostaglandin D2 production . Prostaglandin F2 alpha and prostaglandin D2 synthesis was highest in the first 30 sec after stimulation . Stimulation with the possible cotransmitter ATP (10 mumol/L and 1 mmol/L ATP) also enhanced both prostaglandin F2 alpha and prostaglandin D2 release strongly . The release was highest again during the first 30 sec . Stimulation with noradrenaline and ATP simultaneously did not increase the effects of noradrenaline or ATP alone . Adenosine had no effect on prostaglandin production . The effects of noradrenaline were inhibited specifically by the alpha 1-adrenoreceptor antagonist prazosin but not by the p1-purinoreceptor antagonist 8-phenyltheophylline . The effects of ATP were not antagonized by the inhibitors . Because the metabolic actions of sympathetic hepatic nerves can be inhibited by inhibitors of prostanoid synthesis and mimicked by prostaglandins F2 alpha and D2, and because the Ito cells are well innervated, our results permit the conclusion that Ito cells could be involved in the nervous signal chain: During sympathetic nerve action the neurotransmitter noradrenaline and the cotransmitter ATP cause increases in prostaglandin F2 alpha and prostaglandin D2 release from Ito cells within 30 to 60 sec by way of alpha 1 and p2 receptors, respectively . The released prostaglandins then activate glycogenolysis in the hepatocytes proper.

Arterioscler Thromb, 1994 Jul, 14(7), 1056 - 65
A cell culture system for screening human serum for ability to promote cellular cholesterol efflux . Relations between serum components and efflux, esterification, and transfer; de la Llera Moya M et al.; A cell culture system was employed to test a large number of samples of human serum for the ability to stimulate the efflux of cell cholesterol . The extent of efflux obtained with each specimen was correlated with the serum concentrations of cholesterol, triglycerides, apoprotein (apo) B, apo A-I, apo A-II, and lipoprotein subfractions (ie, high-density lipoprotein2 {HDL2}, HDL3, lipoprotein {Lp} A-I, and LpA-I:A-II) . In addition, the subsequent esterification of the released cholesterol and the distribution of the synthesized exogenous cholesteryl esters between HDL and low-density lipoprotein/very-low-density lipoprotein provided estimates of the lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) activities of each serum . The values for these activities were analyzed for correlations with cell efflux and the various serum parameters . Cell cholesterol efflux best correlated with serum total HDL cholesterol values . HDL2 and HDL3 correlated about equally well with efflux, whereas LpA-I demonstrated a much greater association with efflux than did LpA-I:A-II . Analysis of the data by partial correlation analysis indicated that HDL3 and LpA-I were the HDL subfractions most closely associated with efflux . Esterification of the released radiolabeled cholesterol was strongly and positively correlated with serum triglyceride concentrations and negatively related to the serum concentrations of HDL2 . There was no relation between esterification values, which reflect LCAT activity, and efflux . The transfer of the labeled cholesteryl esters between HDL and apoB-containing lipoproteins was used as a measure of CETP activity and demonstrated a pattern in which all apoB-related parameters were positively correlated to transfer of esterified cholesterol, and all HDL associated parameters, particularly HDL3, were negatively related to transfer . No relations were observed between efflux, esterification, and transfer.

Endocrinology, 1994 Jul, 135(1), 284 - 9
Regulation of insulin-like growth factor-II synthesis in bone cell cultures by skeletal growth factors; Gabbitas B et al.; Insulin-like growth factor-II (IGF-II) is a growth factor secreted by bone cells and presumed to act as an autocrine regulator of bone formation . Although hormones and growth factors regulate the synthesis of skeletal IGF-I, hormones do not seem to modify the synthesis of skeletal IGF-II . We postulated that skeletal IGF-II is regulated by growth factors, and we tested the effects of basic fibroblast growth factor (bFGF), transforming growth factor-beta 1 (TGF beta 1), and platelet-derived growth factor-BB (PDGF-BB) on IGF-II messenger RNA (mRNA) expression and polypeptide concentrations in cultures of osteoblast-enriched (Ob) cells from 22-day-old fetal rat calvariae . Steady state IGF-II mRNA levels were determined by Northern blot analysis, and IGF-II concentrations were determined in acidified and fractionated culture medium by a specific RIA . Treatment of Ob cells with bFGF, TGF beta 1, and PDGF-BB decreased IGF-II mRNA levels after 24-48 h . A continuous 48-h treatment with bFGF at 0.6-6 nM, TGF beta 1 at 0.04-1.2 nM, and PDGF-BB at 0.3-3.3 nM caused a dose-dependent decrease in steady state IGF-II mRNA . The effects of bFGF, TGF beta 1, and PDGF-BB on IGF-II mRNA were dependent on protein synthesis and decreased in the presence of cycloheximide at 3.6 microM, but were independent of cell division, because they were observed in the presence and absence of 1 mM hydroxyurea . Treatment with bFGF, TGF beta 1, and PDGF-BB for 24 h did not cause a change in IGF-II polypeptide levels . PDGF-BB at 3.3 nM and TGF beta 1 at 0.04-0.4 nM for 48 h decreased IGF-II polypeptide levels by about 50%, although bFGF had no effect . In conclusion, bFGF, TGF beta 1, and PDGF decrease skeletal IGF-II transcript levels, and this effect may contribute to their actions on selected aspects of Ob cell function.

Probl Endokrinol (Mosk), 1994 Jul-Aug, 40(4), 45 - 7
{Effect of xenotransplantation of islet cell culture on the course of alloxan diabetes in rats fed a diet varying in protein content}; Sadykova RE et al.; The authors present data on the protective effect of newborn rabbits pancreatic islet cell culture xenotransplantation of Langerhans' islet beta-cells of rats with alloxan diabetes . This effect was the most marked in rats fed diets with normal or increased protein content . The authors discuss a possible stimulating effect of rabbit islet cell culture xenotransplantation on regeneration processes in recipient rat pancreatic islets . This effect was better pronounced in rats kept on rations with increased protein content . Further experiments will help more accurately define the indications for therapy of insulin-dependent diabetes mellitus by xenotransplantations of islet cell cultures.

Hum Immunol, 1994 Jul, 40(3), 210 - 7
Assessment of three methods of evaluating soluble class I HLA molecules in cell culture supernatants and serum samples from the second international workshop on soluble human leukocyte antigens; Nehlsen-Cannarella SL et al.; Three methodologies were compared in assessing sHLA specificities in cell culture supernatants and serum specimens from the Second International Workshop on sHLA: CDC inhibition, FC inhibition, and cellular ELISA inhibition . Initially, the CDC inhibition assay used polyclonal antisera in commercial HLA-phenotyping trays to confirm known specificities and screen for unknown specificities in 31 specimens . Although partly successful, critical limits were imposed by the variable antiserum titers . Thus, using pools of these same antisera and renal transplant recipient antisera, the FC inhibition assay was employed to determine the endpoint serum titers before confirming the known sHLA specificities . Of 25 specimens, four were not confirmed and five gave weak inhibitory reactions . The cellular ELISA inhibition assay, incorporating patient sera and mAbs toward three HLA, successfully confirmed all three known specificities in eight selected workshop specimens . Each methodology had advantages and disadvantages, but all three methods were successful in detecting and identifying sHLA class I specificities . Success, however, was dependent on the initial characterization (specificity and titer) and titration to end point (appropriate for each method's sensitivity) of each antibody preparation.

Poult Sci, 1994 Jul, 73(7), 965 - 74
Use of avian cytokines in mammalian embryonic stem cell culture; Yang Z et al.; Mouse blastocyst-derived embryonic stem (ES) cells are multipotent cells that can be used in vitro as models of differentiation and in vivo can contribute to all embryonic tissues including the germ line . The culture of ES cells requires a source of leukemia inhibitory factor (LIF), often provided by culture with a mouse fibroblast (STO) feeder layer, buffalo rat liver cell-conditioned media (BRL-CM), or the addition of recombinant LIF . To date, all of the ES cell culture systems use mammalian sources of LIF . We found that mouse ES cells can be maintained for over 10 passages in an undifferentiated state with media conditioned by a chicken liver cell line (LMH-CM) or on a feeder layer made with primary chicken embryonic fibroblasts (CEF) . These ES cells can undergo both spontaneous and induced differentiation, which is associated with the disappearance or reduction of the expression of alkaline phosphatase and SSEA-1, similar to that observed for ES cells cultured with BRL-CM or STO feeder layers . The ES cells cultured in LMH-CM did not express cytokeratin Endo-A antigen recognized by TROMA-1, but their differentiated progeny did express this antigen . In contrast to LMH-CM, Endo-A was expressed in ES cells cultured on CEF feeder layers and in differentiated progeny . These results indicate that avian cells can produce a LIF-like cytokine that is active in inhibiting the differentiation of mouse ES cells . This could provide a biological end point for the isolation and characterization of avian LIF.

Kidney Int, 1994 Jul, 46(1), 105 - 12
High glucose elevates c-fos and c-jun transcripts and proteins in mesangial cell cultures; Kreisberg JI et al.; It has been previously shown that rat glomerular mesangial cells synthesized increased amounts of fibronectin, laminin, and type IV collagen when grown in medium containing 30 mM glucose . High glucose exerted its effect at the mRNA level since transcripts for all three extracellular matrix (ECM) proteins were similarly elevated . High glucose appeared to exert its effect on ECM mRNA levels through protein kinase C activation . Using quantitative reverse transcription (RT) PCR, we now report that mRNA levels for c-fos and c-jun were increased approximately twofold after treatment with high glucose . The fos levels were elevated 15 minutes after addition of high glucose and were maintained elevated through 30 minutes; by one hour mRNA levels for fos returned to control levels . c-jun, on the other hand, was increased at two hours and remained elevated at 24 and 48 hours . Fibronectin mRNA levels were increased three- to fourfold at 24 and 48 hours . Immunofluorescence studies with polyclonal antibodies to c-fos and c-jun revealed that high glucose treatment for four hours increased nuclear staining intensity two- to threefold for both proteins . Nuclear staining for fos returned to control levels by 24 hours while staining for jun remained elevated . These determinations were made on images obtained on a confocal laser scanning microscope . Thus, high glucose may effect gene expression of ECM proteins by elevating the transcription factors c-fos and c-jun which complex with one another to form activator protein 1 (AP-1).

Cell Signal, 1994 Jul, 6(5), 513 - 22
Forskolin mimics TSH action on the expression of protein kinase C isozymes in pig thyroid cell cultures; Feliers D et al.; In porcine thyroid cell cultures, phospholipid-dependent protein kinases (PKCs) have the same characteristics as intact glands . The overall PKC activity, presence of PKC isozymes, chromatographic pattern and endogenous substrates specificity were not modified during the two-day culture period . Three PKC isozymes (cPKC epsilon, nPKC epsilon and aPKC zeta) were identified by immunoblot analysis in the two subcellular fractions: cytosol and particulate extract, both in intact glands and two-day-old cultures . In cells cultured for two days in the presence of TSH (0.1 mU/ml), the overall PKC activity was stimulated (ca . 200%) in the two compartments . This stimulation was parallel to the increase in protein expression of the three PKC isoforms (as demonstrated by immunoblot analysis) and was accompanied by a redistribution of cPKC alpha and nPKC epsilon toward the particulate fraction . In TSH-treated cells, hydroxyapatite chromatography of cytosolic PKC revealed an additional peak of PKC activity eluted at 195 mM potassium phosphate . Its elution molarity did not correspond to the molarity of any known PKC isozyme, and it did not cross-react with antibodies directed against cPKC isozymes--: alpha, beta, or gamma . When TSH was replaced by forskolin (10(-5) M), identical quantitative and qualitative modifications were obtained, suggesting that, in thyroid cells, the cyclic AMP-dependent regulatory cascade could be involved in the control of PKC isoforms expression by TSH.

Appl Microbiol Biotechnol, 1994 Jul, 41(5), 560 - 4
Extra- and intracellular amino acid concentrations in continuous Chinese hamster ovary cell culture; Hansen HA et al.; A recombinant DNA Chinese hamster ovary (CHO) cell line that produces tissue-type plasminogen activator (tPA) was cultivated continuously in suspension with a constant dilution rate of 0.5 day with three different asparagine concentrations in the feed (0.05, 2.55 and 7.55 mM) . The up-shift in asparagine concentration caused an up-shift in asparagine consumption {15.7 and 31.4 nmol (10(6) cells)-1 h-1} and intracellular concentration (2.19 and 18.7 mM) . The up-shift was accompanied by an increased production of ammonium, glycine and alanine, and a metabolic shift whereby the cells began to produce aspartate and glutamate, which were consumed before the shift . The tPA production was reduced in the up-shift culture . This might be explained by ammonium inhibition, but alternatively by a surprising down-shift in the intracellular concentration of many amino acids, a down-shift that was not observed in the extracellular concentrations or consumption rates . For efficient physiological engineering of mammalian cells it is necessary to include both extracellular and intracellular measurements and to consider the transport into and out of the cells.

Trends Biotechnol, 1994 Jul, 12(7), 257 - 61
Ensuring safety and consistency in cell culture production processes: viral screening and inactivation; Minor PD; The history of the use of biological products is littered with examples of the iatrogenic transmission of viruses . A major effort of the biotechnology industry is directed towards the elimination of any risk of infection from the products of cell culture . This article summarizes the overall strategy that has been followed, so far with great success, in that there have, as yet, been no reported cases of viral transmission by the products of biotechnology.

J Neurochem, 1994 Jul, 63(1), 118 - 24
Melatonin receptor-mediated inhibition of cyclic AMP accumulation in chick retinal cell cultures; Iuvone PM et al.; Melatonin receptors were characterized in cultured neurons and photoreceptors prepared from chick embryo retina . Cultured cells contained high-affinity 2-{125I}iodomelatonin binding sites (KD = 41.6 pM), similar to those in intact retina . The effects of melatonin and related indoles on cyclic AMP accumulation were examined . Melatonin (10(-7) M) had no effect on basal or K(+)-stimulated cyclic AMP accumulation, but inhibited forskolin-stimulated cyclic AMP accumulation by approximately 50% . Melatonin inhibited forskolin-stimulated cyclic AMP accumulation in the presence or absence of the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, suggesting an effect on cyclic AMP synthesis rather than degradation . Half-maximal inhibition was observed at 5.9 x 10(-10) M melatonin . The relative order of potency among melatonin analogues was 2-iodomelatonin > melatonin approximately 6-chloromelatonin > or = 6-hydroxymelatonin > N-acetylserotonin approximately 5-methoxytryptophol > serotonin . The EC50 value for inhibition of cyclic AMP accumulation by 2-iodomelatonin (36.7 pM) was comparable to the KD value for binding of the radioligand, suggesting that the binding sites represent functional receptors . The inhibitory effect of melatonin was antagonized by the putative melatonin antagonists luzindole, N-acetyltryptamine, and N-(2,4-dinitrophenyl)-5-methoxytryptamine, with estimated KB values of 0.12, 0.17, and 1 microM, respectively . At a concentration of 10 microM, N-(2,4-dinitrophenyl)-5-methoxytryptamine significantly inhibited forskolin-stimulated cyclic AMP accumulation when added alone; at 30 microM, luzindole and N-acetyltryptamine also had significant inhibitory effects . The inhibitory effect of melatonin was blocked by pretreatment with pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

J Neurosci Res, 1994 Jun 15, 38(3), 319 - 26
13C NMR spectroscopy study of cortical nerve cell cultures exposed to hypoxia; Muller TB et al.; Primary cultures of cerebral cortical GABA-ergic neurons growing on top of a preformed layer of astrocytes (co-cultures) were incubated with {1-13C}glucose and exposed to a low oxygen atmosphere (2% O2) for 17 hr . 13C, 1H, and 31P nuclear magnetic resonance (NMR) spectroscopy was performed on perchloric acid (PCA) extracts of cells and of media collected from these cultures . In the control groups incorporation of 13C label into glutamine, citrate, and lactate could be demonstrated in both cell extracts and culture media . Labeled GABA and glutamate were only observed in cell extracts . During hypoxia high energy phosphates decreased but lactate production and glucose consumption increased . There was a decreased amount of citrate and glutamine in cell extracts and media of the hypoxic co-cultures . There was a change in distribution of the 13C label within the GABA molecule, with an increase of labeling in the C-2 position . This change in 13C distribution was not found in glutamine present in the media where it is a precursor for GABA in neurons . Instead a decrease in the corresponding C-4 position was observed . These results suggest that energy depletion during hypoxia leads to reduced export from the astrocytic tricarboxylic acid (TCA) cycle as demonstrated by a decreased amount of citrate and changed distribution of 13C in glutamine . The change in the distribution of label in GABA from cell extracts as compared to glutamine in the medium may indicate that neurons are synthesizing GABA using precursors supplied from their own TCA cycle and not from precursors supplied by astrocytes.

J Physiol, 1994 Jun 15, 477 ( Pt 3), 449 - 58
Potentiation by ATP of the postsynaptic acetylcholine response at developing neuromuscular synapses in Xenopus cell cultures; Fu WM; 1 . Extracellular application of ATP to developing Xenopus neuromuscular synapses in culture resulted in a marked increase in the amplitude and frequency of spontaneous synaptic currents, using whole-cell recording . 2 . The postsynaptic action of ATP was examined by studying the response of isolated muscle cells to iontophoretically applied acetylcholine (ACh) . ATP enhanced the responses of the muscle membrane to ACh . The order of potency for various nucleotides (ATP = ADP >> AMP, adenosine, GTP) suggests that ATP acts through P2-purinoceptors . The effect of ATP on whole-cell currents was also abolished by the protein kinase inhibitor H-7 . 3 . Single-channel measurements indicate that ATP increased the mean open time of low-conductance ACh channels . No change in the conductance of ACh channels was observed . 4 . Local application of ATP to one region of the elongated myocyte surface resulted in potentiated ACh responses only at the ATP-treated region, suggesting that the cytosolic second messengers were effectively confined within the muscle cytoplasm . 5 . The results of the present study suggest that ATP released from the nerve terminals may potentiate the ACh response of developing muscle cells during the early phase of synaptogenesis, and that the action of ATP can be restricted to the subsynaptic region exposed to the secreted ATP.

Clin Immunol Immunopathol, 1994 Jun, 71(3), 287 - 92
Long-term humoral and cellular immunity after vaccination with cell culture rabies vaccines in man; Thraenhart O et al.; To determine the duration of anti-rabies immunity, peripheral blood of 18 vaccinees was obtained between 2 and 14 years after immunization . Peripheral blood mononuclear cells (PBMC) and serum were tested for the presence of either rabies virus-specific antibodies or rabies antigen-specific proliferation . Neutralizing immunoglobulin class G anti-rabies virus antibodies could be detected in sera of all vaccinees, but not in 18 age- and sex-matched controls . Rabies antigen-induced proliferation of PBMCs from vaccinees was significantly higher than that of controls . The anti-rabies T and B cell response showed no time-dependent pattern . These results suggest the induction of a long-term immunity after rabies immunization according to pre- and post-exposure schedules with inactivated cell culture vaccines against rabies.

Endocrinology, 1994 Jun, 134(6), 2541 - 6
Platelet-derived growth factor-AA and -BB (PDGF-AA and -BB) enhance the synthesis of PDGF-AA in bone cell cultures; Rydziel S et al.; Platelet-derived growth factor (PDGF), an agent with important mitogenic effects for bone cells, exists in three isoforms, PDGF-AA, -BB, and -AB . PDGF-AB and -BB are the prevalent circulating isoforms, whereas normal unstimulated cells of the osteoblast lineage synthesize primarily PDGF-AA . We examined the effects of PDGF-BB on PDGF-A mRNA expression and PDGF-AA polypeptide concentrations in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells) . In a selected number of experiments we compared the effects of PDGF-BB with those of PDGF-AA on PDGF-A mRNA levels . Steady state PDGF-A mRNA levels were determined by Northern blot analysis, and PDGF-AA concentrations were determined in acidified and fractionated culture medium by a specific RIA for PDGF-A chains . Treatment of Ob cells with PDGF-AA or -BB at 0.3-3.3 nM caused a dose-dependent increase in steady state PDGF-A mRNA, an effect that was initially observed after 2 h . Treatment with PDGF-BB at 1-3.3 nM for 24 h increased PDGF-AA polypeptide concentrations by 2- to 5-fold . The effects of PDGF on PDGF-A mRNA and polypeptide levels were prevented by the protein synthesis inhibitor cycloheximide at 3.6 microM . Phorbol 12-myristate 13-acetate at 1 microM increased PDGF-A mRNA after 2-6 h and PDGF-AA polypeptide levels after 24 h by 2-fold . However, the protein kinase-C inhibitor staurosporine at 50 nM did not modify basal PDGF-A mRNA levels and did not prevent the stimulatory effect of PDGF-AA or -BB on PDGF-A mRNA or PDGF-AA polypeptide levels . In conclusion, PDGF-BB and -AA increase skeletal PDGF-A synthesis, an effect that reveals autoinduction of PDGF in bone cells.

Infect Immun, 1994 Jun, 62(6), 2600 - 4
Production of alpha interferon in Cowdria ruminantium-infected cattle and its effect on infected endothelial cell cultures; Totte P et al.; Cattle that resisted experimental heartwater infection caused by the rickettsia Cowdria ruminantium produced significant levels of circulating alpha interferon (IFN-alpha), whereas animals that died from heartwater did not . In vitro, recombinant bovine IFN-alpha was found to significantly reduce the yield of Cowdria organisms in bovine endothelial cells, but even at a high concentration (1,000 U/ml), IFN-alpha did not completely prevent the growth of Cowdria organisms in these cells . This limited inhibitory effect of IFN-alpha is in agreement with the in vivo situation where an infectious process has to take place to induce a protective immune response . Our results suggest that IFN-alpha produced in vivo in response to Cowdria infection may represent an efficient way to slow down the infection and allow the animal to mount a protective immune response . IFN-alpha is the first endogenously produced factor shown to have anti-Cowdria activity.

Radiat Res, 1994 Jun, 138(3), 451 - 9
Determination of potential doubling times in human melanoma cell cultures subjected to irradiation and/or hyperthermia by flow cytometry; Zolzer F et al.; The proliferation of human melanoma cells in vitro after irradiation and/or hyperthermia was studied by means of two-parameter flow cytometry . Cultures were incubated with BrdU for 30 min and fixed either immediately or after a delay of several hours . Cells having synthesized DNA were identified with the help of an antibody against BrdU . DNA was stained quantitatively with propidium iodide . In this way the distribution of cells in the phases of cell cycle could be determined and the movement of labeled cells through the phases of the cycle could be analyzed . Experiments in which the cell cycle distribution was studied at 4-h intervals after treatment showed the following: (1) Irradiation (4 Gy X rays) causes the expected G2 block with a maximum after 12-16 h . The proportion of S-phase cells decreases continually during the first 48 h after treatment . (2) Hyperthermia (1 h, 43 degrees C) alone or in combination with irradiation causes a delay in S phase . The cells begin to move into G2 phase only after 12-16 h and accumulate there to some extent . From the progression of labeled cells through the cycle, the duration of S phase could be determined . Experiments and calculations of this kind were done 0, 24 and 48 h after treatment . The duration of S phase was increased only moderately (by 4 h) after irradiation, but a delay of about 30 h occurred after hyperthermia (alone or in combination with X rays) . Smaller delays (up to 9 h) were observed 24 and 48 h after treatment . Two different methods were used to calculate potential doubling times . Both of them gave similar results, but a comparison with the actual population doubling times (determined by cell counting) showed that reasonable estimates could be achieved only for the untreated controls . With cultures subjected to irradiation and/or hyperthermia serious discrepancies were observed . This does not seem to be due to technical problems inasmuch as we are dealing with a whole set of data produced under well-defined in vitro conditions (in contrast to the clinical situation, where potential doubling times have to be estimated from single samples) . Our results certainly do not encourage the extension of the method (which was originally intended for the prediction of unperturbed tumor growth) to a post-treatment setting.

J Neurobiol, 1994 Jun, 25(6), 666 - 93
Postsynaptic regulation of the development and long-term plasticity of Aplysia sensorimotor synapses in cell culture; Glanzman DL; The monosynaptic component of the neuronal circuit that mediates the withdrawal reflex of Aplysia californica can be reconstituted in dissociated cell culture . Study of these in vitro monosynaptic connections has yielded insights into the basic cellular mechanisms of synaptogenesis and long-term synaptic plasticity . One such insight has been that the development of the presynaptic sensory neurons is strongly regulated by the postsynaptic motor neuron . Sensory neurons which have been cocultured with a target motor neuron have more elaborate structures--characterized by neurites with more branches and varicosities--than do sensory neurons grown alone in culture or sensory neurons that have been cocultured with an inappropriate target cell . Another way in which the motor neuron regulates the development of sensory neurons is apparent when sensorimotor cocultures with two presynaptic cells are examined . In such cocultures the outgrowth from the different presynaptic cells is obviously segregated on the processes of the postsynaptic cell . By contrast, when two sensory neurons are placed into cell culture without a motor neuron, their processes readily grow together . In addition to regulating the in vitro development of sensory neurons, the motor neuron also regulates learning-related changes in the structure of sensory neurons . Application of the endogenous facilitatory transmitter serotonin (5-HT) causes long-term facilitation of in vitro sensorimotor synapses due in part to growth of new presynaptic varicosities . But 5-HT applied to sensory neurons alone in culture does not produce structural changes in these cells . More recently it has been found that sensorimotor synapses in cell culture can exhibit long-term potentiation (LTP) . Like LTP of some hippocampal synapses, LTP of in vitro Aplysia synapses is regulated by the voltage of the postsynaptic cell . Pairing high-frequency stimulation of sensory neurons with strong hyperpolarization of the motor neuron blocks the induction of LTP . Moreover, LTP of sensorimotor synapses can be induced in Hebbian fashion by pairing weak presynaptic stimulation with strong postsynaptic depolarization . These findings implicate a Hebbian mechanism in classical conditioning in Aplysia . They also indicate that Hebbian LTP is a phylogenetically ancient form of synaptic plasticity.

Appl Environ Microbiol, 1994 Jun, 60(6), 1921 - 6
In situ detection of hepatitis A virus in cell cultures and shellfish tissues; Romalde JL et al.; An in situ transcription method was developed to detect hepatitis A virus RNA in both cell cultures and shellfish tissues . Radiolabeled cDNA copies were synthesized in situ by reverse transcriptase-directed transcription after annealing with a specific primer to the viral RNA . Both tritium (3H) and 35S were useful in the in situ transcription reaction, but the use of 3H resulted in a lower background and finer detail in the localization of viral particles . Application of the method to different organs of oysters which had bioaccumulated hepatitis A virus allowed the first in situ localization of the virus, specifically in stomach and hepatopancreatic tissues.

Am J Physiol, 1994 Jun, 266(6 Pt 1), C1803 - 11
Method for recovering ATP content and mitochondrial function after chemical anoxia in renal cell cultures; Doctor RB et al.; Cultured renal cells provide a highly reproducible and malleable model to study cellular responses to metabolic perturbations . Nevertheless, there is currently no good method to achieve metabolic inhibition and complete recovery in cultured cells . This study describes a specific method for reversibly inhibiting both glycolytic and oxidative metabolism . Glycolysis was inhibited by removing all glycolytic substrates, and mitochondrial respiration was inhibited with rotenone, a site I inhibitor of the electron transport chain . Within 30 min, ATP values were decreased by 98% . Glycolysis was restored through the reintroduction of glucose . Oxidative metabolism was restored by the addition of heptanoate, a short odd-chain fatty acid, which supplies reducing equivalents to site II of the electron transport chain . Employing Madin-Darby canine kidney and LLC-PK1 cell lines, this protocol caused the immediate and complete recovery of mitochondrial respiration and, by 60 min, the complete recovery of cellular ATP levels . Application of this protocol should allow the investigation of the cellular effects and alterations that occur within cells recovering from sublethal energy depletion.

Int J Dev Biol, 1994 Jun, 38(2), 287 - 99
Mechanisms of the proliferation and differentiation of plant cells in cell culture systems; Fukuda H et al.; Plant cell functions have been investigated in various cell culture systems . In this review, we summarize results obtained from investigations of gene expression during the cell cycle in synchronized cultures of Catharanthus roseus during somatic embryogenesis in suspension cultures of Daucus carota, during organogenesis in tissue cultures of Arabidopsis thaliana and during the transdifferentiation of isolated mesophyll cells to tracheary elements in single-cell cultures of Zinnia elegans.

Xenobiotica, 1994 Jun, 24(6), 487 - 93
Induction of zebrafish (Brachydanio rerio) P450 in vivo and in cell culture; Collodi P et al.; 1 . Induction of zebrafish P450 by 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) was studied in liver tissue, primary liver cell culture and multipassage cell culture derived from zebrafish haploid and diploid embryos and liver . 2 . TCDD induced two hepatic proteins (54 and 50 kDa) in vivo which were recognized by anti-trout P4501A1 IgG . The 54-kDa protein was induced by TCDD in primary and multipassage hepatocyte cultures and in haploid and diploid embryo-derived cells . The proteins in liver homogenates were not induced by aqueous exposure of zebrafish to beta-naphthoflavone (BNF) . 3 . Homogenates of zebrafish liver, cultured hepatocytes and embryo-derived cells also exhibited increased ethoxyresorufin O-deethylase (EROD) and 7-12-dimethyl-benz{a}anthracene (DMBA) hydroxylase activity following TCDD exposure.

Eur J Clin Invest, 1994 Jun, 24(6), 406 - 15
Repression of ALA synthase by heme and zinc-mesoporphyrin in a chick embryo liver cell culture model of acute porphyria; Russo SM et al.; We characterize a liver cell culture model for acute hepatic porphyrias that recapitulates the biochemical features of the human syndrome . In chick embryo liver cells in primary culture exposed to glutethimide and 4,6-dioxoheptanoic acid, heme alone produced a transient dose-dependent decrease in delta-aminolevulinate synthase and a concomitant increase in heme oxygenase . The addition of low concentrations of zinc-mesoporphyrin (50-200 nM), an inhibitor of heme oxygenase, led to more prolonged decreases in activity of the synthase and to an additive effect with heme . These effects of zinc-mesoporphyrin were associated with prolonged inhibition of heme oxygenase . These results suggest that the treatment of choice of acute porphyric syndromes may be the combination of low doses of heme and zinc-mesoporphyrin or another similarly non-toxic inhibitor of heme oxygenase.

Mol Mar Biol Biotechnol, 1994 Jun, 3(3), 131 - 40
Myogenesis in primary cell cultures from larvae of the abalone, Haliotis rufescens; Naganuma T et al.; Myogenesis culminating in the differentiation of contracting myocytes occurs in primary cell cultures derived from premyogenic trochophore and early veliger larvae of Haliotis rufescens (red abalone, gastropod mollusc) . No detectable muscle cells were present at the start of the primary cultures . Onset and organization of myofibrillogenesis in culture (revealed by histochemical and immunohistochemical detection of filamentous actin, myosin, and desmin) generally paralleled those of smooth muscle development in vivo, although development of cells with striated sarcomeres was occasionally observed in culture, but not in the intact larvae . Most of the muscle cells that developed in culture were mononucleate, although some multinucleate syncytia were observed . Dissociated larval cells remained viable up to 12 weeks, exhibiting an average of one to two divisions in the first six days, and attachment of approximately 6 to 8% of the original population . Cell culture and in vitro myogenesis of Haliotis myoblasts and myocytes will facilitate studies of the molecular mechanisms controlling early muscle development, and should provide a useful model system for biotechnological improvement of the abalone.

Biologicals, 1994 Jun, 22(2), 161 - 9
Purified protein products of rDNA technology expressed in animal cell culture; Lubiniecki AS et al.; During the past 7 years, 14 versions of 7 rDNA proteins have been licensed which are derived from animal cell culture expression systems . These medically useful products have included hormones, coagulation factors, enzymes and a vaccine . Aspects of the molecular complexity, manufacture, control and utilization of these products are discussed . In contrast to previous generations of biological production technology, the technology for production of rDNA-derived proteins in animal cells appears to be safe.

J Mol Endocrinol, 1994 Jun, 12(3), 293 - 302
Translocation of protein kinase C isozymes in lactotroph-enriched rat anterior pituitary cell cultures: differential effects of substance P and phorbol ester; Mau SE et al.; Translocation of protein kinase C (PKC) from the cytosol to the plasma membranes is believed to reflect activation of the enzyme . We have studied translocation of PKC in lactotroph-enriched anterior pituitary cell cultures by measuring the incorporation of gamma-32P from {gamma-32P}ATP into a synthetic peptide substrate, MBP4-14, and by immunoblotting of PKC isozymes . Using cells permeabilized with digitonin the effects of PKC cofactors on the distribution of the enzyme were studied . Ca2+ (50 nM) and dioctanoyl-sn-glycerol had no effect when tested alone, but in combination they caused a redistribution of PKC from the soluble to the particulate fraction . Arachidonic acid needed Ca2+ to induce translocation of PKC, while being ineffective under Ca(2+)-free conditions . Western blot analysis of partly purified PKC from lactotroph-enriched pituitary cells revealed the presence of the alpha, beta, delta and zeta isozymes . 12-O-Tetradecanoylphorbol 13-acetate (TPA) and substance P displayed different patterns of redistribution of PKC isozyme immunoreactivity from soluble to membrane-attached forms . Thus, TPA induced time- and dose-dependent (mean effective concentration (EC50) = 1 nM) translocation of the alpha, beta and delta species, while substance P stimulated time- and dose-dependent (EC50 = 1 nM) redistribution of the alpha and beta isozymes . zeta subtype immunoreactivity could not be translocated by either agonist; neither could the immunoreactivity of zeta be down-regulated by long-term treatment (24 h) with TPA . The results indicate that simultaneous activation of phospholipases C and A2 induces a synergistic activation of PKC . Finally it is suggested that substance P may exert some of its effects in lactotrophs by translocation of PKC isozymes alpha and beta.

Antimicrob Agents Chemother, 1994 Jun, 38(6), 1428 - 32
Novel mutation (V75T) in human immunodeficiency virus type 1 reverse transcriptase confers resistance to 2',3'-didehydro-2',3'-dideoxythymidine in cell culture; Lacey SF et al.; We have selected a human immunodeficiency virus type 1 (HIV-1) mutant strain with a moderate (sevenfold) level of resistance to the nucleoside analog 2',3'-didehydro-2',3'-dideoxythymidine (D4T or stavudine) . After serial passage of the HXB2 strain of HIV-1 in MT4 cells, a novel mutation involving two nucleotide substitutions in codon 75 of the viral reverse transcriptase, altering valine to threonine, was seen . When introduced into a wild-type HIV-1 background by site-directed mutagenesis, the T-75 mutation conferred cross-resistance to the dideoxynucleosides dideoxyinosine and dideoxycytosine as well as to 2',3'-didehydro-2',3'-dideoxycytosine.

Biochim Biophys Acta, 1994 May 26, 1222(1), 37 - 44
Cyclic AMP causes differentiation and decreases the expression of neutral glycosphingolipids in cell cultures derived from a malignant glioma; Hoffman LM et al.; Cultures derived from a malignant glioma (U-87 MG) were treated with 3 mM dibutyryl cAMP . The treatment resulted in morphological differentiation of the cultures and a decrease in cell proliferation . Biochemically, dibutyryl cAMP treatment caused a general reduction in the concentration of neutral glycosphingolipids in the U-87 MG cells . The concentration of individual neutral glycosphingolipids in the untreated cells was 1.8- to 3.0-fold higher than in cells treated for 72 h with 3 mM dibutyryl cAMP . Cells were labeled with {3H}galactose to monitor synthesis of the neutral glycosphingolipids . Decreased synthesis was noted in cells treated with dibutyryl cAMP as compared with untreated cells as indicated by decreased uptake of {3H}galactose label . The ganglioside composition of the cells was essentially unchanged after dibutyryl cAMP treatment.

Brain Res, 1994 May 23, 646(2), 315 - 8
Carbon monoxide modulates secretion of corticotropin-releasing factor from rat hypothalamic cell cultures; Parkes D et al.; The present study examined the actions of the putative gaseous neurotransmitter carbon monoxide (CO) on secretion of corticotropin-releasing factor (CRF) from cultured primary rat hypothalamic cells . 6 h treatment of cells with 100% gaseous CO, or with the heme analog, hematin (100 microM) which produces CO as a by-product of its metabolism, increased basal secretion of CRF to 207 +/- 8% and 200 +/- 65% of control respectively . Zinc protoporphyrin IX (ZnPPIX) (0.3-100 microM), a selective inhibitor of CO formation, decreased CRF secretion in a dose-dependent manner . The changes in CRF secretion observed with hematin were attenuated during concurrent treatment with ZnPPIX . These studies suggest that basal secretion of CRF in the rat hypothalamus may be regulated by CO.

Biochem J, 1994 May 15, 300 ( Pt 1), 125 - 31
Gene expression of GLUT3 glucose transporter regulated by glucose in vivo in mouse brain and in vitro in neuronal cell cultures from rat embryos; Nagamatsu S et al.; This study was designed to determine whether glucose regulates the gene expression of glucose transporter GLUT3 in neurons . We examined the regulation of GLUT3 mRNA by glucose in vivo in mouse brain and in vitro by using neuronal cultures from rat embryos . Hypoglycaemia (< 30 mg/dl), produced by 72 h of starvation, increased GLUT3 mRNA in mouse brain by 2-fold . Hybridization studies in situ demonstrated that hypoglycaemia-induced increases in GLUT3 mRNA expression were observed selectively in brain regions including the hippocampus, dentate gyrus, cerebral cortex and piriform cortex, but not the cerebellum . Primary neuronal cultures from rat embryos deprived of glucose for 48 h also showed an increase (4-fold over control) in GLUT3 mRNA, indicating that glucose can directly regulate expression of GLUT3 mRNA . In contrast with hypoglycaemia, hyperglycaemia produced by streptozotocin did not alter the expression of GLUT3 mRNA . We also confirmed previous findings that hypoglycaemia increases GLUT1 mRNA expression in brain . The increase in GLUT1 expression was probably limited to the blood-brain barrier in vivo, since GLUT1 mRNA could not be detected in neurons of the mouse cerebrum . Thus we conclude that up-regulation of neuronal GLUT3 in response to glucose starvation represents a protective mechanism against energy depletion in neurons.

Brain Res Dev Brain Res, 1994 May 13, 79(1), 128 - 31
Alteration in oxidative metabolism of alanine in cerebellar granule cell cultures as a consequence of the development of the ability to utilize alanine as an amino group donor for synthesis of transmitter glutamate; Peng L et al.; Formation of 14CO2 from labeled alanine was measured in cultured cerebellar granule cells grown in the combined presence of alanine, alpha-ketoglutarate and glutamine or in the presence of glutamine alone . This was done in order to study whether the utilization of alpha-ketoglutarate plus alanine as precursors of transmitter glutamate, induced by culturing in the presence of these compounds, is reflected by an increase of CO2 production from alanine during stimulation with an elevated extracellular potassium concentration . Potassium stimulated CO2 production from alanine was present only in the cells grown in the combined presence of alanine, alpha-ketoglutarate and glutamine . This stimulation was abolished by glutamine, but not by ouabain, indicating that the depolarizing-induced stimulation of alanine metabolism is a consequence of increased release of transmitter glutamate formed from alanine, not a simple result of an increased metabolic rate.

Biochemistry, 1994 May 10, 33(18), 5607 - 13
Glycosylation of shaker potassium channel protein in insect cell culture and in Xenopus oocytes; Santacruz-Toloza L et al.; We have studied the glycosylation of Shaker K+ channel protein made in two expression systems: an insect cell culture line and amphibian oocytes . In both systems, two potential sites for N-linked glycosylation were modified . The modified sites were located between the first and second putative transmembrane segments, S1 and S2 . Although the same sites appeared to be glycosylated in both systems, the fraction of protein glycosylated and the size, structure, or composition of the oligosaccharide chains added were quite different . The results indicate that the S1-S2 loop is extracellular, consistent with a cytoplasmic location for the N-terminus and a transmembrane disposition for hydrophobic segment S1 . We have also shown that glycosylation occurs in two stages in oocytes, generating an immature and a mature form of Shaker protein . However, glycosylation is not required either for the assembly of functional channels or for their transport to the cell surface.

Am J Hematol, 1994 May, 46(1), 54 - 6
Spontaneous remission of polycythemia vera: clinical and cell culture characteristics; Cowan DH et al.; A 20-year-old woman presented with polycythemia vera and was treated with phlebotomy alone for eleven years, following which all clinical manifestations of the disease disappeared . The clinical remission with normal physical findings and normal peripheral blood counts has persisted for a further 11 years . Erythroid colony culture results have paralleled the clinical state . Initial bone marrow cultures revealed spontaneous growth of erythroid burst-forming units (BFU-E) . Subsequent cultures of peripheral blood cells throughout most of the period of spontaneous clinical remission have revealed little or no spontaneous growth of BFU-E . This suggests a suppression of the abnormal stem cell clone during the period of remission.

J Infect Dis, 1994 May, 169(5), 1092 - 6
Cytomegalovirus replication in murine microglial cell cultures: suppression of permissive infection by interferon-gamma; Schut RL et al.; The pathogenesis of encephalitis due to cytomegalovirus (CMV), particularly the role of microglial cells in the spread or control of infection, remains incompletely defined . In this study, microglial cells were isolated from the brains of newborn mice and infected in vitro with murine CMV (MCMV) . Microglial cells supported productive MCMV replication, and the MCMV-infected microglia manifested a cytopathic effect (CPE) characteristic of CMV infection . Exposure of microglia to interferon-gamma (IFN-gamma) 24 h before infection markedly suppressed virus production and resultant CPE in a dose-dependent fashion . Furthermore, the addition of IFN-gamma 2 h after infection demonstrated an antiviral effect equivalent to that achieved when IFN-gamma was administered 2 h before infection . These results demonstrate that murine microglial cells are fully permissive to MCMV replication and that IFN-gamma markedly suppresses virus expression in these cells.

J Neurochem, 1994 May, 62(5), 1863 - 9
Nicotine tolerance in chromaffin cell cultures: acute and chronic exposure to smoking-related nicotine doses; Bullock AE et al.; Nicotine tolerance and dependence are key aspects of tobacco addiction; however, the cellular mechanisms underlying these phenomena are poorly understood . Adrenal chromaffin cells release catecholamines upon exposure to nicotine and with repeated exposure this response exhibits nicotine tolerance . Using bovine adrenal chromaffin cells in culture, we have demonstrated acute and chronic nicotine tolerance at doses relevant to that in the blood and tissues of smokers (10(-7) M to 10(-6) M) . Chromaffin cells are preexposed to low doses of nicotine for time periods ranging from 10 min to 7 days and then subsequently challenged with a maximally stimulating dose of nicotine (10(-5) M) for 10 min, all at 37 degrees C . Preexposure to nicotine results in a depression of 45Ca uptake and catecholamine release upon subsequent nicotine challenge . Acute tolerance or desensitization of nicotine-stimulated catecholamine release begins to occur in minutes after preexposure to 10(-6) M nicotine at 37 degrees C . The depression of catecholamine release upon preexposure to nicotine is both dose and temperature dependent and is not seen with potassium-evoked release . Chronic exposure to 10(-7) M nicotine for 3 days led to a depression of the secretory response to approximately 70% of control responses . There was a trend toward recovery of full response by days 5 and 7 of 10(-7) M nicotine preexposure . Nearly complete depression of the nicotine-evoked release occurs within the first day of exposure to 10(-6) M nicotine and persists for at least a week of nicotine exposure at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

J Neurocytol, 1994 May, 23(5), 279 - 95
Characterization of GABAergic neurons in hippocampal cell cultures; Benson DL et al.; The morphological characteristics of GABAergic neurons and the distribution of GABAergic synaptic terminals were examined in cultures of hippocampal neurons from 4-35 days in vitro . Neurons expressing GABA immunoreactivity represented about 6% of the total number of cultured neurons at all time points . Although the morphological characteristics of GABAergic cells suggested a heterogeneous population, GABAergic cells as a class were notably different from the non-GABAergic, presumably pyramidal cells . Most GABAergic cells had more fusiform or polygonal shaped somata, non-spiny and less tapering dendrites and appeared more phase-dense than nonGABAergic cells . Quantitative analysis revealed that GABAergic cells had fewer primary dendrites, more elongated dendritic arbors, and longer dendritic segments than non-GABAergic neurons-characteristics that are similar to GABAergic cells in situ . Double immunostaining revealed that GAD65-positive varicosities were also immunopositive for synapsin I, suggesting that GAD65-positive varicosities that contacted somata and dendrites represented presynaptic specializations . Confocal microscopy revealed the proportion of the synaptic specializations on the cell soma that were GAD65-positive was greater than on the dendrites, suggesting that somata and dendrites differ in their ability to induce the formation of presynaptic specializations by GABAergic axons . These data indicate that the GABAergic cells that develop in culture exhibit distinctive morphological characteristics and participate in different synaptic interactions that nonGABA cells . Thus many of the features that distinguish GABAergic neurons in culture are reminiscent of the characteristics that distinguish GABAergic neurons in situ.

Anticancer Res, 1994 May-Jun, 14(3A), 793 - 8
UDP-N-acetylhexosamines and hypotaurine in human glioblastoma, normal brain tissue and cell cultures: 1H/NMR spectroscopy study; Sonnewald U et al.; NMR spectroscopy was used to analyse perchloric acid extracts of normal human brain, murine brain cell cultures, glioblastoma tissue and the glioblastoma cell line U-87 . 1H NMR spectra revealed the presence of elevated levels of UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine in glioblastoma extracts and the glioblastoma cell line U-87, in comparison with normal brain tissue and primary cell cultures of neurons and astrocytes . UDP-N-acetylhexosamines appear to accumulate in cells that are unable to differentiate . Furthermore, it was found that the culture medium had an effect on the concentration of UDP-N-acetygalactosamine in the glioblastoma cell line . Hypotaurine, previously only associated with oligodendrocytes, has been identified in astrocyte cultures and in cerebellar granule cells . In normal brain it was not observed by NMR spectroscopy, but was easily detectable in glioblastoma tissue extracts . UDP-N-acetylhexoseamines and hypotaurine might be useful markers for brain pathology and play a role in cell differentiation and cell division.

Glia, 1994 May, 11(1), 11 - 7
Development of microglia in mouse neopallial cell cultures; Neuhaus J et al.; Microglia develop in cultures initiated from disaggregated neopallial cells of newborn C3H/HeJ mice when the cultures are subjected to nutritional deprivation for 10 or more days (Hao et al: Int J Dev Neurosci 9:1-14, 1991) . In the present experiments, the cultures were pulsed with BrdU for 3 hours at different times during incubation and then the cells were immunoreacted with antibodies against BrdU, GFAP, and CR3 receptor . The dividing cells (BrdU+) were found to be either GFAP+ or GFAP-, but not Mac-1+/BrdU+ . Infection of proliferating cells after 2 or more days of incubation with replication-deficient retroviral vector containing E . coli lacZ reporter gene resulted in many labeled astroglia cell clones but no labeled microglia . However, when cells were infected right after disaggregation of neopallium, labeled Mac-1+ microglia were found . When Mac-1+ cells in a suspension of disaggregated neopallial cells were killed using complement mediated lysis before setting up the cultures, Mac-1+ microglia developed, in spite of the treatment . We conclude that in cultures initiated from mouse neopallium there are MAC-1-/GFAP- microglia progenitor cells which do not divide in nutritionally deprived cultures but can transform into Mac-1+ microglia under the influence of astroglia-derived trophic factors . Microglia, which become Mac-1+ (i.e., express CR3 receptor), proliferate extensively in the presence of CSF-1 (which is produced by astroglia).

Mol Gen Mikrobiol Virusol, 1994 May-Jun, (3), 16 - 20
{Use of U3 promotor from the LTR region of bovine leukemia virus for expressing genes for antiviral antisense RNAs in cell cultures}; Borisenko AS et al.; The possibility was studied to use the U3 promoter from LTR region of bovine leukemia virus to control the expression of the antisense RNA genes directed against the BLV genome in cell cultures FLK-BLV, CC81, and HeLa . The genetic engineering constructions were obtained carrying the reporter beta-galactosidase gene and the genome of antisense RNA to R-U5 region of the viral genome under the control of the promoter . The functioning of the viral promoter was studied in three cell lines . Its specific activation and kinetics of inhibition of BLV virus reproduction in cell culture CC81 have been demonstrated . The maximal 75% level of the viral reproduction inhibition was achieved at threefold molar excess of the plasmid containing the antisense RNA gene over the pregenomic viral DNA.

J Clin Microbiol, 1994 May, 32(5), 1406 - 7
Performance of three commercially available monoclonal reagents for confirmation of Chlamydia pneumoniae in cell culture; Montalban GS et al.; We evaluated the performance of three commercially available monoclonal antibodies for confirmation of the presence of Chlamydia pneumoniae in cell culture by examining their abilities to stain inclusions of eight strains of C . pneumoniae . The antibodies tested were two unconjugated C . pneumoniae-specific monoclonal reagents and one conjugated genus-specific reagent . All three produced similar intensities of staining of C . pneumoniae, with some strain-to-strain variation . Methanol appeared to be a better choice of fixative than acetone, which greatly reduced the intensity of fluorescence with one of the species-specific antibodies.

J Eukaryot Microbiol, 1994 May-Jun, 41(3), 223 - 8
Genetic diversity of Pneumocystis carinii derived from infected rats, mice, ferrets, and cell cultures; Weinberg GA et al.; The degree of strain and/or species diversity among Pneumocystis carinii isolates is unknown . As a first approach to the study of P . carinii genetic relatedness, we compared the pulsed field gel electrophoretic karyotypes of P . carinii derived from lung homogenates of three immunosuppressed host animals: rats transtracheally inoculated with P . carinii-infected mouse lung; and ferrets which developed reactivated latent P . carinii pneumonia . Rat P . carinii propagated on HEL299 cells was also examined . Karyotypes of P . carinii DNA from both rat lung homogenate and cell culture were identical (14 bands, 315-680 kb) . In contrast, mouse and ferret P . carinii DNA karyotypes were each distinctly different from the rat P . carinii samples (mouse P . carinii 15 bands, 315-610 kb; ferret P . carinii nine bands, 410-760 kb) . Three distinct rat P . carinii gene probes reacted with both Southern-transferred rat and mouse P . carinii DNA but not with ferret P . carinii DNA . Thus, P . carinii from rat, mouse, and ferret are genetically diverse . The results are consistent with recently reported antigenic and nucleic acid sequence differences among P . carinii isolates recovered from different hosts.

Biofizika, 1994 May-Jun, 39(3), 490 - 5
{The mechanism of synchronizing yeast cell cultures with EHF-radiation}; Golant MB et al.; We investigated the development of a Saccharomyces carlsbergensis yeast cell culture irradiated with EHF electromagnetic waves . Short-term EHF action was found to produce change of the budding period . The cell-cycles synchronization occurred after longer irradiation . Extraordinary prolonged synchrony of cycles (more then 20 cycles) was explained as result of the culture self-organization connected with activation of intercellular interaction.

Nippon Rinsho, 1994 May, 52(5), 1389 - 95
{Glomerular endothelial cell culture and its application}; Nitta K et al.; Glomerular endothelial cells (GEN) have recently been cloned, cultivated and characterized . They have as same biological characteristics as expressed in endothelial cells derived from different sources . Because of the difficulties of cloning of GEN, we first transfected plasmid DNA containing large T antigen of SV-40 (NH-GEN1) . Culture method of GEN and NH-GEN1 and its application are discussed.

J Prosthet Dent, 1994 May, 71(5), 505 - 10
Effects of RTC-silicone maxillofacial prosthetic elastomers on cell cultures; Polyzois GL et al.; The use of a wide variety of materials in the construction of maxillofacial prostheses makes biocompatibility testing a necessity . However, the dental literature contains few reports of biocompatibility testing of maxillofacial prosthetic materials . The cytotoxic profiles of five room-temperature cross-linking (RTC)-silicone elastomers were investigated by means of two in vitro cell culture techniques . Mouse fibroblast cells (L929) were used, and the results indicated that RTC-silicone elastomers adversely affected cells in culture and that storage of samples for 1 week in saline solution did not alter this effect . Clinical follow-up of patients wearing prostheses made of these silicone materials is warranted to evaluate host reactions in long-term contact with human mucous membrane and skin tissue.

Alcohol Alcohol, 1994 May, 29(3), 303 - 14
Acetaldehyde induces c-fos and c-jun proto-oncogenes in fat-storing cell cultures through protein kinase C activation; Casini A et al.; Hepatic fibrosis is an important morphological feature of alcohol-induced liver injury . We previously reported that acetaldehyde stimulates collagen I and fibronectin gene transcription in rat fat-storing cell (FSC) culture . We here evaluated whether acetaldehyde increases Col I and FN gene transcription through the induction of c-fos and c-jun proto-oncogenes and studied the possible role played by protein kinase C (PKC) and c-AMP . FSCs, isolated from rat liver on a Nycodenz density gradient, were exposed to acetaldehyde for 1/2, 1, 3, 6, 12, 24 hr and for 10, 20, 30, 45, 60, 90 min in the experiments for jun and fos expression, respectively . Acetaldehyde produced a rapid and transient induction of fos mRNA (undetectable at t = 0, peak at t = 45 and still evident at t = 90) . Jun mRNA was weakly expressed in unstimulated FSCs; acetaldehyde induced a prolonged activation of jun expression up to 24 hr with a peak at 3 hr . To study the role of PKC were repeated the experiments in the presence of Staurosporine and H-7 . These inhibitors of PKC activity blocked the stimulatory effect of acetaldehyde on fos and jun mRNA expression . Furthermore, they abolished the stimulatory effect of acetaldehyde on collagen I and fibronectin gene expression by FSCs . Acetaldehyde increased the cell membrane PKC activity in FSC cultures in a dose-dependent way . Intracellular cAMP levels were not significantly modified by acetaldehyde in the first 30 min of incubation . We conclude that acetaldehyde increases procollagen I and fibronectin gene transcription in FSCs, possibly through c-fos and c-jun expression, and that PKC may play a regulatory role in this chain of events.

Cell Mol Biol (Noisy-le-grand), 1994 May, 40(3), 373 - 80
Comparative IL-6 effects on FSH and hCG-induced functions in porcine granulosa cell cultures; Machelon V et al.; Gonadotropin regulation of granulosa cell (GC) differentiation can be modulated by non-steroidal factors, including cytokines . Interleukin-6 (IL-6), a broad spectrum cytokine, has been previously demonstrated to be produced by GCs and to directly influence follicle stimulating hormone (FSH) differentiated functions of ovarian GCs . In the present study, primary cultures of GCs were prepared from prepubertal sow ovaries . No significant amount of biological active IL-6 was detected in these cultures using the B9 cell growth bioassay . Although our findings suggest that GCs are not source of IL-6 in the porcine ovary, this cytokine may be released by leukocytes present in the ovary and modulate ovarian functions by acting on GCs . Here, adding recombinant human (rh)IL-6 to GC cultures inhibited differentiated functions induced by FSH such as aromatase activity, LH receptor (LHr) expression measured by specific 125I-hCG binding and progesterone (P) production . On the opposite, rhIL-6 did not modulate stimulatory human chorionic hormone (hCG) effects on P release by GCs and did not prevent hCG binding to LHr . These preliminary results clearly showed that IL-6 acted differently on FSH and hCG induced functions although these gonadotropins act primarily through the same transduction pathway involving generation of cyclic AMP . We suggest that IL-6 might act more likely by reducing FSH binding capacity than by modulating transduction pathways . Inhibitory IL-6 effects on FSH-induced functions were not neutralized by adding to culture media a monoclonal antibody against the human IL-6 signal transducer gp130, previously reported to inhibit IL-6 mediated effects in human cell lines.

J Am Soc Nephrol, 1994 May, 4(11), 1908 - 11
Metabolic substrates alter attachment and differentiated functions of proximal tubule cell culture; Tang MJ et al.; Proximal tubules cultured in vitro gradually lose their differentiated functions . Because standard culture media lacks several substrates important for renal proximal tubule oxidative metabolism, whether a mixture of substrates including butyrate, alanine, and lactate (BAL) would modify growth and/or differentiated function of proximal tubular cells in culture was examined . Tubules cultured in media supplemented with 2 mM butyrate, alanine, and lactate exhibited enhanced attachment but did not exhibit an altered growth rate . Higher levels of phosphoenolpyruvate carboxykinase and leucine-amino peptidase were sustained, although these activities were still diminished in comparison with that in fresh tubules . Sodium-dependent glucose uptake and dome formation--other reflections of epithelial cell differentiated function--also were enhanced . These studies demonstrate that the substrates used to culture proximal tubules can modify both their attachment and their manifestation of differentiated function in culture.

Izv Akad Nauk Ser Biol, 1994 May-Jun, (3), 468 - 71
{The antithrombotic and thrombolytic activity of a plasminogen activator isolated from a monolayer cell culture}; Liapina LA et al.; Plasminogen activator has been isolated from the culture of pig kidney cells by gel-filtration on Sephadex G-75 . The plasminogen activator (181.9 MU/200 g body weight) protected the animals against thrombosis hazard in case of provocation by thromboplastin and exerted a thrombolytic effect in case of thrombus appearance.

Res Virol, 1994 May-Aug, 145(3-4), 239 - 44
Detection of SIV in rhesus monkey thymus stroma cell cultures; Muller JG et al.; To clarify the pathogenesis of SIV-induced thymus atrophy, the presence of SIV within thymus stromal cell cultures (epithelial cells, IDC, macrophages or fibroblasts) was investigated . The material studied consisted of 15 thymus specimens of rhesus macaques infected with SIVmac251 (2-4 months postinoculation) . No viral antigen was detected, either in the cultures, by immunohistochemistry, or in cell culture supernatants, by ELISA (p17 antigen), and no viral RNA was detected by in situ hybridization . Only after coculture with the C8166 cell line, was virus detected in 2 out of 15 stroma cultures . The fact that the virus could only be detected after several passages of coculture with the C8166 cell line indicates that the virus exists in the thymus stroma cells in the form of proviral DNA . The infection of thymus stromal cells may contribute to the destruction of the thymus microenvironment and to the SIV-induced thymus atrophy.

Neurochem Int, 1994 May, 24(5), 473 - 83
NMR spectroscopic study of cell cultures of astrocytes and neurons exposed to hypoxia: compartmentation of astrocyte metabolism; Sonnewald U et al.; Primary cultures of murine cerebral cortical astrocytes or cerebellar granule neurons were exposed to 7 h of hypoxia (3 h in some cases) . The culture medium was analyzed at the end of the hypoxic or normoxic period by 1H NMR spectroscopy and intracellular components were analyzed as perchloric acid extracts by 31P and 1H NMR spectroscopy . Lactate production in astrocytes increased only marginally, whereas high energy phosphate concentrations were reduced, during 7 h of hypoxia and after 17 h of reoxygenation . After 3 h of hypoxia full recovery was possible during reoxygenation . Citrate and glutamine secretion was reduced or unchanged, respectively, during 7 h of hypoxia . Succinate secretion was only observed during normoxia, whereas pyruvate was secreted during hypoxia . Cerebellar granule neurons were more efficient in increasing glycolysis and were, therefore, more resistant to the effects of hypoxia than astrocytes . In the neurons lactate production was doubled and no effects on levels of high energy phosphates were seen after 7 h of hypoxia . Astrocytes were reoxygenated for 17 h after hypoxia or normoxia in a medium containing {2-13C}acetate in order to access if astrocytes were still capable of supplying neurons with essential precursors . The media were subsequently analyzed by 13C NMR spectroscopy . After shorter periods of hypoxia (3 h) full recovery was possible . Citrate and glutamine production remained however decreased during reoxygenation after 7 h of hypoxia . 13C incorporation into glutamine was greatly reduced but that into citrate was unchanged . These results suggest that under the present conditions, neurons are more efficient than astrocytes in switching the energy metabolism from aerobic to anaerobic glycolysis and that astrocytes may suffer long term damage to mitochondria from longer periods of hypoxia . Furthermore, evidence is presented for the existence of several TCA cycles within astrocytes based on labeling ratios . During normoxia the labeling ratios in the C-2/C-4 positions in glutamine and in the equivalent positions in citrate were 0.27 and 0.11, respectively.

Res Virol, 1994 May-Aug, 145(3-4), 251 - 9
Establishment and characterization of two new Kaposi's sarcoma cell cultures from an AIDS and a non-AIDS patient; Benelli R et al.; We have established and characterized two new Kaposi's sarcoma (KS) cell lines derived from skin biopsies: AIDS-KSISTIV (from an AIDS-associated KS) and KSISTVIII (from a sporadic KS) . AIDS-KSISTIV and KSISTVIII are composed mostly of spindle-shaped cells . They show similar patterns of immunohistochemical staining and are positive for smooth muscle (smooth muscle alpha-actin) and fibroblastoid (TE7) markers . Neither of these lines express the endothelial marker von Willebrand factor VIII . These immunohistochemical patterns are similar to numerous other KS lines that we and others have established . When seeded on a reconstituted basement membrane ("Matrigel"), AIDS-KSISTIV and KSISTVIII cells form branching colonies and invade into the Matrigel, as do other KS cultures that we have previously examined . This behaviour on Matrigel is similar to that of malignant sarcoma cells of different origin . The expression of vimentin and the morphology of the invasive colonies on Matrigel suggest that KS-derived cells are poorly differentiated mesenchymal cells . KS lesions are characterized by a conspicuous neovascularization, which appears to be derived from host cell recruitment . We tested the capability of the KS-cell supernatants to induce an angiogenic response in vitro . The new lines are able to stimulate human endothelial cell chemotaxis and invasion through Matrigel-coated filters . No differences in angiogenic potential in vitro were observed between the AIDS and the non-AIDS case, as we previously noted for other established cultures . Our new lines have the properties of true KS cells and confirm that KS spindle cells from HIV-positive or -negative patients have identical phenotypic and behavioural characteristics in vitro.

Cell Signal, 1994 May, 6(4), 433 - 8
Lindane affects phosphoinositide turnover through a different mechanism of the phosphatidylinositol synthesis inhibition in rat renal proximal tubule cell culture; Senar S et al.; The ability of lindane to change the metabolism of inositol phospholipids was investigated using rat renal proximal tubular cell cultures labelled with {3H}inositol . Lindane addition to the culture medium caused labelling of inositol trisphosphate, inositol bisphosphate and inositol tetrakisphosphate to decrease and that of the inositol monophosphate pool to increase . A depletion of radioactivity in phosphatidylinositols was also observed after lindane addition . Most strikingly, the addition of lindane considerably increased the levels of glycerophosphoinositol in a dose-dependent manner . The effect of lindane follows a different pattern from that of bradykinin, and it is suggested to act by stimulating phospholipase A activity(ies).

Biotech Histochem, 1994 May, 69(3), 152 - 6
Hoechst 33258 staining for detecting mycoplasma contamination in cell cultures: a method for reducing fluorescence photobleaching; Battaglia M et al.; DNA fluorochrome staining with Hoechst 33258 bisbenzimide is commonly used for detection of mycoplasma contamination in cell cultures . Photobleaching of Hoechst 33258 is pronounced under the conditions of intense illumination, high magnification and resolution required for detection of mycoplasmas . To reduce photobleaching we investigated the effects of some antioxidant molecules, p-phenylenediamine (PPD), n-propyl gallate (NPG) and 1,4-diazabicyclo(2,2,2)octane (DABCO), which are known to reduce the fading rate of fluorescein . Mycoplasma-contaminated cell monolayers were stained with Hoechst 33258 and mounted in glycerol containing different amounts of antioxidant additives . The cells were examined in an epifluorescence microscope, and the emitted light intensity was recorded . Results showed that PPD and, to a lower degree, NPG, retarded the photobleaching of Hoechst 33258-stained cells, whereas DABCO was not effective . However, fluorescence half-life was increased about three-fold by NPG and almost 20-fold by PPD . The rate of fluorescence fading of Hoechst 33258 can therefore be retarded by PPD, with obvious advantages for reading and photographic recording of results.

Toxicol Appl Pharmacol, 1994 May, 126(1), 191 - 4
Effect of lead acetate on nitrite production by murine brain endothelial cell cultures; Blazka ME et al.; One of the mechanisms by which lead may cause a perturbation in the nervous system is the alteration of endothelial cell function . This study investigated the effect of lead acetate on constitutive and cytokine-induced production of nitrite, a marker of nitric oxide, in brain microvascular endothelial cells . Nitric oxide synthase may be a target for lead and changes in its function can result in a cascade of physiological effects seen in vivo . Concentrations of 10, 100, and 1000 nM lead acetate, in the presence or absence of 100 ng lipopolysaccharide/ml, 400 U interferon-gamma/ml and 100 U tumor necrosis factor-alpha/ml, were added to confluent cultures of brain microvascular endothelial cells . Concentrations of lead acetate as low as 10 nM decreased constitutive levels of nitrite by 50% without inhibiting the inducible levels . Addition of 1 microM lead acetate had no effect on {3H}L-leucine incorporation, lactate dehydrogenase release, or cellular morphology, indicating that the effect was selective . Increasing the concentration of extracellular calcium to 2 mM abolished the inhibitory effect of lead acetate on the constitutive production of nitrite . These studies suggest that low concentrations of lead are capable of inhibiting nitrite produced by the calcium-dependent constitutive form of nitric oxide synthase while the calcium-independent, inducible form of nitric oxide synthase is not affected . These data provide another testable hypothesis for the as yet undetermined mechanisms of lead neurotoxicity.

Cancer Lett, 1994 Apr 29, 79(1), 17 - 26
Effects of micronutrients and antioxidants on lipid peroxidation in human plasma and in cell culture; Franke AA et al.; Plasma levels of triglycerides, retinol, cholesterol, lipid-phase antioxidants (alpha-, gamma-tocopherols, beta-carotene, alpha-carotene, lycopene, beta-cryptoxanthin and lutein/zeaxanthin), and thiobarbituric acid-reactive substances (TBA-RS), as an indicator of lipid peroxidation, were repeatedly determined in nine individuals over a 3-month period . Levels of TBA-RS were positively correlated with plasma triglycerides and gamma-tocopherol, and negatively correlated with plasma carotenoids . These results were consistent with in vitro cell culture studies which showed increased TBA-RS for cells supplemented with linolenic acid and decreased levels when treated with beta-carotene . We conclude that TBA-RS measurements in plasma accurately reflect the level of peroxidizable substrate as modified by the presence of a variety of dietary antioxidants, particularly carotenoids . Although the inter- and intra-individual variabilities for TBA-RS are comparable with the micronutrients and antioxidants measured in this study, high interassay variability and the strong association with the more commonly measured plasma triglycerides suggest the TBA-RS assay to be of limited use in epidemiologic studies . However, this assay does appear to be useful in cell culture studies where experimental conditions can be better controlled . Low ratios of inter- to intra-individual variability in some of the plasma micronutrient and lipid-phase antioxidants measured suggest that multiple samples may be required to characterize individuals in studies evaluating the relation between these plasma constituents and disease incidence.

Int J Cancer, 1994 Apr 15, 57(2), 254 - 8
CD4+ T cells recovered from a mixed immune lymphocyte-tumor cell culture induce thymidine incorporation by naive rat lymphocytes in response to tumor cells; Reisser D et al.; Spleen cells from tumor-immune rats incorporate thymidine when co-cultured for 4 days with syngeneic cancer cells . Non-adherent cells, recovered from a 7-day mixed culture with cancer cells, had lost their capacity for incorporating thymidine when exposed again to the same tumor cells; however, in these conditions, the non-adherent cells induce thymidine incorporation by fresh naive spleen cells . This response is restricted to tumor lines which originate from the same tumor as the cells used for immunization and is due to memory and/or activated CD45RC- CD4+ T cells . Our results indicate that tumor-specific T cells maintain their capacity to respond to tumor antigens, even after an extended culture time with tumor cells . In another study, in the same conditions, spleen cells from normal or tumor-bearing rats did not evoke significant responses.

J Cell Physiol, 1994 Apr, 159(1), 121 - 30
Depleted level of heparan sulfate proteoglycan in the extracellular matrix of endothelial cell cultures exposed to endotoxin; Colburn P et al.; Exposure of cultured endothelial cells to endotoxin causes an increase in the amount of cellular heparan sulfate proteoglycan and a depletion of this molecule in the extracellular matrix . Concomitant with the decrease in the extracellular matrix is the appearance of a fraction of proteoglycan bearing altered carbohydrate moieties in the culture medium . beta-mercaptoethanol, mannitol, and dimethyl sulfoxide bring back to normal the structural properties of the proteoglycan in the medium and restore the matrix content in proteoglycan to a level comparable to that of control cells but do not affect the increase in the amount of proteoglycan on the cell . This "uncoupling" suggests that two independent chains of events underlie the synthetic and structural changes of the proteoglycan triggered by endotoxin in the endothelial cell.

J Infect Dis, 1994 Apr, 169(4), 746 - 53
Human immunodeficiency virus type 1 causes productive infection of macrophages in primary placental cell cultures; McGann KA et al.; To characterize the role of the placenta in vertical transmission of human immunodeficiency virus type 1 (HIV-1), the susceptibility of primary human placental cultures and of transformed trophoblast cell lines to infection by several HIV-1 isolates was examined . Placental cultures supported the replication of all strains tested, including lymphocyte-, macrophage-, and amphotropic isolates . All viruses replicated to modest levels, with production of both viral antigen and infectious virus in the culture supernatants . Placental cells demonstrated a pattern of permissiveness for HIV-1 isolates distinct from that seen with lymphocytes, blood-derived macrophages, or T cell lines . Immunofluorescent staining showed that 5%-10% of the cultured placental cells expressed viral antigens, and double labeling revealed that the HIV-positive cells were macrophages not trophoblasts . None of the trophoblast cell line (JEG-3, Jar, BeWo, HP-W1) could be infected by HIV . These results support the hypothesis that infection of the placenta could play a role in maternofetal transmission of HIV-1 and suggest that the placental macrophage is likely to be the primary cell type responsible.

Pathology, 1994 Apr, 26(2), 194 - 7
Comparison of Giemsa and fluorescent monoclonal antibody staining of inoculated cell cultures for diagnosis of Chlamydia trachomatis; Chan SW et al.; Three methods of isolation and identification of Chlamydia trachomatis inclusions in cell monolayers were compared: Giemsa staining followed by darkfield microscopy of cycloheximide treated Buffalo green monkey kidney monolayers (BGM), fluorescent monoclonal antibody staining of cycloheximide treated BGM and of HeLa 229 monolayers . In an unselected group of 895 patients with suspected chlamydial infections including contacts of symptomatic patients, significantly more positive chlamydia isolates were detected with immunofluorescent monoclonal antibody staining of BGM and HeLa 229 than with Giemsa staining followed by darkfield microscopy on BGM . However, there was no disparity between the 2 staining methods with specimens from 156 patients with untreated non-gonococcal urethritis . In all chlamydia positive specimens, significantly more inclusions were seen after BGM-immunofluorescence than after HeLa-immunofluorescence or BGM-Giemsa . The extra cost associated with immunofluorescence staining was justified by the time saved in scanning.

Immunobiology, 1994 Apr, 190(3), 255 - 62
HIV-1 gp41 binds to two proteins in cell culture supernatant of human B cell line Raji and monocyte cell line U937; Chen YH et al.; Based on our findings that HIV-1 gp41 can bind independently of CD4 to the human T cell line H9, B cell line Raji and monocytic cell line U937, as well as human peripheral blood mononuclear cells preferentially to B lymphocytes and monocytes, we examined whether soluble protein for HIV-1 gp41 binding exists in the cell culture supernatant of Raji and U937 . Using HIV-1 recombinant soluble gp41 (sgp41; Env amino acid 539-684) attached to sepharose beads, the cell culture supernatants of Raji and U937 were absorbed . By SDS-PAGE of sgp41-eluates of these cell culture supernatants three protein bands of 70, 75 and 92kD were stained with Coomassie blue . By Western blot (ligand blot) analysis using sgp41, two protein bands of 70 and 75kD were observed in sgp41-eluates from Raji and U937 cell culture supernatants . These sgp41-eluates could inhibit the sgp41 binding to Raji and U937 cells . Our data indicate that Raji and U937 cells produce two soluble binding proteins for HIV-1 gp41.

Mol Mar Biol Biotechnol, 1994 Apr, 3(2), 78 - 86
Basic fibroblast growth factor stimulates proliferation and suppresses melanogenesis in cell cultures derived from early zebrafish embryos; Bradford CS et al.; We are attempting to develop methods for in vitro culture of zebrafish embryonal stem cells . Primary cultures were initiated from wild-type zebrafish early embryos in basal nutrient medium supplemented with insulin, selenite, leukemia inhibitory factor, trout serum, fetal bovine serum, and trout embryo extract . In this medium, melanocytes appeared on the second day of culture . Basic fibroblast growth factor (bFGF) was mitogenic when cells were plated at low densities . bFGF suppressed melanogenesis is a dose-dependent fashion, with maximal effect at 20 ng/mL . Cultures initiated and maintained with bFGF for 24 hours and then incubated without bFGF for as long as 8 days did not contain pigmented cells . Experiments in which bFGF was added or removed at various times after initiation of cultures indicated that maximum sensitivity to bFGF occurred during the first 12 hours of culture . When wild-type cells from cultures without bFGF were injected into albino blastula-stage embryos, melanocytes subsequently developed in host embryos: no melanocytes appeared when cells from cultures with bFGF were injected into albino hosts.

In Vitro Cell Dev Biol Anim, 1994 Apr, 30A(4), 202 - 8
Establishment of pure neuronal and muscle precursor cell cultures from Drosophila early gastrula stage embryos; Hayashi I et al.; Primary cultures of Drosophila gastrula stage embryonic cells will divide and terminally differentiate into morphologically recognizable neurons and muscles . The phenotypically mixed nature of this primary culture system has made it difficult to effectively analyze various parameters of cell growth and differentiation for individual cell types . We report here a simple and economic method to separate early embryonic precursors for different cell types, using a shallow linear reorienting Ficoll gradient at unit gravity . The separated cells were collected into fractions, cultured, and analyzed for their growth and differentiation patterns . The larger and denser cells of the first fractions differentiated to yield pure neuronal cultures, as judged by morphologic, immunologic, and biochemical criteria . Cells in the last fractions differentiated into a predominantly muscle-enriched cell population, which also contained a very small percentage of neurons morphologically distinct from those in the pure neuronal fractions . Approximately 35% of the early gastrula stage embryonic cells differentiate into neuronal cells, and 65% of the non-neuronal lineage cells later develop into predominantly muscle population . The method is highly reproducible, can process 3 x 10(7) cells per procedure, and the recovery is > 90% of the input cells . The separated cells are suitable for cell biological analyses as well as for biochemical and molecular studies of neuron and muscle precursors.

J Vet Diagn Invest, 1994 Apr, 6(2), 153 - 5
Identification of African horse sickness virus in cell culture using a digoxigenin-labeled RNA probe; Brown CC et al.; A digoxigenin-labeled RNA probe was synthesized from a plasmid containing a portion of the African horse sickness virus (AHSV) serotype 4 genome segment coding for nonstructural protein 1 . In an in situ hybridization procedure, this probe hybridized successfully to Vero cells infected with each of the 9 serotypes of AHSV . There was no hybridization with noninfected cell cultures or cell cultures infected with bluetongue virus.

J Vet Diagn Invest, 1994 Apr, 6(2), 143 - 7
Detection of epizootic hemorrhagic disease virus serotypes 1 and 2 in cell culture and clinical samples using polymerase chain reaction; Aradaib IE et al.; The potential for the use of the polymerase chain reaction (PCR) in detecting epizootic hemorrhagic disease virus (EHDV) ribonucleic acid in cell cultures and clinical samples was studied . Using oligoribonucleotide primers selected from genome segment 6 of EHDV-2 (Alberta strain), the PCR-based assay resulted in a 387-base pair (bp) PCR product . EHDV RNA from US prototype serotypes 1 and 2 and a number of EHDV field isolates, propagated in cell cultures, were detected by this EHDV PCR-based assay . Amplification products were visualized on ethidium bromide-stained agarose gels or detected by chemiluminescent hybridization . The sensitivity of the PCR assay was 100 fg of virus RNA (equivalent to 6 x 10(3) virus particles) with ethidium bromide-stained agarose gels . Chemiluminescent hybridization increased the sensitivity of the PCR assay 1,000 times, and specific signals were detected from 0.1 fg of virus RNA (equivalent to 6 virus particles) . Amplification product was not detected when the PCR-based assay was applied to RNA from the US bluetongue (BT) virus prototype serotypes 2, 10, 11, 13, and 17 or total nucleic acid extracts from uninfected baby hamster kidney-21 cells, Vero cells, and blood cells from deer that were EHDV seronegative and virus isolation negative . Application of this EHDV PCR-based assay to clinical samples resulted in detection of EHDV RNA from blood and spleen samples from a deer in California with clinical hemorrhagic disease . This EHDV PCR-based assay could provide a rapid, sensitive, and specific assay for detection of EHDV infection in susceptible ruminants.

J Virol Methods, 1994 Apr, 47(1-2), 203 - 16
Rapid and efficient purification of hepatitis A virus from cell culture; Bishop NE et al.; Hepatitis A virus (HAV) characteristically remains strongly cell-associated when grown in culture, with only small yields in the culture supernatant . Cell factories (6000 cm2) of BS-C-1 cells infected with the cytopathic HM175A.Z strain of HAV for 3, 4 or 7 days were harvested using trypsin to disperse the infected cell monolayer, and cells were collected by low speed centrifugation . More than 70% of the yield of virus and viral antigen can thus be obtained in the packed cell pellet . Packed cell pellets were resuspended in 5 volumes of isotonic buffer and cell membranes lysed by the addition of a non-ionic detergent . After removal of nuclei by centrifugation, ionic detergent was added to the clarified cytoplasmic extract . Under these conditions, HAV particles (virions and empty capsids) are the only particulate material remaining in the sample, and were recovered in a single ultracentrifugation step through discontinuous sucrose/glycerol density gradients . In one day, this method yields viral antigen with minimal cellular contaminants, in a concentrated volume suitable for subsequent biochemical, vaccine or diagnostic uses . The yield of viral antigen over numerous batches varied from 200 to 1600 vaccine-equivalent doses per cell factory, with a titre of up to 1 x 10(10) infectious particles per ml.

Alcohol Clin Exp Res, 1994 Apr, 18(2), 403 - 9
Acetaldehyde-induced stimulation of collagen synthesis and gene expression is dependent on conditions of cell culture: studies with rat lipocytes and fibroblasts; Maher JJ et al.; Acetaldehyde has been proposed as a mediator of fibrogenesis in alcoholic liver disease, based in part on its ability to stimulate collagen synthesis by hepatic lipocytes in late primary or passaged culture . In this study, we examined the effect of acetaldehyde on rat lipocytes and fibroblasts at various stages of culture, in an effort to determine whether culture-related events influence responsiveness to this compound . Lipocytes from normal rat liver were studied in primary culture at 3 and 7 days after plating; fibroblasts were studied in subculture, at subconfluent and confluent densities . Both cell types were incubated with 100 microM acetaldehyde for 24 hr followed by measurement of collagen synthesis and type I collagen gene expression . Acetaldehyde had no effect on lipocytes at either 3 or 7 days in primary culture . The inability of acetaldehyde to stimulate collagen synthesis in primary culture was not attributable to toxicity, because cell morphology and total protein synthesis were identical in both treated and untreated cultures . Fibroblasts exhibited a variable response to acetaldehyde that was dependent on cell density: subconfluent cells contained similar amounts of type I collagen mRNA in both the presence and absence of acetaldehyde, whereas confluent cells exhibited a 2- to 3-fold increase in collagen mRNA levels upon acetaldehyde exposure . To determine whether quiescent lipocytes would respond to acetaldehyde in a culture system that mimics the hepatic environment in vivo, lipocytes were plated in coculture with hepatocytes on a basement membrane gel and incubated with 20 mM ethanol for 72 hr . Direct communication between these two cell types did not provoke lipocyte activation, even in the setting of ethanol oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue Cell, 1994 Apr, 26(2), 209 - 21
Cockroach glial cell cultures: morphological development and voltage-gated potassium channels; Keen L et al.; Insect glial cells derived from the embryonic brain of Periplaneta americana cockroaches have been kept in primary culture conditions for up to 3 weeks . Under the culture conditions used, the glial cells differentiated and formed a complex cellular network on the floor of the culture vessels from which glial-glial and glial-neuronal contacts could be seen . Single-channel currents from cell-attached glial membrane patches were recorded using the gigaseal technique . Depolarisation of membrane patches activated outward currents, which were abolished in the presence of extracellular 50 mM tetraethylammonium or 5 mM 4-aminopyridine, and were insensitive to 1 microM tetrodotoxin, 10 microM picrotoxin and 2 mM Cd2+ . The amplitude of the outward currents increased linearly with depolarisation, and amplitude histograms obtained at several pipette potentials could be reasonably fitted with a single Gaussian corresponding to a single channel type with a slope conductance of 37 +/- 11 pS . The extrapolated equilibrium potential of the outward current was about 5 +/- 10 mV positive to the resting potential and both the channel open time constant and relative open time probability were sensitive to membrane potential, increasing markedly with depolarisation . The results presented in this paper show the presence of a cadmium-insensitive, voltage-dependent outward potassium channel in insect glial cells in vitro.

Plant Physiol, 1994 Apr, 104(4), 1151 - 7
Analysis of the rolC promoter region involved in somatic embryogenesis-related activation in carrot cell cultures; Fujii N et al.; In cell cultures of carrot (Daucus carota L.), somatic embryogenesis can be induced by transferring cells from a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) to one devoid of 2,4-D . Previous analysis of transgenic carrot cells containing the 5' non-coding sequence of the Ri plasmid rolC and a structural gene for bacterial beta-glucuronidase (uidA) has shown that the chimeric gene is actively expressed after induction of somatic embryogenesis . In this study, we demonstrate that activation of the rolC promoter is dependent on the process of embryo development but not on the duration of the cell culture in 2,4-D-free medium . We also analyzed the cis region of the rolC promoter that is responsible for somatic embryogenesis-related activation (SERA), namely relatively low beta-glucuronidase (GUS) activity in calli and proembryogenic masses (PEM) and high GUS activity in heart- and torpedo-stage embryos . When the -255-bp region of the rolC gene was used, SERA was retained . Internal deletions within this -255-bp region did not alter SERA by the rolC promoter . Furthermore, when a rolC promoter fragment (-848 to -94 bp) was fused to the cauliflower mosaic virus (CaMV) 35S core region (-90 to +6 bp), it conferred relatively low GUS activity in calli and PEM but high GUS activity in heart and torpedo embryos . When -848 to -255-bp or -255- to -94-bp fragments of the rolC promoter were fused to the same CaMV 35S core region, GUS activity patterns were not related to somatic embryogenesis . These results suggest that the combination of several regulatory regions in the rolC promoter may be required for SERA in carrot cell cultures.

Int J Biochem, 1994 Apr, 26(4), 479 - 85
The effect of lactogenic hormones on protein synthesis and amino acid uptake in rat mammary acinar cell culture at various physiological stages; Roh SG et al.; 1 . The activities of protein synthesis and amino acid uptake at various physiological stages were determined by the incorporation of radioactive materials ({3H}-lysine, {14C}-cycloleucine) in rat mammary epithelial cell cultures . The activity of protein synthesis and amino acid uptake was higher in early lactation than in virgin, pregnant and late lactation stages . 2 . Lactogenic hormones (prolactin, hydrocortisone and insulin) treatment related with mammary growth and differentiation increased the activities of protein synthesis and amino acid uptake . But increase of these activities was different at each physiological stage . 3 . The effect of prolactin and hydrocortisone on the activities were greater in virgin, pregnant and late lactation than in early lactation . And effect of insulin was greater in pregnant and early lactation than in virgin and weanling.

Eur J Neurosci, 1994 Apr 1, 6(4), 531 - 7
Localization of aminopeptidase N and dipeptidyl peptidase IV in pig striatum and in neuronal and glial cell cultures; Barnes K et al.; The subcellular distribution of the plasma membrane ectoenzymes, aminopeptidase N (aminopeptidase M) and dipeptidyl peptidase IV, has been examined by fractionating homogenates of porcine striata by a discontinuous Percoll gradient centrifugation procedure which distinguishes fractions containing pre- and post-synaptic elements . The two enzymes showed different distributions--dipeptidyl peptidase IV did not show a significant pre-synaptic location, whereas aminopeptidase N was present on both pre- and post-synaptic fractions . Immunofluorescent staining on mixed and neuron-enriched primary cultures of pig striatal tissue using affinity purified antibodies to the aminopeptidase and to the dipeptidyl peptidase revealed the ectoenzymes on distinct populations of cells . The astrocytic identity of the aminopeptidase N-staining cells was established by correlation with immunostaining for glial fibrillary acidic protein and for vimentin by confocal microscopy . Ultracryosections of striatum immunostained with gold-labelled immunoglobulins of differing diameters demonstrated aminopeptidase N on pericytes and confirmed its location on endothelial and astrocytic glial cells . Thus, several independent approaches indicated that aminopeptidase N, in addition to being present on endothelial and synaptic membranes, is found on astrocytes and pericytes in the perivascular neuropil, whereas dipeptidyl peptidase IV is less widely distributed on microvessels and appears not to have a synaptosomal location.

Bull Cancer, 1994 Apr, 81(4), 297 - 302
{Nucleotide metabolism in human glioma: comparative study of primary tumors, grafted tumors on nude mice and cell cultures}; Bardot V et al.; A study developed to test the hypothesis of a possible relationship between metabolic modifications and chromosomal imbalances in solid tumors leads us to investigate the metabolism of purine nucleotides in human gliomas . In order to assess the representativeness of experimental models frequently used, the activities of nine enzymes involved in the synthesis and in the catabolism of purine nucleotides were measured on samples of normal brain, primary and xenografted glial tumors and cell cultures established from human gliomas . In parallel, two enzymes involved in pyrimidine metabolism were also studied on the same samples . The results highlight the low activity of the purine metabolism in human gliomas when compared to normal brain, tissue with low proliferative activity . On the contrary, the pyrimidine metabolism in human gliomas is increased by comparison to normal brain . For the purine metabolism, few differences are observed between enzyme activities calculated in primary glial tumors, xenografts and cells in culture . In grafted tumors and cell cultures, the activity of this metabolism is similar or lower than in normal brain, except for inosine monophosphate dehydrogenase . However, for the pyrimidine metabolism, significantly differences are observed between primary glial tumors, grafted glial tumors and cell cultures . The thymidine kinase/thymidylate synthetase ratio depends on the model studied . These results point out the problem of the representativeness of these models, especially when used for experimental therapeutic studies . This metabolic study also underlines that all results should be interpreted carefully and that the limits for the use of these two experimental models should always be clearly exposed.

Int J Dev Neurosci, 1994 Apr, 12(2), 99 - 106
Neuronal phenotypes in mouse dorsal root ganglion cell cultures: enrichment of substance P and calbindin D-28k expressing neurons in a defined medium; Ninomiya T et al.; The primary sensory neurons in mouse dorsal root ganglia consist of diversified subpopulations which express distinct phenotypic characteristics such as substance P or calbindin D-28k . To determine whether neuronal phenotypes are altered or not in in vitro cultures carried out in a defined synthetic medium, dissociated dorsal root ganglion cells from newborn mice were grown in the alpha-modified minimum essential medium either supplemented with 10% fetal calf serum or serum-free . About 80% of the neurons survived after 5 days of culture in both media, but only 35% or 65% were rescued after 12 days in serum-free or fetal calf serum supplemented medium, respectively . The neuronal subpopulations expressing substance P or calbindin D-28k displayed similar morphological properties in both media and a higher resistance to culture conditions than the whole neuronal cell population, especially in serum-free medium . It is therefore concluded that a defined synthetic medium offers reproducible conditions to culture dorsal root ganglion cells for at least 5 days, stimulates the expression of substance P and enriches preferentially neuronal phenotypes expressing substance P or calbindin D-28k, for a longer period of culture.

Mutat Res, 1994 Apr 1, 306(1), 61 - 70
Spontaneous chromosomal aberrations in cell cultures from patients with neurofibromatosis 1; Kehrer H et al.; Neurofibroma-derived cell cultures, skin fibroblast strains from NF1 patients, melanocyte cultures from cafe-au-lait spots and melanocytes from skin overlying neurofibroma were investigated with regard to the frequencies of spontaneous chromosomal aberrations . A 3.4-fold increased rate of stable and unstable chromosome aberrations in 34 neurofibroma cultures of 18 NF1 patients was noticed in comparison to 17 skin biopsy-derived cultures of healthy probands . Fibroblast strains from the unaffected skin of nine NF1 patients revealed a 2.6-fold higher rate of chromosome breakage compared with the control cultures . Likewise, an increase of spontaneous chromosomal instability by a factor of 13.5 was found in cultured melanocytes from cafe-au-lait spots and by a factor of 11.9 in the skin-melanocyte cultures of six NF1 patients in comparison to foreskin-derived melanocyte strains of five unaffected persons . Analyses of the distribution of chromatid and chromosome breaks along the chromosomes revealed a significant clustering of these events in the centromeric regions specifically in the four kinds of NF1-derived cultures.

J Neurochem, 1994 Apr, 62(4), 1285 - 95
Expression and effects of hyaluronan and of the hyaluronan-binding protein hyaluronectin in newborn rat brain glial cell cultures; Marret S et al.; Hyaluronan (HA) is a polymerized nonsulfated extracellular matrix glycosaminoglycan that may be involved in brain development . We have tested the expression of HA and the HA-binding protein hyaluronectin (HN) in glial cell cultures from newborn rat brain . HA was secreted into the culture medium by type 1 astrocytes in the first stages of the primary cultures . The secretion was high during cell proliferation, reached a maximum when they were confluent, and then decreased . HA was not secreted at a detectable level by total O-2A lineage cell-enriched cultures . HA labeled small O-2A progenitor cells (GFA-, A2B5+, HA+), small O-2A progenitorlike (GFA-, A2B5-, HA+) cells, and type 2 astrocytes (GFA+, A2B5+, HA+), but not mature oligodendrocytes (Galc+, HA-) . In contrast to HA, hyaluronectin labeled oligodendrocyte membranes (i.e., more mature cells) from day 8 . A2B5+ GFA- cells were found to be either HA+ or HN+ at days 7-9, suggesting intermediary stages . The addition of HA to primary cultures and to O-2A progenitor-enriched cultures decreased significantly the increase in the number of O-2A progenitors, of mature (Galc+) oligodendrocytes proportionally to the decrease of the O-2A progenitor number, and of BrdU+ cells, suggesting that HA acts (directly or indirectly) on O-2A cell proliferation . This effect, which was seen for concentrations as low as 0.1 micrograms/ml, was HA specific and was not observed with other glycosaminoglycans . When primary cultures were performed in the presence of hyaluronidase-digested or HA-depleted (by passage on a HN column) fetal calf serum, the total number of O-2A lineage cells was dramatically increased (100%, p < 10(-4)) in comparison with control cultures in standard fetal calf serum . Platelet-derived growth factor increased the total number of O-2A lineage cells and of (Galc+) oligodendrocytes . This effect was opposed by HA dose dependently . The effect of HA was significantly inhibited by HN (30%, p < 10(-4)) . HN had, however, no effect when it was added to culture in the presence of hyaluronidase in fetal calf serum, suggesting its effect was only due to its binding to HA . During cell maturation, HA disappears as HN appears . This and the fact that HA and PDGF have opposite effects suggest an effect of these factors, or of their balance, on myelination.

Biochem Biophys Res Commun, 1994 Mar 15, 199(2), 954 - 61
Activation of the gene for type-b natriuretic factor in mouse stem cell cultures induced for cardiac myogenesis; Boer PH; We assessed the temporal transcriptional activity profiles of the genes for type-B natriuretic factor, BNF, the isoform ANF, and other cardiac muscle proteins in differentiating cultures derived from multipotential mouse cell lines . P19 embryonal carcinoma cells and D3 embryonic stem cells were induced for in vitro cardiac myogenesis; RNA was isolated at regular intervals throughout the differentiation programs, and mRNAs were detected by reverse transcriptase mediated polymerase chain reactions . The transcriptional activation profiles of the ANF and BNF gene were similar, but there were quantitative differences that were best assayed by use of competitive internal DNA standards . The levels of induced BNF transcripts were highest in the P19 developmental system reaching approximately 10% of adult mouse ventricular muscle levels; those for ANF were lower, but also readily detected . The cell lines may be used to define the regulatory control elements for natriuretic factor gene expression, in stably transfected cell lines, during cardiac muscle growth.

Brain Res, 1994 Mar 14, 639(2), 202 - 6
Progesterone 5-alpha-reduction in neuronal and in different types of glial cell cultures: type 1 and 2 astrocytes and oligodendrocytes; Melcangi RC et al.; Progesterone, like testosterone, can be converted in the brain into 5-alpha-reduced metabolites (5-alpha-pregnan-3,20-dione, DHP; 5-alpha-pregnan-3-alpha-ol-20-one, THP) . Recently we have shown that testosterone is 5-alpha-reduced to DHT mainly in neurons, while glial cells possess this enzymatic activity only in limited amounts . On the other hand, a glial cell type (type 1 astrocytes) is almost exclusively responsible for the further metabolism of DHT into 3-alpha-diol . The aim of the present studies was that of evaluating the formation of the 5-alpha-reduced metabolites of progesterone in cultures of neurons, type 1 and 2 astrocytes and oligodendrocytes . The data here presented indicate that, similarly to what happens when testosterone is used as the substrate, the 5-alpha-reductase which metabolizes progesterone shows a significantly higher activity in neurons than in glial cells; however, also type-1 and type-2 astrocytes as well as oligodendrocytes possess some ability to 5-alpha-reduce progesterone . On the contrary, the 3-alpha-hydroxysteroid dehydrogenase (3-alpha-HSD), the enzyme which converts DHP into THP, appears to be mainly present in type-1 astrocytes; much lower levels of this enzyme are present in neurons and in type-2 astrocytes . At variance with the previous results obtained utilizing androgens as precursors, oligodendrocytes show a considerable 3-alpha-HSD activity, even if this is statistically lower than that present in type-1 astrocytes . The existence of isoforms of the enzymes involved in androgen and progesterone metabolism may explain these data.

J Bone Miner Res, 1994 Mar, 9(3), 395 - 400
Effect of ipriflavone on expression of markers characteristic of the osteoblast phenotype in rat bone marrow stromal cell culture; Notoya K et al.; The effects of ipriflavone on cellular proliferation and differentiation of osteoblasts were investigated using stromal cells isolated from the femoral bone marrow of young rats . To induce the formation of mineralized bone-like tissue in vitro, the cells were cultured in the presence of beta-glycerophosphate and dexamethasone . Ipriflavone was added when subculturing was started . After 14 days of culturing with ipriflavone (10(-7)-10(-5) M), increases in both the alkaline phosphatase activity and the hydroxyproline content per culture dish and a slight decrease in the saturated cell density were observed . Furthermore, continuous treatment with ipriflavone for 14-33 days resulted in an increase in the area of bone-like mineralized tissue accompanied by an increase in the secretion of osteocalcin . When culture medium lacking dexamethasone was used, rat bone marrow stromal cells neither differentiated into osteoblasts nor formed bone-like tissue, and under these conditions, ipriflavone had no effect on the proliferation or the phenotypic expression of the cells . These results suggest that ipriflavone directly stimulates markers of the osteoblast phenotype at a certain stage in bone formation without affecting undifferentiated cells that have not been committed to the osteogenic lineage.

Am J Clin Pathol, 1994 Mar, 101(3), 318 - 20
Monoclonal antibodies to Coxiella burnetii for antigenic detection in cell cultures and in paraffin-embedded tissues; Raoult D et al.; The authors developed monoclonal antibodies to Coxiella burnetti, the agent of Q fever . The selected monoclonal antibody, Cox1D8, did not cross-react with other bacteria and was used for early detection of C burnetti in shell vial cell cultures and for staining C burnetii in paraffin embedded tissues . Formalin or Bouin fixation did not alter the reactivity of the antigen with the antibody . This monoclonal antibody could be useful in the pathologic diagnosis of Q fever hepatitis and endocarditis.

J Cell Physiol, 1994 Mar, 158(3), 555 - 72
Cellular expression of bone-related proteins during in vitro osteogenesis in rat bone marrow stromal cell cultures; Malaval L et al.; Rat bone marrow stromal cells comprise a heterogeneous mixture of cell lineages including osteoblastic cells . When grown in the presence of ascorbic acid, beta-glycerophosphate and 10(-8) M dexamethasone, osteoprogenitor cells within the population divide and differentiate to form bone nodules (Maniatopoulos et al., 1988, Cell Tissue Res., 254:317-330; Aubin et al., 1990, J . Bone Miner . Res., 5:S81) providing a useful model to investigate temporal and spatial changes in expression of osteoblastic markers . Immunocytochemistry was combined with Northern blotting, enzymatic assay, and radioimmunoassay to analyze the expression of bone-related proteins during the growth and differentiation sequence . By mRNA levels, protein production and/or enzymatic activity, expression of osteocalcin, bone sialoprotein, and alkaline phosphatase increased concomitantly with the development of bone nodules, while osteopontin mRNA levels decreased and those of SPARC/osteonectin did not change significantly . In older cultures with mineralizing nodules, mRNA levels for alkaline phosphatase and bone sialoprotein, but not osteocalcin, declined . Immunolabeling revealed that cells in early cultures stained poorly for SPARC/osteonectin and strongly for thrombospondin . Later, SPARC/osteonectin staining increased in most cells, while thrombospondin staining could be seen in both matrix and in cells, but with marked intercellular variability in intensity . At all time points studied, osteoblasts within bone nodules stained homogeneously for thrombospondin and alkaline phosphatase, and with marked heterogeneity of intensity amongst cells for SPARC/osteonectin and osteocalcin . Labelling with RCC455.4, a monoclonal antibody raised against rat calvaria cells which intensely labels osteoblasts and osteocytes (Turksen et al., 1992, J . Histochem . Cytochem., 40:1339-1352), co-localized with osteocalcin . Alkaline phosphatase activity and the amount of osteocalcin determined by both radioimmunoassay and immunolabelling decreased in very late cultures, a time corresponding to appearance of fully mineralized nodules . These studies indicate that the bone marrow stromal cell system is a useful model to study the temporal and spatial expression of bone-related proteins during osteogenesis and formation, mineralization, and maturation of bone nodules . Further, immunolabelling at the individual cell and single bone nodule level allowed discrimination of marked variability of expression of osteoblast markers during the differentiation sequence.

J Neurochem, 1994 Mar, 62(3), 1015 - 24
Opioid effects on {3H}norepinephrine release from dissociated embryonic locus coeruleus cell cultures; Raymon HK et al.; The acute and chronic effects of opioid exposure on {3H}norepinephrine ({3H}NE) release were examined in cell cultures of embryonic rat locus coeruleus (LC) . Initial morphological and biochemical characterization of the cultures indicated that the cells exhibited properties similar to those observed in situ . Specific {3H}NE uptake was saturable with a Km value of 222 +/- 52 nM . {3H}NE accumulated by LC cells was released in response to 20 mM K+ stimulation, in a calcium-dependent manner . Both components of neurotransmitter release, spontaneous and K+ evoked, were significantly inhibited by beta-endorphin, with the latter being maintained in the presence of tetrodotoxin . The pharmacology of the opioid effect was consistent with that of mu-receptor activation . The effect of chronic exposure to the mu-selective agonist fentanyl (1 microM) was examined following 4 days of drug treatment . Although there was no significant effect of fentanyl on K(+)-evoked {3H}NE release, these cells were tolerant to the acute inhibitory effect of beta-endorphin . These results indicate that this is an appropriate system for examining the effects of acute and chronic opioid treatment on noradrenergic cells in vitro . In addition, this system may be useful as a CNS model for examining mechanisms that underlie tolerance and dependence following chronic opioid exposure.

Matrix Biol, 1994 Mar, 14(2), 159 - 70
Patterns of type VI collagen compared to types I, III and V collagen in human embryonic and fetal skin and in fetal skin-derived cell cultures; Smith LT; The distribution of type VI collagen was examined in human embryonic and fetal skin and in cultured cells and matrix from this tissue . Frozen sections were immunolabeled with primary antibodies against type VI collagen and types I, III or V collagen, and processed further for fluorescence microscopy and immunoelectron microscopy . At 6 weeks estimated gestational age (EGA), type VI collagen was identified by positive fluorescence and by immunogold staining of filaments and fibers beneath the dermal-epidermal junction (DEJ), weaker fluorescence in the fine matrix of the dermis, and stronger fluorescence in the subcutis . At progressive stages of gestation, immunolabeling for type VI collagen increased in the dermis in parallel with increased deposition of types I and III collagen . By 15 weeks EGA, type VI collagen stained intensely throughout the dermis . At 13 weeks EGA, type VI collagen appeared diminished from the growing tips of invaginating hair buds, but as the hair peg developed, type VI collagen accumulated in adnexal sheaths . Cell cultures were derived from fetal skin at 7.5 to 12 weeks EGA . In primary explant cultures containing both keratinocytes and fibroblasts, mats of type V collagen were present beneath keratinocytes and associated with dense spots that co-labeled for both type VI and type V collagen . In passaged cultures of fibroblasts, individual cells with or without pretreatment with monensin were positive for type VI and/or types I, III or V collagen . Fibrous matrix that was labeled for type VI collagen was also immunopositive for type I or III collagen, while filamentous matrix that was type VI collagen positive tended to exclude types I and III collagen but in some areas to overlap with type V collagen . These findings support the hypothesis that type VI collagen present in both filamentous and fibrous matrix and networks of type VI collagen may serve as a fine scaffolding that facilitates the integration of types I and III collagen into developing fibrous matrix.

Br J Pharmacol, 1994 Mar, 111(3), 880 - 6
Potentiation by endogenously released ATP of spontaneous transmitter secretion at developing neuromuscular synapses in Xenopus cell cultures; Fu WM et al.; 1 . Previously we have shown that extracellular application of ATP, a substance co-stored and co-released with acetylcholine (ACh) in the peripheral nervous system, markedly potentiated the frequency of spontaneous synaptic currents (SSCs) produced by ACh . In the present study, we have further characterized the purinoceptor which mediates the potentiation effect of ATP and the role of endogenously released ATP . 2 . Pretreatment with a P2-purinoceptor antagonist, suramin (0.3 mM), but not a P1-purinoceptor antagonist, 8-phenyltheophylline (0.1 mM), prevented the potentiating effect of ATP . 3 . We studied the role of endogenously released ATP by examining the effect of a specific P2-purinoceptor antagonist on the frequency of spontaneous synaptic events at high-activity synapses (> or = 3 Hz) and found that suramin, but not 8-phenyltheophylline markedly reduced the frequency of SSCs at these high-activity synapses . In addition, desensitizing the P2-purinoceptor with alpha,beta-methylene ATP also produced similar effects to suramin . 4 . Extracellular application of the L-type Ca2+ channel blockers, verapamil, nifedipine or diltiazem (10 microM each) reduced SSC frequency of high-activity synapses, while the N-type Ca2+ channel blocker, omega-conotoxin had no appreciable effect . The potentiating effect of ATP was further prevented by pretreatment with the L-type Ca2+ channel blockers . On the other hand, Bay K 8644, which is a depolarization-dependent L-type Ca2+ channel agonist, potentiated SSC frequency at these high-activity synapses.(ABSTRACT TRUNCATED AT 250 WORDS)

J Pineal Res, 1994 Mar, 16(2), 77 - 84
Multiple circadian oscillators in the photosensitive pike pineal gland: a study using organ and cell culture; Bolliet V et al.; The fish pineal organ contains typical and, in some species, modified photoreceptor cells involved in the photoperiodic control of melatonin production . In the majority of species studied, the rhythm in melatonin production is driven by an intra-pineal circadian oscillator synchronized by the light:dark cycle . In the present study, it is shown that the endogenous rhythm in melatonin release of superfused pike pineals maintained under constant darkness is expressed at temperatures of 19 degrees C, 20 degrees C, 25 degrees C, and 30 degrees C (period > 24 hr), but not at temperatures of 10 degrees C and 15 degrees C . Under constant darkness, pineal fractions containing either typical photoreceptors, modified photoreceptors, or both behaved like total organs . Dissociated pike pineal cells, cultured statically at 20 degrees C, expressed a high amplitude rhythm in melatonin secretion under a light:dark cycle . Under constant darkness, circadian oscillations, which appeared better sustained than in organ culture, were also observed . This study provides the first evidence that the rhythmic production of melatonin, by a fish pineal, is driven by a population of circadian oscillators or clocks . It is hypothesized that each typical and modified photoreceptor might be the locus of a circadian clock . Damping of the overall rhythm under constant darkness might reflect the desynchronization (uncoupling) between these clocks and/or damping of individual oscillators.

Cell Mol Biol (Noisy-le-grand), 1994 Mar, 40(2), 201 - 9
The proopiomelanocortin (POMC) gene expression of AtT20 mouse pituitary cells is dependent on cell culture conditions; Fickel J et al.; We have compared the properties of AtT20 cells cultivated in a Dulbecco's modified Eagles medium containing 10% fetal calf serum (FCS) and of AtT20 cells adapted to a chemically better defined medium with transferrin, albumin, insulin, sodium selenit and 0.2% FCS . Our interest was focused on the hypothalamo-pituitary-adrenal axis (HPA) involved adrenocorticotropic hormone (ACTH) and the potent opioid peptide beta-endorphin (beta-END) . There were no differences in basal secretion of ACTH and beta-END by cells cultivated in medium containing 10% or 0.2% serum, respectively . In combination to the decreased proliferation activity of AtT20 cells, grown in the serum-reduced medium we found a strongly enhanced ACTH secretion activity stimulated by the corticotropin releasing hormone (CRH) in contrast to normally cultivated AtT20 cells (10% serum) . In addition, the proopiomelanocortin (POMC) gene expression was significantly down regulated in serum-reduced medium and was normalized again after further cultivation in a 10% serum containing medium . This leads to the conclusion that under standard conditions (10% serum) the gene transcription is increased by hitherto uncharacterized modulators present in the serum . The unexpected unchanged amounts of ACTH and beta-End could be the result of increased protein convertases activities . These enzymes are responsible for the POMC precursor processing into beta-End and ACTH.

J Neurosci, 1994 Mar, 14(3 Pt 2), 1584 - 95
NMDA receptor activation in differentiating cerebellar cell cultures regulates the expression of a new POU gene, Cns-1; Bulleit RF et al.; POU/homeobox genes encode transcription regulatory proteins that are important in defining cellular phenotypes . Expression of these genes may be critical for to the regulation of CNS cellular differentiation . We have identified a cDNA corresponding to a new member of the POU/homeobox gene family . Expression of RNA encoded by this new gene occurs predominantly in the CNS . Thus, this new gene was designated Cns-1 . Cns-1 transcripts are expressed in differentiating cells cultured from the early postnatal cerebellum . Treatment of these cultured cells with NMDA results in an increase in the level of Cns-1 RNA . This increase is blocked by simultaneous treatment with the specific NMDA receptor antagonist amino-5-phosphonovaleric acid . Continued activation of the NMDA receptor allows maintenance of this new steady state level of Cns-1 mRNA for at least 5 d in these cultured cells . A transcription runoff assay suggests that this increase in the level of RNA is due, at least in part, to an increase in transcription from the Cns-1 gene . The NMDA-induced increase in Cns-1 mRNA was reduced by pretreatment with calcium chelators EGTA or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) tetrakis(acetoxymethyl) . These studies suggest that specific activation of the NMDA receptor in cultures of differentiating cerebellar cells increases Cns-1 gene expression and that calcium entry through the NMDA channel may be required for this response . This change in Cns-1 expression may modify phenotypic characteristics of these cultured cells.

J Assist Reprod Genet, 1994 Mar, 11(3), 165 - 71
Peptides extracted from Vero cell cultures overcome the blastocyst block of mouse embryos in a serum-free medium; Chen HF et al.; PURPOSE: The aim of this work was to evaluate the effect of a Vero cell coculture system on the development of mouse embryos . METHODS: Mouse embryos were randomly divided and cultured in human tubal fluid (HTF) medium with/without Vero cell monolayers, conditioned medium (CM) obtained from Vero cell cultures, and HTF medium supplemented with peptides extracted from CM . The concentrated CM was examined by SDS/PAGE . RESULTS: The development of mouse embryos was blocked at the blastocyst stage in pure HTF medium (1.4% hatching at day 5) . This "blastocyst block" was overcome by coculture with Vero cell monolayers (48.1% hatching at day 5; 1.4 vs 48.1%; P < 0.001) . CM and the addition of 5% fetal bovine serum (24.1 and 34.9% hatching, respectively, at day 5) were also able to enhance the process of hatching . In the other experiment, the addition of peptides extracted from Vero cell cultures also overcame the blastocyst block (12.5%) compared with pure HTF medium (2.1%) (P < 0.05) . Electrophoretic separation revealed several classes of polypeptides consistently secreted into CM obtained from Vero cell cultures . Most peptides occurred in the M(r) range between 6.5 kd and 35.9 kd . CONCLUSION: A developmental block (blastocyst block) of mouse embryos in a serum- and protein-free medium (HTF) was discovered in this study . This block was effectively overcome by HTF plus serum and coculture with Vero cell monolayers and also by the peptides extracted from Vero cell-conditioned medium . We speculate that certain factors secreted or converted by Vero cells may be critical in hatching of mouse embryos . Further study of these factors may be helpful in delineating its mechanism.

Ren Physiol Biochem, 1994 Mar-Apr, 17(2), 62 - 72
Influence of cell culture conditions and passage number on the response of membrane voltage to ATP and angiotensin II in rat mesangial cells; Gloy J et al.; The influence of passage number and different culture conditions on the effect of ATP and angiotensin II (A II) on membrane voltage (Vm) of rat mesangial cells (MC) was examined with the patch clamp technique in slow and fast whole cell recordings . MC were characterized immunologically and grown in standard medium in primary culture (PC) and long-term culture up to passage 21 in the presence of 90 g/l fetal calf serum (LTC/+FCS) or without or with 5 g/l FCS for 1-3 days (LTC/-FCS) . In all three series the studies were performed in a FCS-free Ringer-like solution . Vm of MC did not differ in the series (PC: -49 +/- 1 mV, n = 151; LTC/+FCS: -52 +/- 1 mV, n = 49; LTC/-FCS: -51 +/- 1 mV, n = 44) . In primary culture and long-term cultured MC up to passage 8, FCS (ED50 approximately 5 g/l), ATP (ED50 approximately 2 x 10(-6) mol/l) and A II (ED50 approximately 5 x 10(-10) mol/l) induced a depolarization of Vm . Reduction of extracellular Cl- concentration (from 145 to 32 mmol/l) had no effect on Vm but led to an increased depolarization of Vm by FCS, ATP and A II . In long-term cultured MC above passage 8 grown with 90 g/l FCS both ATP and A II induced a concentration-dependent hyperpolarization of Vm, which was attenuated in increased extracellular K+ concentration (from 3.6 to 33.6 mmol/l) . In long-term cultured MC beyond passage 8, grown without or with a reduced FCS concentration of 5 g/l, ATP and A II led to a transient depolarization of Vm, which was increased in the presence of 32 mmol/l extracellular Cl- . The depolarization was followed by a hyperpolarization, which was attenuated in the presence of increased extracellular K+ . The data indicate that vasoactive agents depolarize Vm of MC in primary culture by activating a Cl- conductance, whereas they hyperpolarize Vm by activation of a K+ conductance in long-term cultured MC grown with FCS . The latter effect was partially reversed when FCS was omitted.

Mutat Res, 1994 Mar, 320(4), 335 - 41
Comparative investigation of anticlastogenic effects in cell cultures of healthy donors and patients with nettle-rash; Arutyunyan RM et al.; In cultures of lymphocytes from 12 healthy donors and 12 patients with nettle-rash (NR) the anticlastogenic effect of the antimutagens WR-2721 (WR), bemitil (BM), tomerzol (TM) and interferon (IF) on the induction of chromosomal aberrations by photrin (PT) and dioxidine (DX) was investigated . There were no statistically significant differences between healthy donors and patients with NR in the levels of chromosomal aberrations that were spontaneous or induced by PT or DX . Statistically significant protective effects of BM, WR, TM and IF were demonstrated in cells of healthy donors after treatment with PT or DX, and after modification of the clastogenic action of PT in lymphocytes of NR patients . There was no protective effect of any of the anticlastogens after treatment of the lymphocyte cultures from NR patients with DX . That observation allows us to suggest the test of anticlastogenic action as a measure of sensitivity of the chromosomal apparatus in groups with different genetic risks.

Neurosci Lett, 1994 Feb 28, 168(1-2), 175 - 80
Heparin treatment increases 9-kb MAP2 mRNA levels in neuronal cell cultures; Klosen P et al.; When neuronal adhesion is reduced by heparin, cultured neurons detach from the substratum and form clusters connected by neuritic fascicles . 24 h after treatment, Northern blots analysis revealed an increase in the expression of the 9-kb MAP2 mRNA in the heparin-treated neuronal cultures . MAP2 being associated with the cross-bridges between microtubules, this increased expression could be an attempt of the cells to rigidify their cytoskeleton to compensate for the reduced attachment . However, the MAP2 protein content was not increased after a 24-h heparin treatment . We discuss possible explanations for this observation and their implications on MAP2 metabolism.

Proc R Soc Lond B Biol Sci, 1994 Feb 22, 255(1343), 113 - 8
Long-term potentiation of Aplysia sensorimotor synapses in cell culture: regulation by postsynaptic voltage; Lin XY et al.; Long-term potentiation (LTP) has been proposed as a cellular mechanism for associative learning in vertebrates . Induction of one type of LTP--observed at synapses in the CA1 region of the mammalian hippocampus--is regulated by the voltage of the postsynaptic cell . To date, a similar form of LTP has not been demonstrated for any invertebrate synapse . We now report that high-frequency stimulation can induce LTP of sensorimotor synapses of the marine mollusc Aplysia in cell culture . Moreover, induction of this form of LTP appears to involve a voltage-dependent postsynaptic mechanism because pairing tetanic stimulation of the presynaptic cell with strong hyperpolarization of the postsynaptic cell blocks the induction of LTP.

Cancer Res, 1994 Feb 15, 54(4), 887 - 90
The potent carcinogen dibenzo{a,l}pyrene is metabolically activated to fjord-region 11,12-diol 13,14-epoxides in human mammary carcinoma MCF-7 cell cultures; Ralston SL et al.; Dibenzo{a,l}pyrene (DB{a,l}P), an environmental hydrocarbon and very potent carcinogen in rodent bioassays, could be activated to DNA-binding intermediates in cells through formation of three different regioisomeric bay- or fjord-region diol-epoxides or other more highly oxidized metabolites . The mechanism of metabolic activation of DB{a,l}P in the human mammary carcinoma cell line MCF-7 was elucidated by analyzing the DB{a,l}P-DNA adducts formed by {35S}phosphorothioate postlabeling, immobilized boronate chromatography, and high-performance liquid chromatography . Six DB{a,l}P-DNA adducts were detected . Comparison with those formed in cells by DB{a,l}P-11,12-diol and by reaction of DNA with syn- and anti-(benzylic hydroxyl and epoxide oxygen cis and trans, respectively) DB{a,l}P-11,12-diol-13,14-epoxide (DB{a,l}PDE) demonstrated that all DB{a,l}P-DNA adducts in MCF-7 cells were formed by these diol-epoxide isomers . Cellular DNA contained large amounts of two syn- and one anti-DB{a,l}PDE-DNA adducts and small amounts of one syn- and two anti-DB{a,l}PDE-DNA adducts . The ability of human cells to activate DB-{a,l}P to its fjord-region 11,12-diol 13,14-epoxides suggests that environmental exposure to DB{a,l}P could pose a risk for humans.

Virology, 1994 Feb 15, 199(1), 81 - 8
Characterization of the prv43 gene of pseudorabies virus and demonstration that it is not required for virus growth in cell culture; Powers L et al.; We have determined the complete DNA sequence of the prv43 gene of a swine herpesvirus, pseudorabies virus . prv43 is 1119 bp in length with a G+C content of 74.5% and is predicted to encode a multiply hydrophobic protein with a molecular mass of 38 kDa . prv43 is colinear with the herpes simplex virus type 1 UL43 locus, and the prv43 and UL43 gene products are predicted to be 47% similar and 23% identical . prv43 is an early gene, producing a 1.2-kb transcript that is easily detected at 2 hr postinfection but not at 6 hr postinfection . We have constructed a prv43 deletion mutant that does not express a prv43 transcript . This mutant did not appear to be defective for viral growth and demonstrated that the gene is nonessential for virus growth in cell culture.

Brain Res, 1994 Feb 4, 636(1), 9 - 27
Immunohistochemical and in situ hybridization study of an insulin-like substance in fetal neuron cell cultures; Schechter R et al.; We studied the ability of fetal neuron cell cultures from different rabbit fetal brain gestational ages to produce and secrete an insulin-like substance (ILS) . Neurons from 22-day gestation were incubated in serum-containing medium or insulin-free/serum-free medium, and 18-day gestation fetal rabbit neurons were also incubated in serum-free/insulin containing medium and serum-containing medium . The 22-day cultures survived in the serum-containing medium and the insulin-free/serum-free medium . The 18-day cultures died when incubated in the insulin-free/serum-free or serum-free/insulin-containing medium, but survived when incubated in serum-containing medium . Using immunohistochemical and in situ hybridization techniques, ILS and insulin-like mRNA were demonstrated within the 22-day cultures incubated in all media compositions, but not within the 18-day cultures incubated in the serum-containing medium . Ultrastructural studies of the 22-day cultures demonstrated an ILS in the endoplasmic reticulum, Golgi and cytoplasm . Northern blots showed the presence of an insulin-like mRNA within the 22-day gestation neuron cell cultures . Insulin receptor was present in the 22-day cultures, but was absent in the 18-day cultures . In addition, we characterized the ILS from the 22-day cultures incubated in the insulin-free/serum-free medium employing high-performance liquid chromatography (HPLC), radioimmunoassay and Western blots . Analysis by HPLC and Western blots demonstrated the presence of an ILS in the extract . We have shown that while 22-day fetal neuron cultures produce and secrete an insulin-like substance indistinguishable from authentic insulin, neuron cell cultures from early brain development do not express this capability.

Brain Res, 1994 Feb 4, 636(1), 49 - 54
Aldosterone modulates glucocorticoid receptor binding in hippocampal cell cultures via the mineralocorticoid receptor; O'Donnell D et al.; The regulation of corticosteroid receptor expression in the rat brain by adrenal steroids remains controversial . The results of in vivo studies {Brinton and McEwen, 1988; Luttge et al., 1989} suggest that activation of type I receptors can modulate both mineralocorticoid (MR; type I) and glucocorticoid (GR; type II) receptor binding in selected brain regions . The present study utilized primary hippocampal cell cultures from rats sacrificed at E19-20 days of gestation to examine the effects of RU 28362, corticosterone (CORT) and aldosterone (ALDO) on GR binding ({3H}dexamethasone +/- RU 28362) . Four days of exposure to 10 nM RU 28362, a highly selective GR agonist, resulted in a robust (approximately 70%) decrease in GR binding . Similar exposure to 10 nM of either CORT or ALDO also produced a significant (50-55%) decrease in GR binding capacity . Scatchard analyses confirmed that the diminished GR binding capacity in response to ALDO was due to a decrease in total number of binding sites (Bmax for Control = 112 +/- 25 fmol/mg vs . ALDO = 48 +/- 12 fmol/mg) with no significant change in the affinity constant . The calculated EC50 from the ALDO concentration response curve was 3.5 nM . Competition studies demonstrated that such low nM concentrations of ALDO were unable to displace specific {3H}dexamethasone +/- RU 28362 binding . Spironolactone, a highly specific MR antagonist, inhibited the ALDO-induced down-regulation of GR binding . These findings support the hypothesis that MR activation can modulate GR binding in hippocampal cells.

Carcinogenesis, 1994 Feb, 15(2), 359 - 63
No tumor-specific expression levels of protein kinase C isoenzymes and of c-fos in human breast cancer cell cultures; Imber R et al.; Epithelial cells derived from 46 human breast tissue samples of patients suffering from breast cancer have been cultivated . Twenty-five of these cell cultures stemmed from normal and 21 from tumor tissues . Moderate to large variations of protein levels of three protein kinase C (PKC) isoenzymes (alpha, delta and epsilon) were found among the various cell cultures . The cell cultures also exhibited very heterogeneous basal as well as inducible levels of c-fos mRNA . However, none of these variations could be correlated with the character of the original tissue nor with any clinical parameter of the respective patient . Our results suggest that altered levels of PKC isoenzymes or of the protooncogene c-fos per se cannot serve as an indication for a transformed behavior of the epithelial cell fraction of human breast tissue.

Am J Vet Res, 1994 Feb, 55(2), 239 - 46
Bovine mammary teat and ductal epithelial cell cultures; Cifrian E et al.; Bovine mammary epithelial cells from teat and ductal tissue were isolated at necropsy and were grown in culture . Cells were characterized by the presence of cytokeratin filaments, cell morphologic features, synthesis of milk proteins, esterase activity, DNA content, and growth patterns on polystyrene, fibronectin, laminin, collagen, and reconstituted basement membrane from the Engelbreth-Holm-Swarm murine sarcoma . Cultured teat and ductal cells stained intensely for cytokeratin and had similar morphologic features . Both cell types synthesized alpha-casein, beta-casein, alpha-lactalbumin, beta-lactoglobulin, and lactoferrin to variable degrees . Cell type and culture conditions did not affect the DNA content of the cells, as indicated by similar amounts of DNA in G0G1 and G2M phases of the mitotic cycle in cultured cells and in cells from freshly isolated mammary explants . Cells cultured on polystyrene, fibronectin, laminin, and collagen formed pavement-like cell monolayers suitable for cytotoxicity and bacterial adherence studies . Cells cultured on the reconstituted basement membrane from the Engelbreth-Holm-Swarm murine sarcoma formed three-dimensional structures closely resembling lactiferous ducts and alveoli, which could be used for studying lactogenesis and galactopoiesis . Freshly isolated cells and cultured cells were stored at -70 C or in liquid nitrogen . The latter storage method affected the cells less than did freezing at -70 C.

Acta Obstet Gynecol Scand, 1994 Feb, 73(2), 119 - 22
The role of cordocentesis in assessment of mosaicism found in amniotic fluid cell culture; Shalev E et al.; Chromosomal mosaicism presents one of the most difficult problems in prenatal cytogenetic diagnosis, requiring the differentiation of true mosaicism from pseudomosaicism . To overcome associated problems and to prevent termination of normal pregnancies, we investigated 23 pregnancies in which true mosaicism has been found in amniotic fluid cell culture . A fetal blood sample was obtained by cordocentesis for rapid karyotyping, and meticulous sonographic examinations were carried out for detecting fetal abnormalities . The 23 cases in which mosaicisms were found in amniocytes involved five cases with sex chromosomal abnormalities, twelve with autosomal trisomy, four with autosomal structural defects, one with a supernumerary marker and one with tetraploidy . The karyotype from fetal leukocytes confirmed the diagnosis of mosaicism in only three out of 23 cases . These three included: two autosomal trisomies (47,XY + 13/47,XY + 21 and 46,XY/47,XY + 21) and one sex chromosome mosaicism (45,X/46,XY) . These were all selected for elective termination of pregnancies by the parents' request . Post abortion karyotype re-confirmed previous karyotype . The other twenty lymphocyte karyotypes were normal, and of these, 19 patients gave birth at term, and one delivered prematurely due to premature rupture of membranes . All 20 born infants were found normal by both neonatal examination and re-karyotypes . We conclude that finding of mosaicism in amniotic fluid culture requires further investigation . Furthermore, in the presence of amniotic fluid cell true mosaicism and normal karyotype in fetal blood, continuation of the pregnancy is safe and to be recommended.

J Clin Invest, 1994 Feb, 93(2), 725 - 30
Retinoic acid suppresses parathyroid hormone (PTH) secretion and PreproPTH mRNA levels in bovine parathyroid cell culture; MacDonald PN et al.; 1,25-dihydroxyvitamin D3{1,25(OH)2D3} suppresses parathyroid hormone (PTH) gene transcription . Recent evidence suggests that retinoid X receptors are involved in 1,25(OH)2D3-mediated transcriptional events . However, little data exists for a role of retinoids in parathyroid function or in PTH expression . In the present study, we observed that all-trans- or 9-cis retinoic acid suppressed the release of PTH from bovine parathyroid cell cultures . Both retinoids were remarkably potent with significant decreases evident at 10(-10) M and a maximally suppressive effect (approximately 65%) at 10(-7) M . All-trans-retinol was considerably less potent in this system . The effect was not evident until 12 h, suggesting that retinoids did not affect the rapid secretion of preexisting PTH stores . PreproPTH mRNA levels were also suppressed by retinoic acid and the retinoid potencies were similar to those observed in the secretion studies . Combined treatment with 10(-6) M retinoic acid and 10(-8) M 1,25(OH)2D3 more effectively decreased PTH secretion and preproPTH mRNA than did either compound alone . These data indicate that retinoic acid: (a) elicits a bioresponse in bovine parathyroid cells; (b) attenuates PTH expression at the protein and mRNA levels, and (c) acts independently of 1,25(OH)2D3 in the control of PTH expression.

Immunobiology, 1994 Feb, 190(1-2), 13 - 22
c-fos and jun gene expression in murine precursor B lymphocytes developed in the interleukin-7-dependent bone marrow cell culture; Fujita K et al.; We analyzed the expression of c-fos and jun family gene in murine pre-B cells developed in the interleukin-7-(IL-7) dependent bone marrow cell culture . The c-fos gene was expressed in the pre-B cells . The c-fos RNA became undetectable after IgM+ B cells were developed in the culture . The c-fos expression in the pre-B cells was IL-7-dependent . However, the c-fos RNA was not induced when the pre-B cells cultured without IL-7 for 6 h were restimulated with IL-7 . The expression of c-jun RNA was slightly induced in the restimulated pre-B cells . The junB and junD RNA were the steady state level in the cells . These gene products formed AP-1 molecule in the pre-B cells . These results suggest that the AP-1 plays a role in the IL-7-dependent pre-B cell development.

Chem Senses, 1994 Feb, 19(1), 25 - 34
Olfactory cell cultures on ensheathing cell monolayers; Chuah MI et al.; Olfactory neurons dissociated from the olfactory mucosa of 4-5-week-old Sprague-Dawley rats are plated on either monolayers of ensheathing cells or cortical astrocytes . It is found that the ensheathing cells support a slightly higher percentage of neurite-bearing olfactory neurons than the astrocytes . Scanning electron microscopy shows that some of the cytoplasmic extensions of the ensheathing cells are closely associated with the olfactory axons while others appear to ensheath them . Olfactory neurons grown on uncoated, poly-L-lysine or laminin-coated glass coverslips in the presence of medium conditioned by ensheathing cells fail to grow neurites, suggesting that interaction between membrane molecules, and not trophic factors, may be required for neurite growth . However, it is unlikely that these membrane molecules are L1 and N-cadherin because immunohistochemical staining shows that only a small proportion of the cultured ensheathing cells express L1 (9%) and N-cadherin (24%).

Xenobiotica, 1994 Feb, 24(2), 95 - 107
Collagen gel immobilization: a useful cell culture technique for long-term metabolic studies on human hepatocytes; Koebe HG et al.; 1 . Primary cultures of human hepatocytes have already been employed in various applications for the study of xenobiotic metabolism . Most of these approaches were performed either on freshly isolated cells or on short-term primary cultures . Standard culture techniques do not maintain functional stability of P450 enzymes for > 1 week in vitro . 2 . The aim of this study was to demonstrate the beneficial effect of an easy to apply, extracellular matrix configuration on the long-term performance of cultured human liver cells . Light microscopical examination of the cultures indicated that the cells remained viable over 1 month . As revealed by electron microscopy, hepatocytes exhibited bile canaliculi and desmosomes and were rich in mitochondria and endoplasmatic reticulum, indicating metabolic activity . 3 . An early culture phase (3 days after isolation) could be described with decreasing DNA content of the cultures, peak values of alanine-amino-transferase (ALAT), and increasing albumin synthesis . After this adaptive period stable levels for DNA content and albumin synthesis were noted; ALAT returned to low values . 4 . Functional activity was monitored by measurements of P450 1A1-dependent O-demethylation of p-nitroanisole to p-nitrophenol, which appeared to be constant over 3 weeks and weakly inducible by 1 mM phenobarbital . Another set-up examined conjugation of acetaminophen at subtoxic concentrations: acetaminophen was metabolized to its glucuronide and sulphate; 3-(glutathione-S-yl)-acetaminophen was not detected . Almost identical metabolism was found, comparing day 3 with 16 of culture . 5 . We concluded that collagen gel immobilization not only provides mechanical support to cultured hepatocytes, but also supports long-term differentiated function of the cells for metabolic studies.

Cell Immunol, 1994 Feb, 153(2), 277 - 86
Suppression of IgE production in unseparated spleen cell cultures; Maeda-Yoshimoto K et al.; When B cells from BALB/c mice were cultured with lipopolysaccharide (LPS) and interleukin-4 (IL-4), a large amount of IgE was detected in the culture supernatants . The IgE production from unseparated spleen cells cultured with LPS and IL-4 was less than the amount of IgE obtained from separated B cells . When syngeneic T cells were added to separated B cells cultures, which were subsequently stimulated with LPS and IL-4, less IgE was produced, as compared to cultures without T cells . The hypothesis that T cells, or factors secreted by these cells, inhibit IgE production is supported by the fact that the degree of suppression of IgE production paralleled the number of T cells added . CD8(+)-enriched T cells were slightly more suppressive than CD4(+)-enriched T cells . Addition of exogenous IL-3 was only partially suppressive . These observations suggest that IL-4 added to unseparated spleen cells in vitro stimulates B cells for IgE production and also stimulates T cells . Lymphokines secreted by these stimulated T cells may in turn act on B cells . Some of these lymphokines, such as interferon-gamma and IL-2, may have a suppressive action on IgE production.

Curr Opin Biotechnol, 1994 Feb, 5(2), 175 - 9
Large-scale mammalian cell culture; Reiter M et al.; Over the past year, progress has been made toward a better understanding of the physiological and biochemical responses of cell cultures to various environmental changes . Much of the theoretical and experimental work that has been reported will help improve expression in mammalian cell culture, which is one of the key steps in biopharmaceutical manufacturing.

Curr Opin Biotechnol, 1994 Feb, 5(2), 165 - 74
Large-scale insect and plant cell culture; Taticek RA et al.; Currently, insect and plant cell cultures are not widely used to make products of commercial interest, largely because the development of large-scale cultivation methods is still in its infancy . With the advances made over the past year, some of the limitations associated with scale-up of these two types of expression system have been addressed . Increasing the oxygen supply and the concentration of various nutrients supplied to insect cells after infection has enabled high specific protein production to be maintained to higher cell densities than ever before, improving overall volumetric yields . Detailed work has focused on the capacity of insect cells to carry out complex post-translational modifications; however, as yet, evidence is conflicting as to the extent of protein processing and complex glycosylation possible in infected cells . In plant cell culture, the accepted axioms concerning large-scale culture have been re-examined . Recent studies have assessed culture at high cell densities and the constraints in reactor design resulting from the 'shear sensitivity' of plant cells . Results show that, as cell densities increase, alterations occur in the pathways of secondary metabolism, leading to decreases in specific productivity . The use of nutrient supplements and a medium cycling strategy shows promise for increasing and sustaining product formation . Furthermore, the importance of dissolved gas composition has been clearly demonstrated by use of a gas recirculation reactor . Reports of taxol and vindoline production in vitro demonstrate the potential and the necessity for further research in scale-up of plant cell culture.

Bioseparation, 1994 Feb, 4(1), 7 - 20
Ion exchange resins for the purification of monoclonal antibodies from animal cell culture; Graf H et al.; We have compared various ion exchangers for monoclonal antibody (MAb) purification using different starting materials such as ascitic fluid and cell culture supernatant . Twelve cation and anion exchange resins were tested so far . Purification of MAbs with regard to the starting material is described . In well-defined conditions of adsorption (20 mM MES buffer, pH 6.50), one purification step based on cation-exchange chromatography is generally sufficient to achieve at least 90% purity of the MAb, even when produced by animal cell culture . Cation-exchange supports exhibit higher capacity for MAbs compared to anion exchangers . Among the cation exchangers tested, we have selected the cross-linked matrix S Sepharose FF for its large specificity and capacity for MAbs . Considering these key parameters and also the good mechanical resistance of the S Sepharose FF, we describe how, by varying the flow rate, sample concentration, and size of the column, the productivity may be improved in a monoclonal antibody purification process . Finally, a general 'gram scale' purification protocol of MAbs produced by animal cell cultures is proposed . This protocol, based on economical adsorption conditions and three steps of elution (100 mM, 200 mM and 1 M NaCl), allows the recovery of highly purified MAbs.

Calcif Tissue Int, 1994 Feb, 54(2), 165 - 9
Effects of lead on osteoclast-like cell formation in mouse bone marrow cell cultures; Miyahara T et al.; To examine an effect of lead (Pb) on the process of osteoclast-like cell formation from its progenitors, we used a mouse bone marrow culture system in which osteoclast-like multinucleated cells (MNCs) were formed in response to bone-resorbing agents . In a 9-day culture period, Pb dose-dependently stimulated MNC formation over the concentration range 2-10 microM, whereas at 40 microM Pb, MNC formation declined . In an 11-day culture period, MNC formation reached a maximum at 5 microM Pb and decreased with increasing concentration of Pb at 10-40 microM . Pb-stimulated MNC formation was inhibited by both indomethacin and SC19220, an antagonist of prostaglandin E2 (PGE2) receptor . Pb stimulated the production of PGE2 in marrow cell cultures, suggesting that Pb-stimulated MNC formation is dependent on the production of PGE2 . 3-Isobutyl-1-methylxanthine potentiated Pb-stimulated MNC formation and 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, inhibited it . A calcium ionophore A23187 increased Pb-induced MNC formation and verapamil, a calcium channel blocker, depressed it . It is possible that a PGE2-induced increase in the levels of cyclic adenosine 3',5'-monophosphate (cAMP) and calcium ions in marrow cells is involved in Pb-induced MNC formation . Pb and parathyroid hormone showed a synergistic stimulation on MNC formation . From these results, Pb is thought to induce osteoclast-like cell formation by a mechanism involving PGE2 which increases the intracellular levels of cAMP and calcium ions.

Biomaterials, 1994 Feb, 15(3), 213 - 22
Mechanism of initial attachment of cells derived from human bone to commonly used prosthetic materials during cell culture; Howlett CR et al.; The suitability of polymeric biomaterials as surfaces for the attachment and growth of cells has often been investigated in cell culture . In this study the contribution that serum fibronectin (Fn) or vitronectin (Vn) make to the attachment and spreading of cells cultured from explanted human bone (bone-derived cells) during the first 90 min of culture was determined for metallic and ceramic surfaces . The requirement for Fn or Vn for attachment and spreading of bone-derived cells onto stainless steel 316 (SS), titanium (Ti) and alumina (Al2O3) and to polyethyleneterephthalate (PET) was directly tested by selective removal of Fn or Vn from the serum prior to addition to the culture medium . Attachment and spreading of bone-derived cells onto SS, Ti and Al2O3 surfaces were reduced by 73-83% when the cells were seeded in medium containing serum from which the Vn had been removed . Cell attachment and spreading on these surfaces when seeded in medium containing Fn-depleted serum (which contained Vn) were not reduced to the same extent as in the medium containing Vn-depleted serum . The bone-derived cells failed to attach to the surfaces to the same extent when seeded in medium containing serum depleted of both Vn and Fn . Our results show that for human bone-derived cells, the attachment and spreading of cells onto SS, Ti and Al2O3 as well as PET during the first 90 min of a cell culture attachment assay are a function of adsorption of serum Vn onto the surface.

Mol Cell Endocrinol, 1994 Feb, 99(1), 103 - 10
TSH action on cAMP binding to the regulatory subunits of cAMP-dependent protein kinases in pig thyroid cell cultures; Ben Abdelkhalek M et al.; This study examines the mechanism of TSH action on the cAMP-dependent protein kinases (PKA) by measuring the catalytic activity of the two PKA isozymes (PKA I and PKA II) and their capacity to bind cAMP to the regulatory subunits (RI and RII) in thyroid cell cultures exposed for two days to different doses of TSH . In TSH-treated cell cultures a selective down regulation (up to 60%) of the catalytic activity was found; the PKA I was down regulated at lower TSH doses (0.1 mU/ml and even 0.05 mU/ml) than was the PKA II (1.0 mU/ml TSH) . At the dose of 1.0 mU/ml the loss of the catalytic activity in PKA I and PKA II was respectively 60% and 40% . No free catalytic activity was found either in control or in TSH-treated cells . Binding of cAMP to regulatory subunits (R) measured under exchange conditions at 37 degrees C, showed that no change in total regulatory subunit protein content occurs in TSH-treated cells . Binding of cAMP to R subunits at 4 degrees C (when only free cAMP binding sites are measured) revealed an important endogenous occupancy of cAMP binding sites of RI and RII isoreceptors under basal conditions (40%) and a significantly increased occupancy after exposure of cells to TSH (60%) . Pools of regulatory subunits with more than 50% of sites occupied, which were devoid of enzyme activity, were found both, in control and TSH-exposed cells . They were identified as RI subunits which represented a mixed population of native and partly degraded molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Nippon Eiseigaku Zasshi, 1994 Feb, 48(6), 1058 - 66
{Studies on growth of brown adipose tissue by means of cell culture and angiogenesis in vitro techniques}; Ueno N et al.; To elucidate the mechanism of brown adipose tissue (BAT) enlargement, we investigated the effects of norepinephrine (NE) and insulin on the in vitro growth of rat brown adipose cells (RBAC) and the capillary growth in angiogenesis in vitro using co-culture of bovine capillary endothelial cells (BCEC) and rat smooth muscle cells . In the primary cell culture, NE significantly enhanced the growth of RBAC in the range of 10(-9)-10(-5)M, whereas it did not stimulate the growth of BCEC . Insulin showed the same trend . Moreover, both NE and insulin appeared to increase the expression of mRNA for basic fibroblast growth factor (bFGF), which is a potent angiogenic factor, in RBAC . At 4 h after NE stimulation, the bFGF mRNA expression was considerably increased but it decreased markedly after 16 h . These results suggest that the bFGF mRNA expression in RBAC is quickly simulated by NE, wih resulting bFGF production . Actually, bFGF stimulated the RBAC growth up to about 170% of the control level . However, neither NE nor insulin stimulated the expression of the bFGF gene in BCEC . On the other hand, NE notably increased the capillary growth in vitro compared with insulin . It is thus possible that NE and insulin contribute to the growth of RBAC and endothelial cells partly through bFGF production by an autocrine mechanism, suggesting that both agonists play an important role not only in the thermogenic function of BAT but also in BAT enlargement.

J Neurochem, 1994 Feb, 62(2), 715 - 23
Increased production of paired helical filament epitopes in a cell culture system reduces the turnover of tau; Vincent I et al.; To investigate the regulation of posttranslational modifications of tau that might be pertinent to the production of the paired helical filament (PHF) of Alzheimer's disease, we incubated human neuroblastoma cells with the protein phosphatase inhibitor okadaic acid . This treatment results in increased immunoreactivity of tau with the monoclonal antibodies Alz-50, PHF-1, T3P, and NP8, a reduction in Tau-1 immunoreactivity, and an elevation in apparent molecular weight of tau . Moreover, our data demonstrate that accumulation of phosphates in tau leads to a decrease in the turnover rate of tau in the neuroblastoma cells . It is suggested that similar build-up of hyperphosphorylated tau in the neuronal perikarya may represent an early event in PHF formation . The present system facilitates the investigation of regulatory mechanisms governing the occurrence of PHF epitopes, their effects on neuronal cell metabolism, and possible pharmacological intervention.

FASEB J, 1994 Jan, 8(1), 43 - 53
Regulation of expression of glucose transporters by glucose: a review of studies in vivo and in cell cultures; Klip A et al.; Glucose transporters are membrane-embedded proteins that mediate the uptake of glucose from the surrounding medium into the cell . Glucose is the main fuel for most cells, and its uptake is rate-limiting for glucose utilization . For this reason, it is expected that glucose transport is tightly regulated . Whereas rapid regulation of glucose transporters by hormones has been known for some time, the regulation of glucose transporters by substrate availability (i.e., by glucose itself) is less well understood . This question has been approached by scientists from two angles: one, by measuring the consequence of diabetic states (in which there is surplus of glucose availability) on the expression of glucose transporter genes, and another one, by measuring the effect of glucose availability and glucose deprivation in cell cultures on glucose transporter gene expression . The results from both camps are unfortunately not coincident, due in part to the coexistence of other variables in the diabetic animals, and to the lack of ideal cell cultures . In spite of these caveats, the profuse literature on both approaches propelled us to find commonalities within each approach . This review concludes that in animal studies, one isoform of glucose transporters, the GLUT4 type, is down-regulated by high levels of circulating glucose in muscle but not in fat cells . This down-regulation of the protein is independent of regulation of transcription . In contrast, in fat cells, high glucose levels depress GLUT4 mRNA levels . In cell culture studies, high glucose levels lead to lower expression of the GLUT1 transporter isoform relative to glucose-deprived cultures . Glucose levels do not affect the amount of GLUT4 transporter isoform . The down-regulation of the GLUT1 transporter protein is caused by pre- and post-transcriptional mechanisms, the prevalence of each being cell-type specific . No glucose-responsive elements have been identified on either the GLUT1 or GLUT4 genes, and no information is available on the glucose metabolites that mediate the response of glucose transporter gene expression to glucose availability.

Cancer Immunol Immunother, 1994 Jan, 38(1), 53 - 60
Poor induction of interleukin-2 receptor expression on CD8bright+ cells in whole blood cell cultures with CD3 mAb . Implications for immunotherapy with CD3 mAb; Janssen RA et al.; To induce better stimulation of T cells during recombinant interleukin-2 (rIL-2) therapy of renal cell carcinoma patients, pretreatment with low-dose CD3 monoclonal antibody (mAb) has been proposed . However, in our clinic, such a treatment did not induce additional activation of T cells . To investigate this we performed whole blood cell cultures with rIL-2 or CD3 mAb as a stimulant . Cultures using isolated blood mononuclear cells were used as a control . When stimulated by the addition of rIL-2, the lymphocyte composition and activation of whole blood cultures did not differ from those of mononuclear cell (MNC) cultures . However, when stimulation was performed with CD3 mAb, CD8bright+ cells in whole blood cultures were not or only minimally induced to express CD25 or IL-2 receptor beta (IL-2R beta) . This is in contrast to the situation found in MNC cultures where all CD8bright+ cells expressed CD25 or IL-2R beta to a high extent at the end of culture . When rIL-2 or recombinant interferon gamma (rIFN gamma) was added to whole blood cultures together with CD3 mAb, significantly more CD8bright+ cells were induced to express CD25 or IL-2R beta . These results suggest that whole blood cultures represent the in vivo situation better than MNC cultures . In addition, the results suggest that, also in vivo, administration of low-dose CD3 mAb alone might not be sufficient to induce IL-2R expression on CD8bright+ cells, and would therefore not induce additional specific T cell activation in rIL-2-based immunotherapy . The presented results suggest that in vivo simultaneous administration of rIFN gamma or rIL-2 with low-dose CD3 mAb might induce better stimulation of CD8+ T cells than CD3 mAb only.

Dev Biol, 1994 Jan, 161(1), 218 - 28
Induction of rapid osteoblast differentiation in rat bone marrow stromal cell cultures by dexamethasone and BMP-2; Rickard DJ et al.; Adult vertebrates require a continuous supply of osteoblasts for both bone remodeling and regeneration during fracture repair . This implies the existence of a reservoir of cells in the body capable of osteogenesis . One source of these osteoprogenitors is the stem cells within the fibroblastic component of bone marrow stroma . Mature osteoblasts are characterized by high alkaline phosphatase and osteopontin levels, combined with expression of the bone-specific matrix proteins osteocalcin and bone sialoprotein and the capacity for matrix mineralization . We have used these markers to define the conditions permitting rapid osteoblast differentiation from cultured bone marrow stromal cells . Osteoblastic differentiation was induced by continuous culture with 10(-8) M dexamethasone (dex) which stimulated alkaline phosphatase (AP) activity and mRNA levels as well as osteopontin, bone sialoprotein, and osteocalcin mRNA by Day 8 of culture; coaddition of 10(-8) M 1,25-dihydroxyvitamin D3 (vitamin D) with dex was essential for high osteocalcin mRNA expression . Recombinant bone morphogenetic protein-2 (BMP-2) exerted similar effects to dex and acted in synergy with dex to yield greatly elevated AP activity as well as increased levels of osteoblastic mRNAs . Using in situ hybridization to detect the presence of mRNAs in individual cells, it was shown that appearance of osteopontin mRNA preceded AP mRNA, and was expressed in dex-treated cell colonies as early as Day 4 . Quantitation of cell surface AP protein by flow cytometry indicated that culture with dex or BMP-2 produced a mixed population of cells with low AP (dim cells) and cells with high AP levels, while the combination of dex + BMP-2 yielded very few dim cells and a population of cells containing higher AP levels than with either inducer alone . When the dim population from dex-treated cells was sorted and recultured with inducers, these cultures developed high AP levels and were able to deposit a mineralized matrix . Thus, treatment of marrow stromal cells with inducer results in a population of mature osteoblasts as well as a population of undifferentiated cells which retains the capacity for osteoblastic differentiation with further exposure to inducers . These data demonstrate that stem cells within the stromal compartment of bone marrow are capable of rapidly acquiring osteoblast features and suggest a potential role for glucocorticoids in combination with BMP-2 and vitamin D in stages of osteogenic development.

J Neurosci, 1994 Jan, 14(1), 368 - 83
Pairing-specific, activity-dependent presynaptic facilitation at Aplysia sensory-motor neuron synapses in isolated cell culture; Eliot LS et al.; Synapses made by Aplysia sensory neurons onto motor- and interneuron followers in the intact nervous system exhibit an associative form of synaptic facilitation that is thought to contribute to classical conditioning of the animal's gill and siphon withdrawal reflex (Hawkins et al., 1983; Walters and Byrne, 1983) . Here we demonstrate that a similar associative facilitation can be induced between individual sensory and motor neurons isolated in culture . Pairing tetanic stimulation with either of two facilitatory transmitters, 5-HT or small cardioactive peptide, considerably prolongs facilitation compared to either tetanus or transmitter alone . When corrected for the depression that occurs simply in response to low-frequency testing, the facilitation produced by one pairing trial does not decay for more than 20 min after training . This facilitation requires the temporal pairing (0.5 sec forward interstimulus interval) of the two stimuli, tetanus and 5-HT . Delivering the same two stimuli in an unpaired fashion (1 min forward interval) fails to produce the long-lasting effect . Measurements of spontaneous transmitter release during either paired or unpaired training reveal no changes in unitary mEPSP or mEPSC ("mini") amplitude, indicating that the facilitation involves a presynaptic mechanism . While both forms of training dramatically increase the initial frequency of spontaneous release, mini frequency does not remain elevated as long as the evoked EPSP following paired training, nor does paired training specifically enhance spontaneous release frequency . Pairing-specific facilitation was not blocked by the protein kinase C inhibitor H7 . In contrast, the same training procedure produced pairing-specific increases of sensory neuron excitability and action potential width, suggesting that cAMP-mediated processes are involved in the paired effect . Although Ca2+ influx is necessary for the associative effect (Abrams, 1985), we find that the facilitation does not require influx through L-type voltage-gated Ca2+ channels, since the effect was not blocked by the dihydropyridine antagonist nitrendipine . Together, these findings indicate that the mechanism underlying associative, activity-dependent facilitation is intrinsic to the sensory neuron synapse, that it is presynaptically mediated by processes unique to evoked synaptic transmission, and that it appears to involve a pairing-specific broadening of the presynaptic action potential, allowing enhanced Ca2+ influx through the dihydropyridine-insensitive channels responsible for release.

J Virol, 1994 Jan, 68(1), 548 - 54
Nucleotide sequence of wild-type hepatitis A virus GBM in comparison with two cell culture-adapted variants; Graff J et al.; In order to study cell tropism and attenuation of hepatitis A virus (HAV), the genome of HAV wild-type GBM and two cell culture-adapted variants, GBM/FRhK and GBM/HFS, were cloned and sequenced after amplification by reverse transcriptase-PCR . During virus cultivation, the HAV variant GBM/FRhK had a strict host range for FRhK-4 cells, in contrast to GBM/HFS, which can be grown in HFS and FRhK-4 cells . The HAV variant GBM/HFS was shown to be attenuated when inoculated into chimpanzees (B . Flehmig, R . F . Mauler, G . Noll, E . Weinmann, and J . P . Gregerson, p . 87-90, in A . Zuckerman, ed., Viral Hepatitis and Liver Disease, 1988) . On the basis of this biological background, the comparison of the nucleotide sequences of these three HAV GBM variants should elucidate differences which may be of importance for cell tropism and attenuation . The comparison of the genome between the GBM wild type and HAV wild types HM175 (J . I . Cohen, J . R . Ticehurst, R . H . Purcell, A . Buckler-White, and B . M . Baroudy, J . Virol . 61:50-59, 1987) and HAV-LA (R . Najarian, O . Caput, W . Gee, S . J . Potter, A . Renard, J . Merryweather, G . Van Nest, and D . Dina, Proc . Natl . Acad . Sci . USA 82:2627-2631, 1985) showed a 92 to 96.3% identity, whereas the identity was 99.3 to 99.6% between the GBM variants . Nucleotide differences between the wild-type and the cell culture-adapted variants, which were identical in both cell culture-adapted GBM variants, were localized in the 5' noncoding region; in 2B, 3B, and 3D; and in the 3' noncoding region . Our result concerning the 2B/2C region confirms a mutation at position 3889 (C-->T, alanine to valine), which had been shown to be of importance for cell culture adaptation (S . U . Emerson, C . McRill, B . Rosenblum, S . M . Feinstone, and R . H . Purcell, J . Virol . 65:4882-4886, 1991; S . U . Emerson, Y . K . Huang, C . McRill, M . Lewis, and R . H . Purcell, J . Virol . 66:650-654, 1992), whereas other mutations differ from published HAV sequence data and may be cell specific . Further comparison of the two cell culture-adapted GBM variants showed cell-specific mutations resulting in deletions of six amino acids in the VP1 region and three amino acids in the 3A region of the GBM variant GBM/FRhK.

J Virol, 1994 Jan, 68(1), 48 - 55
ICP34.5 mutants of herpes simplex virus type 1 strain 17syn+ are attenuated for neurovirulence in mice and for replication in confluent primary mouse embryo cell cultures; Bolovan CA et al.; In a recent report, the neurovirulence of herpes simplex virus type 1 (HSV-1) was mapped to the ICP34.5 gene (J . Chou, E . R . Kern, R . J . Whitley, and B . Roizman, Science 250:1262-1266, 1990) . In this report, specific mutations within ICP34.5 were constructed in HSV-1 strain 17syn+ to determine the effects of these mutations in a fully neurovirulent isolate . It was found that termination of the ICP34.5 gene after the N-terminal 30 amino acids resulted in a mutant, 17termA, which was 25- to 90-fold reduced in neurovirulence . This reduction of neurovirulence was associated with restricted replication of the mutant virus in mouse brain . The reduced replication phenotype was also evident in the trigeminal and dorsal root ganglia following inoculation at the periphery . 17termA was capable of replicating with wild-type kinetics in mouse footpads, and therefore the restriction seen in neural tissues was not due to a generalized replication defect in mouse cells . Significantly, replication of the mutant was also restricted in the mouse cornea in vivo and in confluent primary mouse embryo cells and mouse 10T1/2 cells in vitro . However, 17termA replicated with much greater efficiency in subconfluent mouse embryo cells, suggesting that the physiological state of the cell may be an important factor for productive replication of this mutant . Restoration of the ICP34.5 gene to the mutant resulted in a virus which displayed wild-type neurovirulence and replication kinetics in all cells and tissues tested.

Epilepsy Res, 1994 Jan, 17(1), 1 - 11
Limitation by gabapentin of high frequency action potential firing by mouse central neurons in cell culture; Wamil AW et al.; The investigational anticonvulsant drug, gabapentin (GP; 1-(aminomethyl) cyclohexaneacetic acid) limited repetitive firing of sodium-dependent action potentials of mouse spinal cord and neocortical neurons in monolayer dissociated cell culture . The effect developed slowly over time with sustained exposure . The IC50 was 1.3 x 10(-4) M for exposure times < or = 60 s, 1.9 x 10(-5) M for 10-60 min, and 4.0 x 10(-6) M for 12-48 h . Hyperpolarization restored sustained firing in the continuing presence of GP . Blockade of action potential firing by GP was frequency (use)-dependent . After preincubation with 2.9 x 10(-5) M GP (5 micrograms/ml), trains of brief stimuli at > or = 50 Hz elicited fewer action potentials than in control solution . Also, at 150 Hz, maximal rate of rise of action potentials decreased progressively with repetitive firing in GP-containing, but not control, solution . After overnight incubation in 2.9 x 10(-5) M GP, the absolute refractory period was prolonged from 2.4 +/- 0.6 ms in control solution (n = 11) to 4.7 +/- 0.3 ms (n = 10; P = 0.02 vs . control), and complete recovery from inactivation was prolonged from 8.0 +/- 1.3 ms to 17.0 +/- 2.6 ms (P < 0.001 vs . control) . These findings suggest that GP may alter function of voltage-activated sodium channels, but the mechanism is unproven and may be indirect . Limitation of firing was observed in > or = 50% of neurons at concentrations in the range of those found in plasma and cerebrospinal fluid of patients treated successfully with GP . These results suggest that limiting the rate of firing of sodium-dependent action potentials may contribute to the anticonvulsant efficacy of gabapentin.

Pathology, 1994 Jan, 26(1), 29 - 32
Cell culture studies on human nerve sheath tumors; Kharbanda K et al.; The main controversy about nerve sheath tumors (NSTs) has been their histogenesis . A Schwann cell origin has been proposed by many investigators for both schwannomas and neurofibromas . However Erlandson and Woodruff observed that while schwannomas appeared to be composed predominantly of Schwann cells, neurofibromas consisted of mainly perineurial cells . In addition, variable numbers of fibroblast-like cells and intermediate cells also have been reported in the 2 lesions . Whether these represent distinct cell types or variants of Schwann cells is still debatable . In an attempt to solve this controversy, the present study was undertaken to observe the morphology and the behaviour of these tumors in culture . These studies showed that all nerve sheath tumors are basically of Schwann cell origin and that intermediate cells and fibroblasts are variants of Schwann cell . Tissue culture studies done chiefly on schwannomas showed that the morphological features of schwannomas are preserved in 'in vitro culture' condition and therefore the difference between neurofibroma and schwannoma appears to be due to inherent differentiating property of the Schwann cells along with some environmental stimulus.

Biomaterials, 1994 Jan, 15(1), 63 - 7
Cytotoxicity testing of cyanoacrylates using direct contact assay on cell cultures; Ciapetti G et al.; The use of a tissue adhesive for surgical procedures has prompted a large number of clinical and experimental studies . Alkyl-2-cyanoacrylate esters constitute a family of adhesives with good mechanical properties but their biological compatibility has to be assessed . In this study the cytotoxicity of three commercially available cyanoacrylates and one of unknown composition has been determined . The first part of the study deals with direct contact testing procedures using L 929 cells challenged with drops of adhesives: cell morphology, cell growth and bacterial growth inhibition were assayed . Testing methods included cell viability assay using vital dyes, cell growth measurement using crystal violet staining uptake and bacterial growth assay using S . aureus growth inhibition . All the cyanoacrylate adhesives tested were found to be cytotoxic and to inhibit cell proliferation: differences between the cyanoacrylates were found.

Radiats Biol Radioecol, 1994 Jan-Feb, 34(1), 94 - 9
{The kinetics of polykaryon formation in a HeLa cell culture after irradiation at doses of 5 and 10 Gy}; Kalendo GS et al.; Monolayer culture of HeLa tumor cells irradiated at 5 Gy and 10 Gy doses was followed up in kinetics for 14 days . It was shown that starting with day 3 the cell monolayer is modified in such a way that some cells occur in 2- to 8-cell accumulations . The rate of formation of such groups and the number of cells in them were dose-related . The labeling index in these formations was several times higher than that in the irradiated population on the whole . The analysis of the grade of DNA synthesis synchronization in these accumulations showed that complete cell fusion and polykaryon formation after irradiation at the dose of 10 Gy occurred on day 4 whereas after 5 Gy, only on day 7 . In both cases the status of complete fusion was 3 days in duration.

J Cancer Res Clin Oncol, 1994, 120(6), 359 - 64
A rapid luciferase transfection assay for transcription activation effects and stability control of estrogenic drugs in cell cultures; Meyer T et al.; A rapid assay system for measuring the potential of estrogenic drugs is introduced . Luciferase induction could be measured in estrogen-receptor-positive human MCF-7 breast cancer cells, which had been transfected with a novel luciferase reporter plasmid ERE luc . The minimal requirement was 1 h exposure to the inducing drug and 3.5 h of incubation after removal of the drug . The assay system was used to measure the stability of the drug diaqua-{1,2-bis (2,6-dichloro-4-hydroxyphenyl) ethylenediamine} platinum(II) sulfate, containing an estrogenic ligand and reactive platinum . Luciferase activity was observed only when the drug was in the culture medium and cells for short times, whereas the estrogenic ligand alone remained active . It is assumed that binding of the platinum moiety to macromolecular constituents of the culture or cells renders the drug inaccessible for binding to the estrogen receptor.

Development, 1994 Jan, 120(1), 115 - 22
Expression of the Brachyury gene during mesoderm development in differentiating embryonal carcinoma cell cultures; Vidricaire G et al.; When aggregated and treated with dimethyl sulfoxide (DMSO), P19 embryonal carcinoma cells differentiate into cell types normally derived from the mesoderm and endoderm including epithelium and cardiac and skeletal muscle . The Brachyury gene is expressed transiently in these differentiating cultures several days before the appearance of markers of the differentiated cell types . The expression of Brachyury is not affected by DMSO but is induced by cell aggregation, which requires extracellular calcium . Expression of Brachyury is also induced by various members of the TGF beta family such as activin and bone morphogenetic proteins . D3 is a mutant clone of P19 cells selected for its failure to differentiate when aggregated in DMSO . Aggregated D3 cells express Brachyury mRNA suggesting that the mutation(s) responsible for the phenotype of D3 cells is downstream of the chain of events initiated by Brachyury expression.

Lik Sprava, 1994 Jan, (1), 77 - 80
{The immunological indices of diabetic patients following the transplantation of a pancreatic islet cell culture}; Karabun PM et al.; Some indices of cell-mediated and humoral immunity were studied in 35 patients with insulin-dependent diabetes mellitus before and 7, 14 days, 3, 6, and 12 months after transplantation of pancreatic islet cell culture obtained from newborn pigs into m . rectus abdominis . It was established that the transplantation does not affect number of T- and B-lymphocytes, T-helpers, T-suppressors and level of antibodies to insulin . Clinical effect depended on the base level of transplantation immunity . Favourable results, such as liquidation of hypoglycaemia and reduction of exogenic insulin at the background of improved general condition may be expected in cases with high of heterophilic haemagglutinins low cytotoxicity index and increased number of sensibilized leukocytes.

Vet Microbiol, 1994 Jan, 38(3), 263 - 76
Antiviral activity of interferon against transmissible gastroenteritis virus in cell culture and ligated intestinal segments in neonatal pigs; Jordan LT et al.; Segments of jejunum in 5 to 6 days old piglets were surgically ligated, inoculated with transmissible gastroenteritis virus (TGEV) and 18 hours later the segments were fixed for histology or suspensions were prepared for plaque assay in swine testis (ST) cell cultures to determine the yield of virus . When the virulent Purdue strain of TGEV was used, villous atrophy was seen and TGEV antigen was demonstrated immunohistochemically in the villous enterocytes . The Miller M6 strain of virus produced less extensive lesions in the segments, but since it was titratable by plaque assay it was used in the subsequent yield reduction assays to determine the antiviral activity of interferon . When intestinal segments were inoculated simultaneously with either 3200 units of natural porcine interferon-alpha or up to 1000,000 units of recombinant human interferon-alpha 2 a, and TGEV, there no reductions in virus yield, although the same cytokines exerted an antiviral effect in ST cells treated in a similar way . However, virus yields were significantly reduced in intestinal segments in piglets treated parenterally with the synthetic interferon inducer polyinosinic: polycytidylic acid 6 hours before challenge of the segments with TGEV . There was also a trend for the antiviral effects of interferon induction before challenge to be augmented by the inclusion of interferon with the virus inoculum . It was concluded that interferon would be ineffective as a therapeutic for TGEV, although it might be useful prophylactically.

Biomaterials, 1994 Jan, 15(2), 92 - 6
Toxicity of cyanoacrylates in vitro using extract dilution assay on cell cultures; Ciapetti G et al.; Comparative cytotoxicity testing of four cyanoacrylate adhesives suggested for orthopaedic applications was performed . These substances were placed in complete culture medium with serum and the resulting extraction fluids were tested on L 929 cells and human lymphocytes . Testing procedures include cell morphology assessment using light microscopy and vital dyes, cell counting using a computer-assisted image analysis system, cell growth measurement using total protein content assay and cell viability assessment using the MTT method . Quantitation of the toxicity of the degradation products released by cyanoacrylates in the extracts was achieved and differences in the cytopathic effect related to the chemical composition of the cyanoacrylates were found . A toxicity rating of the assayed cyanoacrylate adhesives was obtained as follows (in order of increasing toxicity): BCA < xCA < ECAg < ECAl.

C R Acad Sci III, 1994 Jan, 317(1), 85 - 9
Enantiomeric 2',3'-dideoxycytidine derivatives are potent human immunodeficiency virus inhibitors in cell cultures; Gosselin G et al.; 2',3'-dideoxy-beta-L-cytidine (beta-L-DDC) and its 5-fluoro derivative (beta-L-5-F-DDC) have been stereospecifically synthesized by a multi-step reaction sequence from L-xylose and their anti-HIV properties examined . Among these two L-enantiomers, the hitherto unknown beta-L-5-F-DDC emerged as a potent anti-HIV-1 and HIV-2 compound in different cell culture systems, with selectivity indices similar or superior to those of the currently licensed drug DDC which has the natural beta-D configuration.

Acta Anat (Basel), 1994, 150(2), 136 - 49
Nodule formation and calcification of mandibular condylar cartilage cell cultures mimic in vivo ultrastructure; Engel FE et al.; Mandibular condylar cartilage (MCC) of growing mammals contains four layers of cells which display a series of increasingly differentiated phenotypes and which culminate in a terminally differentiated cell that produces a calcified matrix . In this study, MCC cells were placed into culture in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 50 micrograms/ml ascorbic acid . After 12 h in culture, transmission electron microscopy revealed the presence of multiple cell types that underwent differentiation with additional time in culture . By 7 days, fusiform-shaped cells were seen that contained numerous actin-like cytofilaments and micropinocytotic vesicles characteristic of myofibroblasts . Chondroblast-like cells were also observed . By 10 days, without addition of beta-glycerophosphate or dexamethasone, these cellular events culminated in the formation of mineralized nodules containing matrix vesicles . The nodular surface at day 13 consisted of two or more layers of myofibroblast-like cells, while the deeper zones of the nodule contained cells displaying a morphology typical of calcified hypertrophic chondroblasts . These ultrastructural observations are consistent with the hypothesis that cells from the MCC are capable of recapitulating in culture the maturational events seen in vivo . This cell culture model may be useful for investigating cell-mediated cartilage calcification without the addition of exogenous phosphate or other regulatory factors.

Urol Res, 1994, 22(2), 85 - 91
Stimulation of intercellular adhesion molecule-1 (ICAM-1) antigen expression and shedding by interferon-gamma and phorbol ester in human renal carcinoma cell cultures: relation to peripheral blood mononuclear cell adhesion; Hansen AB et al.; In the present study we investigated the effect of interferon-gamma (IFN-gamma) and phorbol-12-myristate 13 acetate (PMA) on intercellular adhesion molecule-1 (ICAM-1) antigen expression and shedding in human renal carcinoma cell cultures . We also examined the functional consequences of ICAM-1 antigen expression and soluble ICAM-1 molecules on the adhesion of peripheral blood mononuclear cells (PBMC) . Incubation of the human renal carcinoma cell line CaKi-1 with IFN-gamma or PMA enhanced ICAM-1 antigen expression . The calcium ionophore, 4-bromo-calcium ionophore A23187 (Bromo-A23187) significantly enhanced the IFN-gamma and PMA effect . Soluble ICAM-1 (sICAM-1) was detected in the supernatants of stimulated but not unstimulated cultures, and correlated significantly with cellular expression . Using 51Cr-labelled peripheral blood mononuclear cells in a cell adhesion assay, we demonstrated increased adhesion in IFN-gamma-treated CaKi-1 cultures, which was augmented by Bromo-A23187 . This adhesion was blocked by preincubation of CaKi-1 cells with monoclonal antibody against ICAM-1 or by preincubation of PBMC with either monoclonal antibody against leucocyte function associated antigen-1 alpha (LFA-1 alpha), a major receptor for ICAM-1, supernatants from treated cultures or purified sICAM-1 molecules . Thus, shedding of ICAM-1 may play a role during the escape from immunosurveillance by renal carcinoma cells.

Eye, 1994, 8 ( Pt 2), 238 - 44
The use of xenografting to evaluate the remyelinating potential of glial cell cultures; Targett MP et al.; Experiments in rodents have shown a potential role for glial cell transplantation as a means of influencing repair in the central nervous system of man . A crucial step in developing human therapy is to establish whether knowledge gained from studies in rodents is applicable to larger mammalian species . In order to explore this issue we examined the ability of cat glial cell cultures to remyelinate areas of ethidium-bromide-induced demyelination in the spinal cord of immunosuppressed rats and cats . Transplantation of density-gradient-isolated glial cells obtained from the forebrain of 7-day-old kittens resulted in enhanced oligodendrocyte remyelination in the rat but failed to enhance oligodendrocyte remyelination in the cat . The feasibility of enhancing oligodendrocyte remyelination in the cat lesion was demonstrated by transplanting a rat culture containing a high proportion of cells of the oligodendrocyte lineage . Tissue culture of the density-gradient-isolated cell preparations suggested that the failure of the kitten glial preparation to enhance oligodendrocyte remyelination in the cat was most probably due to its poor oligodendrocyte-generating capacity . However, our lack of understanding of the biology of feline glial cells precludes a full understanding of these experiments.

Radiat Environ Biophys, 1994, 33(2), 125 - 39
Slow clones, reduced clonogenicity, and intraclonal recovery in X-irradiated L5178Y-S cell cultures; Beer JZ et al.; Profound, long-lasting growth disturbances and reduced viability and clonogenicity were observed in suspension cultures of L5178Y-S (LY-S) murine leukemic lymphoblasts exposed to 0.25-6 Gy of X rays . In most cases, uncloned cultures grew at a reduced rate for periods corresponding to at least 100 cell generations, even when viability of such cultures returned to the normal level . These disturbances were analyzed in clones isolated using agar-supplemented medium . A slow phenotype was much more frequent among surviving clones isolated from LY-S cell cultures irradiated with 3 Gy of X rays than among clones isolated from nonirradiated controls . Growth of individual LY-S clones was affected to different extents, regardless of the clone's viability . The slowest clones had doubling time twice as long (22 h) as that of the control (10-12 h) . More than 100 slow clones isolated from irradiated and nonirradiated cultures were followed for prolonged times, and some of them were further subcloned . The slow clones showed a high degree of heterogeneity, and selection for the slowest clone produced clones with increasing proliferative impairment and decreasing cloning efficiency . These results showed that progeny of X-irradiated LY-S cells contained many slowly growing cells, and that their presence affected the growth rate for scores of cell generations . The prolonged impairment of growth rate, viability, and clonogenicity appeared to depend on heritable lesions that were overcome as a result of intraclonal recovery . All slow clones were capable of such recovery, which for clones derived from irradiated cultures typically required periods corresponding to several scores of, but in some cases > 200, cell generations . Intraclonal recovery was much more rapid in slow clones isolated from nonirradiated cultures . This finding indicated that either slow phenotype depended on different cellular changes in the two groups of clones or mechanisms of intraclonal recovery were affected by radiation.

Magy Traumatol Ortop Kezseb Plasztikai Seb, 1994, 37(3), 257 - 60
{Comparative study of the in vitro effects of calcitonin, NaF and ipriflavone in cell culture}; Bucsi L et al.; The aim of these in vitro series of experiments was to state whether the medicaments used in the treatment of loss of bone density the Calcitonin, NaF and Ipriflavon do have a direct effect on the preosteoblast cells . The results show that both the Calcitonin and Fluorid stimulated the development of the fibroblast colonies and the NaF had a role in the increase of the alkaline phosphatase too . In the concentration of Ipriflavon applied no effect could be demonstrated in any parameter.

J Nat Prod, 1994 Jan, 57(1), 116 - 22
New bioactive taxoids from cell cultures of Taxus baccata; Ma W et al.; Four new taxoids were isolated from cell cultures of Taxus baccata . Their structures were elucidated by spectroscopic analyses . Two were the aglycones corresponding to previously isolated 7-O-xylosides of taxol C {1} and 10-deacetyltaxol C {2} . The third {3} had an N-methylated side-chain, while the fourth, named taxcultine {4}, contained an n-propyl group on the side-chain . All four compounds actively promoted tubulin assembly . Taxol C {1} showed potent and selective cytotoxicity in the NCI human cell line screen.

J Neural Transm Suppl, 1994, 44, 47 - 60
An aggregate brain cell culture model for studying neuronal degeneration and regeneration; Chatterjee SS et al.; Rotation-mediated aggregating cell cultures from fetal rat telencephalons containing glial and neuronal cells mature in a fashion comparable to that known to occur in brain in vivo . Large aggregates of 300-500 microM diameters can now reproducibly be cultivated and maintained for more than 40 days in a well defined serum free medium . Validity of the use of such cultures for in vitro studies of various physiological, pharmacological and toxicological phenomenon has already been demonstrated . In this communication some observations suggesting the usefulness of such cultures for pharmacological studies clarifying the possible effects of drugs and other agents on excitatory amino acid induced pathological processes will be presented . The advantages and limitations of the use of aggregated brain cell culture based models for the development of agents potentially useful for the treatment of aging and dementia will also be discussed.

J Clin Lab Anal, 1994, 8(6), 447 - 51
Cytokine production in whole blood cell cultures of patients with Crohn's disease and ulcerative colitis; Elsasser-Beile U et al.; Levels of the cytokines interleukin-1-alpha, -1-beta, and -2 (IL-1-alpha, IL-1-beta, IL-2), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) were measured in the mitogen-stimulated whole blood cell cultures from 96 patients with Crohn's disease (48 untreated, 12 treated with sulfasalazine, 36 treated with corticosteroids), 74 patients with ulcerative colitis (21 untreated, 25 treated with sulfasalazine, 28 steroid treated), and 360 healthy controls . The cytokines were measured 4 days after induction by a sensitive immunoenzyme assay . In the blood cell cultures of the untreated and sulfasalazine treated patients with Crohn's disease and ulcerative colitis higher levels of TNF-alpha, IL-1-alpha and IL-1-beta were found whereas IL-2 production was decreased and IFN-gamma-production was not significantly different as compared to the controls . Leukocytes of the corticosteroid-treated patients with both diagnoses showed a lower production of all measured cytokines compared to the untreated patients . The same results were obtained, when the somewhat different counts of mononuclear cells in the peripheral blood of the patients and controls were taken into account . The elevated production of proinflammatory cytokines in the blood cell cultures suggests a systemic immune activation in patients with inflammatory bowel disease.

Neurotoxicology, 1994 Fall, 15(3), 715 - 9
Long-term treatment of aggregating brain cell cultures with low concentrations of lead acetate; Zurich MG et al.; Aggregating brain cell cultures were used as a model to study the effect of chronic exposure to low levels of lead acetate . Long-term maintenance of cultures could be improved by supplementation of the medium with albumin-bound lipids . Exposure for 9 days to 10(-6)-10(-4) M lead acetate caused a decrease of GABAergic (glutamic acid decarboxylase) and astrocytic (glutamine synthetase) markers which was also found after prolonged treatment (50 days) with 10(-7) M lead acetate . Total protein content and choline acetyltransferase were not changed . The results show that prolonged exposure of aggregating brain cell cultures to a low concentration of lead acetate causes distinct changes of cell type-specific parameters.

Virchows Arch, 1994, 425(5), 521 - 9
Endothelial cell heterogeneity in experimentally-induced rabbit atherosclerosis . Demonstration of multinucleated giant endothelial cells by scanning electron microscopy and cell culture; Tashiro K et al.; We investigated the aortic endothelial cells of cholesterol-fed rabbits, using scanning electron microscopy and a cell culture technique . Rabbits were given a 1% cholesterol diet intermittently for up to 40 weeks . In these animals, the area of endothelial cells was increased and the cells showed polymorphism in relation to the progression of atherosclerosis . In animals fed the cholesterol diet for 12, 28 and 40 weeks, the average area of the endothelial cells was 436 +/- 15, 762 +/- 153, and 836 +/- 165 microns2, respectively . In the cholesterol-fed 40-week group, in particular, giant endothelial cells, measuring more than 1200 microns2, accounted for 14% of the population . In animals fed a standard diet there was no significant difference in endothelial cell morphology between control 0-week and control 40-week groups; in both, the luminal surface of the thoracic aorta formed a homogeneous sheet covered by small rhomboidal endothelial cells, the area of most being less than 400 microns2 . Primary cultured endothelial cells harvested from those control groups were mononuclear typical small cells with a centrally located nucleus; the proportion of binucleated cells was less than 2% and no multinucleated giant cells with three or more nuclei were detected . Endothelial cells from the cholesterol-fed groups, however, contained larger numbers of binucleated cells, with the number increasing in proportion to the duration of cholesterol feeding . The major distinguishing feature of the endothelial cells in the cholesterol-fed groups was the presence of multinucleated giant cells with three or more nuclei; these accounted for 2.3% and 3.3% of the total cell population in the cholesterol-fed 28- and 40-week groups, respectively . No bromodeoxyuridine uptake was found in the nuclei of the cultured multinucleated giant cells . Heterogeneity of endothelial cells, with the concomitant appearance of multinucleated giant cells, emerges with the progression of diet-induced atherosclerosis . The morphological alterations of endothelial cells observed in the present study intimately reflect changes in their function associated with the progression of atherosclerotic lesions.

J Hematother, 1994 Fall, 3(3), 175 - 84
Detection of tumor contamination of peripheral stem cells in patients with lymphoma using cell culture and polymerase chain reaction technology; Chan WC et al.; The important question of whether residual tumor in the bone marrow or peripheral blood stem cell graft contributes to relapse in autologous bone marrow transplantation in patients with non-Hodgkin's lymphoma can be addressed only if there is an accurate and sensitive measurement of tumor cell contamination of the graft . Assays utilizing DNA amplification based on the polymerase chain reaction (PCR) are highly sensitive . Tumor-specific primers and probes can be designed for the clonally rearranged Ig or T cell antigen receptor genes in the original tumors, and these can then be used to detect minimal residual disease in subsequent specimens . Specific translocations can also be exploited as tumor markers, and the t(14;18) translocation has been widely employed for detecting tumor cells in blood and bone marrow samples . Lymphoma cells have also been grown successfully in tissue culture, and the detection of tumor contamination of autologous grafts has been associated with a poorer prognosis in patients with intermediate- or high-grade lymphoma . It is of interest to compare the sensitivity of tumor detection and the predictive value for patient survival of the PCR-based and culture-based assays . The information obtained may help to determine whether minimal tumor contamination of an autologous graft is clinically significant and, if so, the assay(s) that should be employed.

Receptor, 1994 Fall, 4(3), 135 - 41
Quantitative determination of recombinant fibroblast growth factor receptor in baculovirus-infected insect cell cultures; Duke JL et al.; Members of the fibroblast growth factor protein family are involved in several biologically important processes, including angiogenesis, wound healing, and tumor growth and metastasis . Interactions of basic fibroblast growth factor (bFGF) and its receptors are of considerable pharmacological importance . Attempts were made to produce gram quantities of a soluble extracellular form of basic fibroblast growth factor receptor type 1 (bFGFR1) in order to study the energetics of its interaction with bFGF . The aim of the present study was to develop a method for monitoring changes in concentration of bFGFR1 during its production by large-scale baculovirus-infected insect cell culture . A simple reverse-phase high-performance liquid chromatographic assay was developed for direct determination of the soluble receptor secreted into insect cell-culture media . The method permitted cell-culture samples containing varying amounts of fetal calf serum and bFGFR1 (10-30 mg/L) to be analyzed without prior purification . The assay was linear for added receptor in the range of 1-7 micrograms.

Nephrol Dial Transplant, 1994, 9 Suppl 4, 22 - 5
Cell shape changes and detachment in cell culture: models of renal injury; Racusen LC; Loss of tubular cell adhesion may be important in the pathophysiology of injury to renal tubular cell epithelium . In-vitro model systems have been utilized to examine altered cell adhesion in response to hypoxic/anoxic and oxidant-induced cell injury . Alterations in cell cytoskeleton and cell surface adhesion molecules, including L-CAM and integrins, have been demonstrated in these systems, and are probably important in the pathogenesis of altered structure and function in response to injury in renal tubular epithelium.

J Cancer Res Clin Oncol, 1994, 120(12), 700 - 6
Antiproliferative effects of interferon gamma in combination with alpha-difluoromethylornithine on human carcinoma cell cultures; Klouche M et al.; The antiproliferative effects of human recombinant interferon gamma (IFN gamma) in combination with alpha-difluoromethylornithine (DFMO) or as single agents were assessed on human cell cultures derived from carcinomas of the breast (MCF-7), the ovary (EFO-27) or the kidneys (EGI-4) . Results were obtained in proliferation assays by direct cell counting . The cell lines differed considerably in their sensitivities to the antiproliferative effect of IFN gamma as compared by the 50% inhibition doses of the growth (ID50) . In contrast to the findings with IFN gamma, similar antiproliferative effects resulted from the application of comparable doses of DFMO . While IFN gamma induced cytotoxic effects in EGI-4 cells, DFMO produced only cytostatic actions in the cell lines analyzed . Synergistic growth inhibition resulted from the combined application of IFN gamma and DFMO in EFO-27 cell cultures . This finding was most pronounced after treatment with IFN gamma or DFMO doses below the respective ID50 values . However, antagonistic effects occurred in cells of the line EGI-4 after DFMO had been combined with IFN gamma at concentrations below the cytotoxic dose range . Within the sensitivity of our proliferation assay, no synergistic interactions were found in MCF-7 cell cultures . In the cell lines tested, no relation between the sensitivity for the single agents and the effectivity of the drug combination was identified . Despite promising synergistic effects in the moderately IFN gamma-sensitive ovarian carcinoma cell line EFO-27, the efficacy of the IFN gamma/DFMO combination was restrained by possible antagonistic effects as demonstrated in the highly IFN gamma-sensitive EGI-4 renal carcinoma cell cultures . We conclude that the differential interaction patterns in the cell cultures analyzed preclude general suggestions for clinical studies using IFN gamma and DFMO.

Cytotechnology, 1994, 16(3), 137 - 46
Two-dimensional gel electrophoresis for controlling and comparing culture supernatants of mammalian cell culture productions systems; Wimmer K et al.; A recombinant Chinese hamster ovary cell line, producing human erythropoietin, was cultivated in a continuous mode in a stirred tank reactor applying different dilution rates . In order to monitor the stability of this expression system, product and non-product proteins of the cell culture supernatant were analyzed by two-dimensional electrophoresis . The consistency of the isoforms of the recombinant product was determined by western blot combined with specific staining . The same cell line was propagated in a high cell density cultivation system based on macro-cell-aggregates . The patterns of secreted proteins of the cell line cultivated in the different systems were compared in order to detect modifications in protein expression of the product and of non product proteins relevant for cell culture supernatant . Hardly any alterations in two-dimensional pattern were detectable . The isoforms of erythropoietin, as well as the overall pattern of secreted proteins, detectable with the two-dimensional electrophoresis method were remarkably stable under different cultivation conditions.

Cytotechnology, 1994, 16(2), 109 - 20
Identification of media supplements that improve the viability of primarily cell cultures of Crassostrea gigas oysters; Domart-Coulon I et al.; Media supplements have been investigated for their influence on the viability of primary cell cultures from the heart of Crassostrea gigas oysters . Soluble factors of vertebrate origin were tested, belonging to five families of supplements that had proven to increase the viability of insect and mammal cell cultures . Using two-level complete factorial assays, factors and mutual interactions were screened within each family with a MTT reduction assay . Results pointed out the positive influence of hormones, growth factor, antioxidants and lipids on the mitochondrial metabolism of oyster's heart cells . Consequently, a new concentrated complex supplement was developed . At 10% (v/v) final concentration in modified Leibovitz L-15 medium, it increases by 30% the cellular viability of one-week old cultures as compared with non-supplemented medium, a similar improvement as the one obtained with 10% (v/v) fetal calf serum . Combined with fetal calf serum, this new supplement doubles the cellular viability of one-week old cultures and allows networks of cardiomuscular cells to be maintained functional over three months in vitro.

Cytotechnology, 1994, 16(1), 17 - 26
Peptone, a low-cost growth-promoting nutrient for intensive animal cell culture; Jan DC et al.; The effect of addition of peptone to serum-free and serum supplemented media for the growth of hybridoma cells in various systems was studied . Supplementation of defined medium with either proteose peptone or meat peptone resulted in significant increases in cell number and specific monoclonal antibody production in batch culture system . Other peptones were either inactive or less effective . In continuous culture, using medium supplemented with new born calf serum, the addition of peptone resulted in 125% and 150% increases in cell and antibody concentrations respectively . Similar increase in cell number (128%) was also obtained in spin-filter perfusion culture when medium was supplemented with peptone . By comparison, the substitution of a defined 1 x MEM amino acids mixture resulted in only a 50% increase . At higher perfusion rates the cell number maintained in steady state using peptone supplement could be increased to 1.3 x 10(7) cells ml-1 while the serum concentration was reduced from 5% to 1% at a perfusion rate of 2.5 volumes per day.

Cytotechnology, 1994, 14(3), 191 - 204
Modeling of cell culture processes; Tziampazis E et al.; Models of cell processes can be particularly useful in simulating, optimizing and controlling cell culture systems . Models reported in the literature are of various degrees of biological structure and mathematical complexity and describe cell growth, death, metabolism, and product formation, alone or in combination with each other . This paper reviews these modeling efforts, discusses their results, potential and limitations, and identifies areas where future modeling studies may be especially valuable.

Cytotechnology, 1994, 14(3), 177 - 82
Determination of ammonium and L-glutamine in hybridoma cell cultures by sequential flow injection analysis; Campmajo C et al.; A flow injection analytical system based on a gas diffusion membrane module for ammonia and an ammonium flow-through potentiometric detector has been set up for measurement of L-glutamine and ammonium ions in hybridoma cell cultures . The main feature of the system is that the same basic analytical concept and equipment is used in both measurements, the only difference being for the determination of L-glutamine, in which the sample flows through an immobilized glutaminase cartridge . The conditions to enable the performance of both analysis consecutively, avoiding potential interferences by unwanted deamination of other compounds in the samples, have been determined . Finally, the proposed system has been compared with reference analytical methods for batch hybridoma cell culture experiments.

Biotechnol Prog, 1994 Jan-Feb, 10(1), 55 - 9
Heterologous protein expression affects the death kinetics of baculovirus-infected insect cell cultures: a quantitative study by use of n-target theory; Wu SC et al.; The death of cultured insect cells after baculovirus infection is a time-dependent event . Without a quantitative model, it is difficult to characterize its kinetics . Our group has shown that the cell survival rate can be characterized by use of the n-target theory, which involves only two parameters: the number of hypothetical inactivation targets (n) and the first-order death rate (k) . In this study, we used different recombinant viruses to examine the effect of heterologous protein expression on the cell survival rate . The proteins expressed were beta-galactosidase, human T-cell leukemia virus type I p40x, human interleukin-2, and human tissue plasminogen activator (tPA) . The survival rate was affected by protein expression, but the n value remained constant if the protein expression level was high (above 30 mg/L) . Low-level expression of secreted, glycosylated tPA resulted in a reduced n value, which was restored to the normal value when the tPA signal peptide and prosequence were deleted . In addition, if the n value was normal (10-11), the level of protein expression correlated negatively with the death rate . However, if the n value was reduced by unfavorable culture conditions or foreign protein expression, the expression level correlated positively with the death rate . A dimensionless plot with kt as the dimensionless time shows that alteration of the k value while retaining constant n is equivalent to a rescaling of time . Therefore, the survival curves with constant n reduce to a single curve on the dimensionless plot.(ABSTRACT TRUNCATED AT 250 WORDS)

Biotechnol Prog, 1994 Jan-Feb, 10(1), 121 - 4
Influence of ammonium on growth, metabolism, and productivity of a continuous suspension Chinese hamster ovary cell culture; Hansen HA et al.; A recombinant DNA Chinese hamster ovary (CHO) cell line which produces tissue-type plasminogen activator (t-PA) was cultivated continuously in suspension with a constant dilution rate of 0.5 day-1 . The cultivation consisted of four phases with four different ammonium chloride concentrations (0, 2.5, 5, and 7.5 mM) in the feed medium, causing a reactor ammonium concentration of up to 8 mM . Cell growth was not inhibited by these high ammonium concentrations, as cell densities of around 2.3 x 10(6) cells mL-1 were established . In contrast, the production of t-PA was reduced under high ammonium concentration . The decrease in specific t-PA production could be due to either a negative ammonium influence on productivity or a limitation of medium components, e.g., amino acids . Cell metabolism was changed under high ammonium concentrations, seen most clearly by a decrease in specific ammonium production by a factor of 8 and an increase in specific alanine production of 30%.

Phytochemistry, 1994 Jan, 35(2), 353 - 60
Heterologous expression of the plant proteins strictosidine synthase and berberine bridge enzyme in insect cell culture; Kutchan TM et al.; The heterologous expression of cDNAs encoding the alkaloid biosynthetic enzymes, strictosidine synthase {EC 4.3.3.2} from Rauvolfia serpentina and the berberine bridge enzyme {(S)-reticuline: oxygen oxidoreductase (methylene-bridge-forming), EC 1.5.3.9} from Eschscholtzia californica, has been achieved in a cell culture (Sf9) of the fall army worm, Spodoptera frugiperda, using a baculovirus-based expression system . The expression resulted in the overproduction of each plant enzyme in a catalytically active form . The maximal production attained was 4 mg purified, active enzyme per litre cell culture for both the strictosidine synthase and berberine bridge enzymes.

Antisense Res Dev, 1994 Winter, 4(4), 231 - 41
Properties of exonuclease-resistant, psoralen-conjugated oligodeoxyribonucleotides in vitro and in cell culture; Levis JT et al.; We have prepared oligodeoxyribonucleotides that are modified at the 3'-terminal with N4-(4-aminobutyl)deoxycytidine and derivatized at the 5'-end with a 4'-({N-(aminoethyl)amino}methyl)-4,5',8-trimethylpsoralen, (ae)AMT, and whose sequences are complementary to vesicular stomatitis virus (VSV), N-protein mRNA, (ae)AMT-II, or VSV M-protein mRNA, (ae)AMT-III . (ae)AMT-II cross-links exclusively to VSV N-mRNA when a mixture of the oligomer and poly(A+) RNA from VSV-infected cells is irradiated in vitro with long wavelength UV light at either 20 degrees or 37 degrees C . N4-(4-Aminobutyl)deoxycytidine at the 3'-end of (ae)AMT-II does not appear to affect the binding or cross-linking of the oligomer to its target RNA . Oligomer (ae)AMT-II is completely resistant to hydrolysis by the 3'-5'-exonuclease activity found in fetal calf serum whereas a similar oligomer, (ae)AMT-I, which contains a 3'-terminal deoxycytidine, is hydrolyzed within 30 min when incubated at 37 degrees C . Intact (ae)AMT-II was found in both the cell lysate and cell culture medium after 12 hr of incubation with mouse L-cells along with d-(ae)AMTpT, which appears to result from endonuclease degradation of the oligomer . In contrast no intact (ae)AMT-I was found in either the cell lysate or the culture medium after 1 hr incubation . Although 10 microM (ae)AMT-II had no effect on VSV-protein synthesis in either unirradiated or UV-irradiated VSV-infected mouse L-cells, 10 microM (ae)AMT-III inhibited VSV protein synthesis 30% in irradiated cells . These results show that introduction of a N4-(4-aminobutyl)deoxycytidine at the 3'-end of an oligodeoxyribonucleotide significantly increases the resistance of the oligomer to degradation by 3'-5'-exonucleases but does not interfere with its ability to bind selectively to complementary RNA . Further derivatization with psoralen creates an oligomer that can be triggered to cross-link with RNA in a sequence-specific manner, is taken up intact by mammalian cells in culture, and exhibits biological activity . In combination, these two modifications endow the oligodeoxyribonucleotide with novel properties that could be exploited in the design of antisense or antigene reagents for use in controlling gene expression in mammalian cells.

Dev Neurosci, 1994, 16(3-4), 172 - 9
Tumor necrosis factor-alpha potentiates glutamate neurotoxicity in human fetal brain cell cultures; Chao CC et al.; Cytokines may play a pathogenetic role in the brain . Using human fetal brain cell cultures, we investigated whether cytokines released during inflammation modulate neuronal injury . Exposure of human fetal neuronal cells to the excitatory amino acid neurotransmitter, glutamate, for 6 days resulted in a dose-dependent cell loss . Tumor necrosis factor (TNF)-alpha potentiated glutamate neurotoxicity . This TNF alpha-potentiated glutamate neurotoxicity was blocked by the glutamate receptor antagonists, 2-APV and MK-801, suggesting that the potentiating effect of TNF alpha is predominantly mediated by a glutamate receptor mechanism . Exposure of neuronal cultures to TNF alpha for 5 days resulted in a 27% decrease in astrocyte glutamine synthetase and in a 50% inhibition of 3H-glutamate uptake, suggesting that the effect of TNF alpha indirectly involves glutamate metabolism . These findings suggest that under pathologic conditions, TNF alpha may impair embryonic development of the brain by exacerbating excitotoxicity.

Acta Vet Scand, 1994, 35(4), 371 - 6
Immunization of cattle against rabies using inactivated cell culture vaccines; Sihvonen L et al.; Twenty-one heads of cattle were vaccinated with Madibovin, 31 with Rabdomun and 127 with Rabisin on 4 different farms . Rabies neutralizing antibody titre (> or = 0.5 IU/ml) was detected in 80% of 163 animals tested about 1 month and in 42% of 133 animals tested about 1 year after primary vaccination . On 3 of the farms 86 animals received booster vaccination about 1 year after primary vaccination . All these animals had antibody titre (> or = 0.5 IU/ml) about 1 month after booster and antibody levels were higher than after the primary vaccination . Rabies antibody titres (> or = 0.5 IU/ml) were detected in 96% of 50 animals tested 1 year after the booster . No significant differences (p > 0.05) in antibody levels were detected between animals vaccinated with Madibovin or Rabisin (farm C) respectively with Rabisin or Rabdomun (farm D) at any collection time . Responses to rabies vaccines varied considerably between the farms . After primary vaccination of the animals on 2 farms with the same batch of Rabisin, the antibody levels clearly differed (p < 0.0001) between the farms . Our results indicate that booster is always necessary after primary vaccination to ensure that all animals are protected.

Virchows Arch, 1994, 425(4), 391 - 8
Cell culture from rat renal glomeruli; Ringstead S et al.; The primary culture of rat renal glomeruli was found to result in the ready outgrowth of two cells types . One type designated c-cells were cytokeratin positive and exhibited microvilli and cilia . The second type designated f-cells were vimentin positive and showed rugose surfaces . C-cells were polygonal in culture on plastic surfaces and were derived from cells of parietal epithelial origin . F-cells assumed a more extended form on plastic and were judged to be a sub-set of parietal epithelial cells . Neither cell type was derived from the visceral epithelium which was found to have been destroyed during isolation of the glomeruli . When cultured on isolated glomerular basement membrane both the c-cells and f-cells assumed a polygonal morphology but when grown on Matrigel the cells assumed the form of long strands interconnecting the outgrowths between the glomeruli . The appearance of the cells in the strands, judged from scanning electron microscopy, suggested that these were formed from f-cells but other cell types were entrained in the structures . Glomeruli subjected to vigorous proteinase digestion of the basement membrane allowed culture of a wider variety of cells . These included endothelial cells, judged by OX-43 antibody and anti-von Willebrand Factor staining, and mesangial cells . In cultures from glomeruli polygonal cells are often assumed to be visceral epithelial cells, the results from this study indicate that this assumption is unsound . The very different behaviour of cells grown on isolated basement membrane as compared with cells grown on Matrigel suggests that Matrigel may not faithfully mimic basement membrane with respect to cell response in culture.

Microsc Res Tech, 1993 Dec 15, 26(6), 517 - 22
A novel cell culture technique for electron microscopy; Wang F et al.; A simplified technique for the monolayer growth of cultured cells and their in situ embedment on the inner surface of the pyramidal portion of the Beem capsule for electron microscopy has been developed . The results demonstrated that the cell monolayers grew well on the surface of the Beem capsule and could be embedded in situ . Electron micrographs showed cells in their natural state of contact with one another . The plasma membrane and intracellular organelles were well preserved . This method minimizes many difficult steps and eliminates the disruption of cells by scraping, pelleting, or enzymatic reaction to remove them.

Clin Mater, 1994, 15(3), 173 - 90
Cell culture methods for testing biocompatibility; Pizzoferrato A et al.; Cell culture systems may be of value in testing the biocompatibility of prosthetic materials before they are introduced into clinical use . In recent years, in vitro methods for assaying biomaterials have gained in importance owing to the growing concern over the use of animals for biomaterials testing . Significant effort is therefore being focused toward developing predictive and quantitative, but also simple and reliable, methods of testing using cultured cells . At present, a number of methods for measuring both the cytotoxicity and the specific cytocompatibility of different materials are available . The usefulness of these systems is no longer confined to screening new materials; they can be used to study the mechanisms of action of various materials during tissue/material interaction . This paper reviews the published literature on the use of cell culture models in evaluating biocompatibility and reports on the personal experience of the authors, who have been using cell culture systems for many years and for different purposes.

Mol Cell Biochem, 1993 Dec 8, 129(1), 93 - 8
Induction of heme oxygenase in intestinal epithelial cells: studies in Caco-2 cell cultures; Cable JW et al.; Enterally administered, heme is a good source of iron in humans and other animals, but the metabolism of heme by enterocytes has not been fully characterized . Caco-2 cells in culture provide a useful model for studying cells that resemble small intestinal epithelium, both morphologically and functionally . In this paper we show that heme oxygenase, the rate-controlling enzyme of heme catabolism, is present in abundance in Caco-2 cells, and that levels of its mRNA and activity can be increased by exposure of the cells to heme or metal ions (cadmium, cobalt) . Caco-2 cells also contain biliverdin reductase activity which, in the basal state, is similar to that of heme oxygenase (approximately 40 pmole of product per mg protein per minute); however, when heme oxygenase is induced, biliverdin reductase may become rate-limiting for bilirubin production.

J Clin Microbiol, 1993 Dec, 31(12), 3204 - 10
Evaluation of Clearview and Magic Lite tests, polymerase chain reaction, and cell culture for detection of Chlamydia trachomatis in urogenital specimens; Kluytmans JA et al.; The Clearview Chlamydia test (CV; Unipath Ltd., Bedford, United Kingdom), the Magic Lite Chlamydia test (ML; CIBA Corning, Medfield, Mass.), a polymerase chain reaction (PCR), and cell culture (CC) were evaluated for detection of Chlamydia trachomatis in urogenital specimens . Specimens were collected from 283 men and 724 women visiting the outpatient clinic for Sexually Transmitted Diseases at the University Hospital Rotterdam, Rotterdam, The Netherlands . ML, PCR, and CC were all performed on the same sample to prevent swab-to-swab variability . CV was performed on a separate sample . Analysis of discordant results was performed by application of the following confirmatory assays: first, PCR on the CC, second, ML was repeated, and third, PCR was repeated by using a different DNA extraction protocol . If more than one test was positive, the sample was considered true positive . If only one test was positive, which was confirmed by the confirmatory assay, the sample was also considered true positive . By using these interpretations, the following results were obtained . The sensitivity and specificity of CV for samples from men were 60.4 and 86.3%, respectively . For samples from women, these values were 62.3 and 99.7%, respectively . The low specificity for samples from men was caused by unidentified substances in the swab that was used . The use of CV on samples from men is not recommended by the manufacturer . For samples from women, the specificity of CV was high, but the low sensitivity of CV limits its use for diagnostic purposes . The sensitivities of ML were low for samples from both men and women (68.8% and 50.9% respectively), while specificities were excellent for samples from both groups (100 and 99.9%, respectively) . The low sensitivity of ML limits its diagnostic value . The PCR technique was highly specific for samples from both men (99.6%) and women (99.9%) . The sensitivity of PCR, however, was unexpectedly low for samples from both groups (men, 87.5%; women, 79.2%), most likely because of the sample treatment method used . The sensitivity and specificity values of CC for samples from men were 95.8 and 100%, respectively . For samples from women, these values were 100 and 99.9%, respectively . In the present study, CC was the most reliable technique for the detection of C . trachomatis.

Pharm Res, 1993 Dec, 10(12), 1710 - 4
A correlation between the permeability characteristics of a series of peptides using an in vitro cell culture model (Caco-2) and those using an in situ perfused rat ileum model of the intestinal mucosa; Kim DC et al.; In an attempt to establish an in vitro/in situ correlation of intestinal permeability data, the permeability coefficients (Papp) for a series of model peptides, which were determined using an in situ perfused rat ileum model, were compared to the permeability coefficients (Pmono) determined using an in vitro cell culture model (Caco-2) . The model peptides, which were all blocked on the N-terminal (acetyl, Ac) and the C-terminal (amide, NH2) ends, consisted of D-phenylalanine (F) residues (e.g., AcFNH2, AcFFNH2, AcFFFNH2) . To alter the degree of hydrogen bonding potential, the nitrogens of the amide bonds were sequentially methylated {e.g., AcFF(Me)FNH2, AcF(Me)F(Me)FNH2, Ac(Me)F(Me)FNH2, Ac(Me)F(Me)F(Me)} . These peptides were shown not to be metabolized in the in situ perfused rat ileum system . The results of the transport experiments showed that there were poor correlations between the apparent permeability coefficients (Papp) determined in an in situ perfused rat ileum model and the octanol-water partition coefficients (r = 0.60) or the hydrogen bonding numbers (r = 0.63) of these peptides . However, good correlations were observed between the in situ Papp values for these peptides and their partition coefficients in heptane-ethylene glycol (r = 0.96) and the differences in their partition coefficients between octanol-water and isooctane-water (r = 0.86) . These results suggest that lipophilicity may not be the major factor in determining the intestinal permeability of these peptides and that hydrogen bonding potential may be a major contributing factor . These results suggest that lipophilicity may not be the major factor in determining the intestinal permeability of these peptides and that hydrogen bonding potential may be a major contributing factor.(ABSTRACT TRUNCATED AT 250 WORDS)

J Lipid Res, 1993 Dec, 34(12), 2077 - 90
Fatty acid metabolism studies of human epidermal cell cultures; Marcelo CL et al.; Adult human epidermal keratinocytes grow rapidly in medium that is essential fatty acid (EFA)-deficient . In this medium they exhibit decreased amounts of the fatty acids, 18:2, 20:3, 20:4, and contain increased amounts of monounsaturated fatty acids . {14C}- and {3H}acetate and radiolabeled fatty acids, 16:0, 18:2, and 20:4 were used to study the fatty acid metabolism of these cells . Label from acetate appeared in 14- to 20-carbon fatty acids, both saturated and monounsaturated . No label was seen in the essential fatty acid 18:2, 18:3, and 20:4 . Radiolabel from {9, 10-3H}palmitic acid (16:0) was detected in 16:0, 16:1, 18:0, and 18:1 . {14C}linoleic acid (18:2) was converted to 18:3, 20:2, 20:3, and 20:4, demonstrating delta 6 and delta 5 desaturase activity in keratinocytes . Label from acetate, 16:0, or 18:2 was found mostly in the cellular phospholipids while only one third of the label from {14C}arachidonic was found in the phospholipids . {14C}acetate and {14C}18:2 time course data were used to construct a model of the metabolism of these reactants, using coupled, first-order differential equations . The data show that EFA-deficient keratinocytes metabolize fatty acids using pathways previously found in liver; they suggest the positioning of 18:2 desaturase and 18:3 elongase near the plasma membrane; they indicate that for the synthesis of nonessential fatty acids the formation of 18:0 from 16:0 is the rate-determining step; and they show that the conversion of 18:2 to 20:3 is rapid . These experiments demonstrate a method to study lipid enzyme kinetics in living cells.

Biotechniques, 1993 Dec, 15(6), 1106 - 9
Enhanced proliferation of peripheral blood mononuclear cells and tumor-infiltrating lymphocytes in a plasma-derived cell culture fluid; Torres AR et al.; The proliferation and activation of murine and human T-lymphocytes in a high-protein lymphocyte culture fluid (LCF) is compared to that of cells cultured in 10% fetal bovine serum (FBS) . Tumor-infiltrating lymphocytes (TIL) proliferate exponentially in the LCF for up to 46 days and generate cell numbers that are nearly 100,000-fold greater than cells cultured in FBS . This rapid growth of T cells in LCF could have an important impact in adoptive immunotherapy and gene therapy since cell growth is a limiting factor in these technologies.

Biol Reprod, 1993 Dec, 49(6), 1353 - 61
Expression of E-cadherin in immature rat and mouse testis and in rat Sertoli cell cultures; Wu JC et al.; Cadherins are Ca(2+)-dependent cell adhesion molecules that play essential roles in organogenesis . Northern blot analysis was used to determine the levels of epithelial (E)-, neural (N)-, and placental (P)-cadherin expression in developing rat testes from 1-, 2-, 3-, and 4-wk-old rats . Highest expression of all three cadherins occurred during the first two postnatal weeks, prior to the establishment of the blood-testis barrier . Transcripts of all three cadherins were also detected in RNA isolated from Sertoli cells cultured from 3-wk-old rat testes . Immunoblot analysis demonstrated levels of E- and P-cadherin protein in the testis and Sertoli cells that corresponded to the levels of their respective RNA transcripts . Immunohistochemical reactivity of E-cadherin in 8-day-old mouse testis was associated with germ cells situated basally in the seminiferous tubules . This report establishes the expression of E-cadherin in the testis and the simultaneous expression of E-, N-, and P-cadherin in both testis and Sertoli cell cultures.

Cell Biochem Funct, 1993 Dec, 11(4), 257 - 61
PTH induces modification of transductive events in otosclerotic bone cell cultures; Fano G et al.; We studied the effect of PTH (10-100 nM) on transductive mechanisms (adenylate cyclase activity, Ca2+ metabolism, IP3 levels) in cell cultures derived from normal and otosclerotic human bone fragments . The cultured cells were osteoblast-like but with calcitonin-receptors still present and with PTH receptors coupled with the adenylate cyclase system . The results showed that PTH activated adenylate cyclase and increased the intracellular Ca2+ levels with qualitative and quantitative differences between the two cellular populations . In particular, otosclerotic cells responded less to hormone stimulation, which is in accord with the current hypothesis of a desensitization of the receptor/enzyme complex associated with the pathological status.

Prenat Diagn, 1993 Dec, 13(12), 1101 - 10
Filtration and recirculation of early amniotic fluid . Evaluation of cell cultures from 100 diagnostic cases; Sundberg K et al.; Due to the low cell concentration, cultures from early amniotic fluid specimens usually require 2-3 weeks in culture prior to karyotyping . The purpose of this study was to evaluate the culture quality of amniotic fluid cells from early pregnancy, obtained by a new filter technique . The hypothetical advantage of the technique was that the increased cell yield might reduce the culture time before karyotyping . Culture quality was assessed by the number of colonies, the percentage of colonies containing mitoses in filter and control cultures, and the culture time . The setting was a consecutive clinical trial . One hundred samples were obtained from ongoing pregnancies at 11-14 weeks of gestation (mean 12.8 weeks) . By circulating a mean of 26 ml of amniotic fluid through a cell filter system leading the cell-free fluid back to the amniotic cavity, the cell yield was increased in the sample of 7 ml corresponding to the dead space of the filter system . The culture results were compared with control cultures from 5 ml samples drawn from the same pregnancies prior to recirculation . The cultures from the first flushing of the filter system yielded 2.6 times more colonies and in total 4.2 times more colonies were found in the three cultures grown from each filter sample when compared with the control cultures . Moreover, the filter cultures showed significantly more colonies with mitoses . The mean culture time was 8.0 days for the filter cultures, from which the karyotypes were analysed . The controls would have needed more time in culture to fulfil the diagnostic criteria for karyotyping . One case of 47,XY,+21 was found; the rest had normal karyotypes . We conclude that the filter technique improves the culture quality of early amniotic fluid samples and allows early arrest of the cultures.

In Vitro Cell Dev Biol Anim, 1993 Dec, 29A(12), 943 - 9
Rat Sertoli cell aromatase cytochrome P450: regulation by cell culture conditions and relationship to the state of cell differentiation; Papadopoulos V et al.; Primary cultures of immature rat Sertoli cells in plastic dishes are highly responsive to follicle stimulating hormone (FSH) and its second messenger, cAMP, in metabolizing testosterone to estradiol, thus indicating the presence of an active, hormone-regulated aromatase cytochrome P450 (P450arom) . However, in vivo studies indicated that P450arom is FSH-responsive only in very young animals, where the cells have not yet differentiated, but they lose this ability later on in development . Sertoli cells grown on Matrigel (a reconstituted basement membrane), laminin (a basement membrane component), or in bicameral chambers coated with Matrigel, assume structural and functional characteristics more similar to that of in vivo differentiated Sertoli cells . When the cells were cultured on laminin or Matrigel, the FSH- and cAMP-induced estradiol production was greatly reduced by 30 and 60%, respectively . When Sertoli cells were cultured in bicameral chambers coated with Matrigel, no induction of testosterone aromatization by FSH or cAMP was observed . However, FSH-induced cAMP formation was greater when the cells were cultured on basement membrane or in the chambers than on plastic dishes . These results suggest that culture conditions favoring the assumption by Sertoli cells of a phenotype closer that of the differentiated cells in vivo (tall columnar and highly polarized) suppress the induction of P450arom by FSH and cAMP . We then examined the mechanism(s) by which cell phenotype affects p450arom activity . Northern blot analyses of Sertoli cell RNA revealed one major band of 1.9 Kb and two minor bands of 3.3 and 5.2 Kb . However, there were no changes at the level of the expression of P450arom messenger RNA under the different culture conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Int J Dev Neurosci, 1993 Dec, 11(6), 721 - 9
Neuropeptides in dissociated cell cultures of mammalian spinal cord and dorsal root ganglion; Matthew E; Immunohistochemical studies of leucine-enkephalin, somatostatin, vasoactive intestinal polypeptide and neurotensin were carried out in dissociated cell co-cultures of embryonic mouse spinal cord and dorsal root ganglion, using the peroxidase-antiperoxidase technique . Leucine-enkephalin immunoreactivity exceeded that of the other peptides in these coculture preparations . Leucine-enkephalin, substance P and somatostatin were also studied in spinal cord cultures (without dorsal root ganglia) and in dorsal root ganglia cultures (without spinal cord) . Each of these peptides was present in only a small percentage (< 10%) of perikarya and processes in spinal cord cultures . No leucine-enkephalin immunoreactivity was seen in dorsal root ganglion cultures; a considerable proportion of the processes were immunoreactive for substance P or somatostatin . These observations suggest that co-cultures of spinal cord and dorsal root ganglia can provide a simplified in vitro "model" of the nervous system for the study of peptidergic interactions.

Naunyn Schmiedebergs Arch Pharmacol, 1993 Dec, 348(6), 582 - 5
COMT inhibitors and metabolism of fluorodopa enantiomers in aggregating cell cultures; Wiese C et al.; Organotypic primary cell cultures of fetal rat brain were used as a model system to study the effect of COMT inhibitors on the cerebral metabolic conversions of fluoro-DOPA enantiomers . The selective COMT inhibitors OR 486 and CGP 28014 were used in conjunction with 5F-L-DOPA, 6F-L-DOPA and 6F-D-DOPA as substrates . Methylation can be clearly reduced by application of OR 486 at nanomolar level, without inhibition of AADC and MAO . The uptake of the substrate is unchanged . CGP 28014, already known to be active only in vivo, has no influence on the metabolic conversion rates of the fluoro-DOPA isomers . These results show that use of this culture system allows statement concerning the in vitro activity of COMT inhibitors . It has not been possible to show an increase of absolute levels of decarboxylation products due to inhibition of COMT, however, but the reduction in levels of methylated product itself may have significance for PET studies of the human brain.

Endocr J, 1993 Dec, 40(6), 699 - 704
Effect of calyculin-A, phosphatase inhibitor, on 20 alpha-hydroxysteroid dehydrogenase activity in rat luteal cell culture; Yamanouchi K et al.; The effects of calyculin-A (CL-A), a phosphatase inhibitor, on 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity were studied in rat luteal cell cultures to examine whether protein phosphatases were involved in the regulation of the enzyme activity . Luteal cells were harvested from the rats on day 6 of pseudopregnancy . In the absence of prolactin (PRL), the stimulatory effect of CL-A (10(-9) M) on 20 alpha-HSD activity was observed during the first 3 h period of the culture, but was not discernible thereafter . Since the enzyme activity of luteal cells during pseudopregnancy is known to be suppressed by PRL, the results suggest that this effect of CL-A could be manifested only as long as the effect of endogenous PRL was remained . On the other hand, CL-A enhanced the 20 alpha-HSD activity of the cells cultured for 24 h, where a spontaneous increase in 20 alpha-HSD activity was blocked by exogenously supplementing the culture medium with PRL . These results suggest that a CL-A sensitive phosphatase(s) may be involved in the action of PRL in suppressing the increase in 20 alpha-HSD activity in rat luteal cells.

Neuroscience, 1993 Dec, 57(4), 1147 - 57
Neurotransmitter properties of guinea-pig sympathetic neurons grown in dissociated cell culture--II . Fetal and embryonic neurons: regulation of neuropeptide Y expression; Matsumoto SG et al.; We report here the neurotransmitter characteristics of neurons cultured from the same ganglia of fetal and embryonic guinea-pigs . Both the celiac ganglion and the superior mesenteric ganglion were examined . In a previous paper we described the neurotransmitter properties of adult guinea-pig prevertebral sympathetic neurons grown in dissociated cell culture, including the expression by these cells of immunoreactivity for tyrosine hydroxylase, neuropeptide Y and somatostatin . Tyrosine hydroxylase immunoreactivity was ubiquitously expressed in all fetal embryonic cultures, as was the case for adult neurons . Fetal-derived celiac and superior mesenteric gangli neurons displayed neuropeptide Y and somatostatin immunoreactivity in the same percentage of neurons as in adult cultures but at markedly lower levels . Embryonic neurons also expressed somatostatin immunoreactivity in roughly the same proportion of neurons as in adult and fetal cultures; however, the expression of neuropeptide Y immunoreactivity in both celiac and superior mesenteric gangli cultures was significantly different . Specifically, neuropeptide Y immunoreactivity in embryonic celiac cultures was greatly reduced in both the number of positive-labeled neurons and the amount of immunoreactive product, while neuropeptide Y immunoreactivity in embryonic superior mesenteric gangli cultures was markedly increased compared to their adult and fetal counterparts . The expression of neuropeptide Y immunoreactivity in celiac neurons was found to be specifically elevated by culturing the neurons in medium conditioned by disassociated vascular cells, this treatment having no effect on tyrosine hydroxylase or somatostatin immunoreactivity . Heart cell-conditioned medium did not effect neuropeptide Y or somatostatin immunoreactivity, although it did result in a significant reduction of tyrosine hydroxylase immunoreactivity and an increase in 5-hydroxytryptamine immunoreactivity . We conclude that the expression of neuropeptide Y immunoreactivity develops independently in cultures of adult and near-term fetuses but that embryonic neurons require interactions with target cells to express this phenotype . Neuropeptide Y immunoreactivity can be induced in embryonic sympathetic neurons by a target-derived factor(s).

Neuroscience, 1993 Dec, 57(4), 1135 - 45
Neurotransmitter properties of guinea-pig sympathetic neurons grown in dissociated cell culture--I . Adult neurons; Matsumoto SG et al.; The autonomic nervous system of mammals displays extensive neurotransmitter diversity . The guinea-pig sympathetic nervous system has served as a model for in vivo studies of neurotransmitter co-expression . We have developed methods for the dissociation and long-term culture of adult guinea-pig prevertebral sympathetic ganglia . The neurotransmitter properties of cultured adult guinea-pig sympathetic neurons from the celiac and superior mesenteric ganglia were examined . Cultured principal neurons were found to display many of their in vivo neurotransmitter characteristics, including catecholamine-specific histofluorescence and immunoreactivity for tyrosine hydroxylase and the neuropeptides, neuropeptide Y, somatostatin and vasoactive intestinal polypeptide . In addition, the cultures of both ganglia displayed the various neurotransmitter characteristics in approximately the same percentage of the cultured neurons as reported in in vivo studies . A small percentage of principal neurons and many small, intensely fluorescent-like cells labeled with antibodies against 5-hydroxytryptamine . Many of the principal neurons were found to bear 5-hydroxytryptamine3 receptors, suggesting a possible role for this neurotransmitter in neuron-neuron and small, intensely fluorescent cell-neuron transmission . We conclude that adult guinea-pig sympathetic neurons retain their neurotransmitter phenotypes when grown in dissociated cell culture . These properties include the co-expression of the classical transmitters, norepinephrine, 5-hydroxytryptamine and neuropeptides . This culture preparation will prove to be valuable in future studies on the functional properties of these neurons and their development.

J Interferon Res, 1993 Dec, 13(6), 387 - 95
Does the gender difference in interferon production seen in picornavirus-infected spleen cell cultures from ICR Swiss mice have any in vivo significance?
Curiel RE, Miller MH, Ishikawa R, Thomas DC, Bigley NJ.
Splenocyte cultures from female ICR Swiss mice produced greater interferon (IFN) levels, particularly IFN-gamma, than did cultures from males by 12 h post-infection (pi) with the D variant of encephalomyocarditis virus (EMCV-D) . This early IFN-gamma is produced by natural killer (NK)-like cells and is dependent on plastic adherent cells and IFN-alpha/beta . In this study, we evaluated the significance of this observation on the innate resistance of ICR Swiss females to EMCV-D-mediated disease . Treatment of females with rabbit anti-mouse IFN-alpha/beta serum rendered them susceptible to the diabetogenicity of EMCV-D . Although sera from both sexes of ICR Swiss mice exhibited peak IFN levels day 3 pi, IFN-gamma was present in the sera of males at only 1 day pi and in the sera of females at days 1-3 pi . Females cleared virus from the circulation by day 2 pi, 1 day earlier than did males . Flow cytometric evaluations of lymphoid cell phenotypes in spleens and pancreata of infected mice revealed that percentages of L3T4+ cells were significantly decreased only in spleens from males at day 1 pi and were diminished along with Ly2+ cells in pancreata of males at 7 days pi, suggesting that T-cell responses were impaired in virus-infected males.

J Immunol Methods, 1993 Nov 5, 166(1), 85 - 91
The concentrations of glutamine and ammonia in commercially available cell culture media; Heeneman S et al.; The amino acid glutamine is an essential nutrient for cells in culture . In aqueous solutions such as liquid culture media, glutamine spontaneously decomposes into ammonia . In this study, we examined the toxicity of ammonia for two different cell lines . In mouse hybridoma cell cultures, viable cell counts were reduced at exogenous ammonia concentrations of 1000 microM . In the human promyelocytic cell line however, viable cell counts were shown to be reduced at exogenous ammonia concentrations of 300 microM . Next, we determined ammonia and glutamine levels in 11 commercially available media on the day of delivery . It was found that all media contained significantly less glutamine than prescribed . Ammonia was found in all media with concentrations ranging up to 1000 microM . Storage at both 4 degrees C and 20 degrees C caused a further degradation of glutamine and significant accumulation of ammonia in all media . The degradation curves of the various media were used to calculate the first order degradation constant k, which can be used to determine the kinetics of the spontaneous decomposition in culture media . These results suggest that precautions must be taken to avoid the deterioration of commercially available culture media, because of the decay of glutamine . Long storage times lead to a rapid decay of glutamine and an accumulation of the toxic degradation product ammonia.

Endocrinology, 1993 Nov, 133(5), 2379 - 90
A primary cell culture system of luteinizing hormone releasing hormone neurons derived from embryonic olfactory placode in the rhesus monkey; Terasawa E et al.; The purpose of this study is to establish a primary LHRH cell culture system using embryonic olfactory placode and to examine whether LHRH cells derived from olfactory placode and the migratory pathway of LHRH neurons mature in vitro . Six monkey fetuses at the ages of E34-E36 were delivered surgically and the area including the olfactory placode (PL) and the areas that encompass the migratory pathway (MP) were dissected out . The tissues were cut into small pieces and plated on collagen- or poly-L-lysine-coated glass coverslips in medium M199 . Cultures were maintained for up to 33 days and immunostained for LHRH, GnRH-associated peptide, neurofilament protein, neuron-specific enolase, and glial fibrillary acidic protein . LHRH positive cells were also positive for neurofilament proteins neuron-specific enolase, and GnRH-associated peptides, but negative for glial fibrillary acidic protein . In the first week of culture, LHRH cells remained within the explants of PL, were rounded (average dimensions: 13.0 x 11.3 microns) and stained lightly . By the second week a number of LHRH cells (15.7 x 13.6 microns) with neurites started to migrate out from PL explants, whereas some still remained in the PL . By the third week a large number of LHRH cells (19.3 x 9.4 microns) had migrated out from the PL . They were fusiform in shape with clear nuclei and extended long varicose neurites up to 500 microns in length . A few "pioneer" LHRH cells appeared to lead the migration of 100-400 LHRH cells forming 1-3 major migratory paths . In contrast, LHRH cells from MP explants migrated out sooner than those from PL explants . LHRH cells from the ventral part of the MP, which is close to the PL, migrated out by 1-2 weeks and formed several migratory paths, whereas LHRH cells from the dorsal part of the MP, which is farther from the PL, were scattered widely around explants and their neurites were extended tortuously . Cultured LHRH cells released LHRH into the media and responded to challenge with high K+ . The results indicate that 1) primary LHRH neurons can be obtained from the embryonic PL and their migratory pathway, 2) these neurons migrate and mature in culture and 3) they are accessible for cellular and molecular studies.

J Dermatol, 1993 Nov, 20(11), 684 - 90
Dispersed cell culture of human sweat duct cells under serum-free conditions; Uchida N et al.; Human eccrine gland duct cells were successfully cultured using a serum-free medium, K-GM medium . Eccrine sweat ducts were isolated from dispase treated skin specimens from palms or soles . After treatment of the isolated ducts with trypsin and EDTA, dispersed cells were cultured in K-GM medium . In primary cultures, small colonies were seen 3 to 4 days after inoculation . Then the cells rapidly proliferated and formed large colonies with a paving stone-like cell arrangement . During the culture, small dome shaped areas were sometimes formed in the centers of colonies . Cultures multiplied for a maximum of 7 passages . The plating efficiencies of the 1st to 6th passage cells were about 20% to 30% . Immunocytochemically, cultured cells were positively stained with anti-carcinoembryonic antigens, K8.37 and K8.13, but not with anti-S100 protein, anti-HLA-DR, 34 beta B4, or PKK3 . An electron micrograph of the cultured cells showed a multilayer of flattened cells linked by desmosomes . These results indicate that the cultured cells possessed the staining properties compatible with those of the ductal portion of eccrine sweat glands . No contamination by other mesenchymal cells, such as fibroblasts, was seen during the culture.

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