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,