Cell cultures

Biochem Biophys Res Commun, 1999 Dec 9, 266(1), 15 - 8
TSH control of PKA catalytic subunit activity in thyroid cell cultures; Ben Abdelkhalek M et al.; The protein expression and the enzyme activity of the catalytic subunit (C) of the cAMP-dependent protein kinases were studied in porcine thyroid cell primary cultures stimulated with two doses of TSH (0.1 mU/ml and 1 mU/ml) for 1 to 3 days . In TSH-stimulated cells the desensitization of the catalytic subunit activity was accompanied by a simultaneous and parallel decrease of its immunoreactivity . The loss of catalytic subunit was rapid and reached its maximum after 1 day of culture . It is similar in the two subcellular compartments: cytosol and particulate extracts . Contrary to the observed loss of the C subunit protein molecules in TSH-stimulated cells, the expression of the Cbeta subunit mRNA in these cells was increased fivefold compared to controls, while no significant change was observed on the Calpha subunit mRNA . These results suggest that TSH controls the Cbeta subunits of PKA at two levels: at the transcriptional level it increases Cbeta mRNA expression, and at the translational or posttranslational level TSH decreases the amount and the activity of the Cbeta protein molecules .

Biotechnol Bioeng, 1999, 66(4), 231 - 7
Development of a bioartificial pancreas: II . Effects of oxygen on long-term entrapped betaTC3 cell cultures; Papas KK et al.; Tissue-engineered pancreatic constructs based on immunoisolated, insulin-secreting cells are promising in providing an effective, relatively inexpensive, long-term treatment for type I (insulin-dependent) diabetes . An in vitro characterization of construct function under conditions mimicking the in vivo environment is essential prior to any extensive animal experimentation . Encapsulated cells may experience hypoxic conditions postimplantation as a result of one or more of the following: the design of the construct; the environment at the implantation site; or the development of fibrosis around the construct . In this work, we studied the effects of 3- and 4-day-long hypoxic episodes on the metabolic and secretory activities and on the levels of intracellular metabolites detectable by phosphorus-31 nuclear magnetic resonance ((31)P NMR) of alginate/poly-L-lysine/alginate entrapped betaTC3 mouse insulinomas continuously perfused with culture medium . Results show that, upon decreasing the oxygen concentration in the surrounding medium, the encapsulated cell system reached a new, lower metabolic and secretory state . Hypoxia drove the cells to a more anaerobic glycolytic metabolism, increased the rates of glucose consumption (GCR) and lactate production (LPR), and reduced the rates of oxygen consumption (OCR) and insulin secretion (ISR) . Furthermore, hypoxia reduced the levels of intracellular nucleotide triphosphates (NTP) and phosphorylcholine (PC) and caused a rapid transient increase in inorganic phosphate (P(i)) . Upon restoration of the oxygen concentration in the perfusion medium, all parameters returned to their prehypoxic levels within 2 to 3 days following either gradual unidirectional changes (ISR, NTP, PC) or more complicated dynamic patterns (OCR, GCR, LPR) . A further increase in oxygen concentration in the perfusion medium drove OCR, ISR, NTP, PC, and P(i) to new, higher levels . It is concluded that (31)P NMR spectroscopy can be used for the prolonged noninvasive monitoring of the bioenergetic changes of encapsulated betaTC3 cells occurring with changes in oxygen tension . The data also indicate that the oxygen-dependent states might be related to the total number of viable, metabolically active cells supported by the particular oxygen level to which the system is exposed . These findings have significant implications in developing and non-invasively monitoring a tissue-engineered bioartificial pancreas based on transformed beta cells, as well as in understanding the biochemical events pertaining to insulin secretion from betaTC3 insulinomas .

Biotechnol Bioeng, 1999, 66(4), 219 - 30
Development of a bioartificial pancreas: I . long-term propagation and basal and induced secretion from entrapped betaTC3 cell cultures; Papas KK et al.; Bioartificial pancreatic constructs based on immunoisolated, insulin-secreting cells have the potential for providing effective, long-term treatment of type I (insulin-dependent) diabetes . Use of insulinoma cells, which can be amplified in culture, relaxes the tissue availability limitation that exists with normal pancreatic islet transplantations . We have adopted mouse insulinoma betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate (APA) beads as our model system for a bioartificial pancreas, and we have characterized the effects of long-term propagation and of glucose concentration step changes on the bioenergetic status and on the metabolic and secretory activities of the entrapped cells . Cell bioenergetics were evaluated nonivasively by phosphorus-31 nuclear magnetic resonance ((31)P NMR) spectroscopy, and metabolic and secretory parameters by assaying cell culture medium . Data indicate that net cell growth occurred between days 3 and 10 of the experiment, resulting in an approximate doubling of the overall metabolic and secretory rates and of the intracellular metabolite levels . Concurrently, a reorganization of cell distribution within the beads was observed . Following this growth period, the measured metabolic and secretory parameters remained constant with time . During glucose step changes in the perfusion medium from a high concentration of 12 to 15 mM to 0 mM for 4.5 h to the same high glucose concentration, the oxygen consumption rate was not affected, whereas insulin secretion was always glucose-responsive . Intracellular nucleotide triphosphates did not change during 0 mM glucose episodes performed early in culture history, but they declined by 20% during episodes performed later in the experiment . It is concluded that the system of APA-entrapped betaTC3 cells exhibits several of the desirable characteristics of a bioartificial pancreas device, and that a correlation between ATP and the rate of insulin secretion from betaTC3 cells exists for only a domain of culture conditions . These findings have significant implications in tissue engineering a long-term functional bioartificial endocrine pancreas, in developing noninvasive methods for assessing construct function postimplantation, and in the biochemical processes associated with insulin secretion .

Biotechnol Bioeng, 1999, 66(4), 238 - 46
Improvement of the primary metabolism of cell cultures by introducing a new cytoplasmic pyruvate carboxylase reaction; Irani N et al.; Continuous mammalian cell lines are important hosts for the production of biological pharmaceuticals . However, these cell lines show some severe disorders in primary metabolism, which they have in common with many cancer cells . This leads to a high throughput of substrates giving a low energy yield and ample toxic side products such as lactate and ammonia . Because the enzymatic connection between glycolysis and the tricarboxylic acid cycle (TCA) is very poor, glucose is mainly degraded via oxidative glycolysis . It will be shown that introducing a pyruvate carboxylase gene expressed in the cytoplasma into a continuous BHK-21 cell line, and thus reconstituting the missing link between glycolysis and TCA, can reduce this problem . Thus, glucose consumption could be reduced by a factor of four and glutamine utilization up to a factor of two, compared with control . Moreover, a 1.4-fold-higher adenosine triphosphate (ATP) content was achieved . The flux of labeled {(14)C}-glucose into the TCA is shown to be enhanced, indicating a higher rate of oxidative glucose degradation . Host cell lines with an improved energy metabolism will therefore result in better exploitation of substrates, an increasing yield by the more efficient use of carbon source, and higher product integrity combined with lower production costs .

J Leukoc Biol, 1999 Nov, 66(5), 822 - 8
Modulatory effects of human herpes virus-7 on cytokine synthesis and cell proliferation in human peripheral blood mononuclear cell cultures; Atedzoe BN et al.; Human herpes virus-7 (HHV-7) infects cells of the immune system and thus may modulate their function . To investigate the potential immunomodulatory effects of this virus, we performed an in vitro study in which we investigated effects of HHV-7 on the synthesis of several key immunomodulatory cytokines, i.e . tumor necrosis factor alpha (TNF-alpha), interleukin-2 (IL-2), interferon-gamma (IFN-gamma), IL-4, IL-6, and transforming growth factor beta (TGF-beta) . This was examined after in vitro infection of human peripheral blood mononuclear cells (PBMC) with HHV-7 . We found elevated levels of TNF-alpha, TGF-beta, and IFN-gamma in the supernatants of HHV-7-infected cells . By reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, using cytokine-specific primers, we found that levels of TNF-alpha, TGF-beta, and IFN-gamma mRNA were increased in the infected cells . The HHV-7 infection also significantly (P < 0.05) decreased the production of IL-2 from activated, IL-2-producing PBMC . Furthermore, mitogen- and cytokine-induced cellular proliferative responses of human PBMC were found to be significantly (P < 0.05) down-regulated by this virus . On the other hand, HHV-7 did not affect IL-4 and IL-6 synthesis . Overall, our results indicate that HHV-7 infection causes significant immunomodulatory effects with regard to cytokine synthesis in these cells as well as inhibiting lymphocyte proliferation by various stimuli.

Plant J, 1999 Oct, 20(1), 109 - 17
Millisecond UV-B irradiation evokes prolonged elevation of cytosolic-free Ca2+ and stimulates gene expression in transgenic parsley cell cultures
Frohnmeyer H, Loyall L, Blatt MR, Grabov A.
Chalcone synthase (CHS) is a key enzyme leading to the generation of protective flavonoids in plants under environmental stress . Expression of the CHS gene is strongly upregulated by exposures to UV light, a response also observed in heterotrophic parsley cell cultures . Although there are hints that the stimulus for CHS expression may be coupled to UV-B irradiation through a rise in cytosolic-free Ca2+ ({Ca2+}i), the temporal relationship of these events has never been investigated critically . To explore this question, we have used a CHS promoter/luciferase (CHS/LUC) reporter gene fusion and recorded its expression and {Ca2+}i elevation in a transgenic parsley cell culture following millisecond light pulses . Luciferase expression was enhanced maximally seven- (+/- 2) fold by 30 10 ms flashes of UV-B light . The response was specific to wavelengths of 300-330 nm and could be inhibited in the presence of the Ca2+ channel blocker nifedipine . In parallel measurements, using Fura-2 fluorescence ratio microphotometry, we found that 10 ms UV-B flashes also evoked a gradual and prolonged rise of {Ca2+}i in the parsley cells which was irreversible within the timescale of these experiments, but could be prevented by prior treatment with nifedipine . These, and additional results, indicate a remarkably high temporal sensitivity to, and specificity for, UV-B light in CHS gene expression independent of UV-mediated DNA damage by thymine dimerization . The ability of transient UV-B stimulation to evoke prolonged elevations of {Ca2+}i suggests a functional coupling between the initial light stimulus and subsequent gene expression that takes place many tens of minutes later.

Acta Physiol Scand, 1999 Oct, 167(2), 161 - 6
Visualization of nitric oxide formation in cell cultures and living tissue; Wiklund NP et al.; We have visualized nitric oxide (NO) released from cell cultures and living tissue . NO was visualized by a reaction with luminol and hydrogen peroxide to yield photons which were counted using a microscope coupled to a photon counting camera . Murine macrophages were activated with interferon-gamma (IFN-gamma) and endotoxin (LPS) . Cultured endothelial cells were stimulated with bradykinin, and neurones in the guinea-pig myenteric plexus and the rabbit hypogastric nerve trunk were electrically stimulated . There was a marked increase in photons emitted from the cultured cells as well as from the living tissues during stimulation . The stimulation-induced photon emission was markedly reduced by inhibition of nitric oxide synthase (NOS); removal of L-arginine from the medium also decreased photon counts . The present method allowed integration times in the order of minutes to improve signal-to-noise ratio . However, the high sensitivity of this method also makes it possible to generate an image in seconds, allowing the production of real time films . Photon emission was enhanced under conditions known to increase NO production, and diminished in the presence of NO inhibitors . Thus, this method has demonstrated specificity for the L-arginine:NO pathway from a wide range of biological sources such as cultured cells and living tissues, and has the potential for real time imaging of NO formation, with high temporal and spatial resolution.

Int J Tissue React, 1999, 21(2), 43 - 9
Plasma cytokine concentration and the cytokine producing ability of whole blood cell cultures from healthy females with pharmacologically induced hyperprolactinemia; Rovensky J et al.; We investigated the in vitro effect of domperidone-induced hyperprolactinemia on plasma cytokine concentration and blood leukocyte cytokine production in healthy female volunteers . No changes were found in the plasma concentration of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-4, IL-10, IL-6 and IL-13 during hyperprolactinemia when compared with control values . Using unseparated blood leukocytes, we found that the spontaneous production of IL-6 (4-8 h) and transforming growth factor (TGF)-beta 1 (2-4 h) was significantly decreased and that the in vitro stimulated production of IFN-gamma (2-8 h) and TNF (4 h) was significantly increased compared with control . Our data concerning the increased IFN-gamma and TNF producing capacity of unseparated leukocytes during pharmacologically induced hyperprolactinemia strongly support the possibility that the lymphocyte production of these cytokines can be rapidly amplified by prolactin via a priming mechanism.

J Clin Microbiol, 1999 Dec, 37(12), 3971 - 4
Comparison of four clinical specimen types for detection of influenza A and B viruses by optical immunoassay (FLU OIA test) and cell culture methods; Covalciuc KA et al.; Although laboratory diagnosis of respiratory viruses has been widely studied, there is a relative insufficiency of literature examining the impact of specimen type on the laboratory diagnosis of influenza A and B . In a clinical study comparing the FLU OIA test with 14-day cell culture, clinical specimens from nasopharyngeal swabs, throat swabs, nasal aspirates, and sputum were obtained from patients experiencing influenza-like symptoms . A total of 404 clinical specimens were collected from 184 patients . Patients were defined as influenza positive if the viral culture of a specimen from any sample site was positive . Patients were defined as influenza negative if the viral cultures of specimens from all sample sites were negative . By this gold standard, culture and FLU OIA test results for each sample type were compared . For each of the four specimen types, the viral culture and FLU OIA test demonstrated equal abilities to detect the presence of influenza A or B virus or viral antigen . Sputum and nasal aspirate samples were the most predictive of influenza virus infection . Throat swabs were the least predictive of influenza virus infection, with both tests failing to detect influenza virus in nearly 50% of the throat samples studied.

Pharmacol Toxicol, 1999 Oct, 85(4), 162 - 8
Growth-modulating effects of dichloro myristic and dichloro stearic acid in cell cultures; Hostmark AT et al.; Chloro-containing fatty acids are a major fraction of extractable, organically bound chlorine in fish . It has been suggested that dichloro stearic acid (9,10-dichlorooctadecanoic acid) (C18) is metabolized to dichloro myristic acid (5,6-dichlorotetradecanoic acid) (C14) which accumulates in tissues . Hence, the biological effects of the C18 dichloro fatty acid could be due to formation of the C14 dichloro fatty acid . In this study we have compared the effects of dichloro stearic and dichloro myristic acid on growth of three widely differing cell lines . Both fatty acids inhibited cell growth; however, dichloro myristic acid had a weaker growth inhibitory effect than dichloro stearic acid . Dichloro myristic acid had a biphasic effect (i.e . growth was stimulated at low concentrations, followed by inhibition at higher concentrations) on the growth of human hepatoma cells and immortalized human kidney epithelial cells, but no such effect on human microvascular endothelial cells . The order of potency for growth inhibition by dichloro myristic acid was consistently human hepatoma cells>immortalized human kidney epithelial cells >human microvascular endothelial cells, whereas the relative potency of dichloro stearic acid was variable . Albumin alone stimulated cell growth and had a stronger protective effect against growth inhibition by dichloro myristic acid than against that of dichloro stearic acid . It seems unlikely that a major part of the effect of dichloro stearic acid on cell growth is caused by conversion to dichloro myristic acid.

J Virol, 1999 Dec, 73(12), 9891 - 8
Ability of foot-and-mouth disease virus to form plaques in cell culture is associated with suppression of alpha/beta interferon; Chinsangaram J et al.; A genetic variant of foot-and-mouth disease virus lacking the leader proteinase coding region (A12-LLV2) is attenuated in both cattle and swine and, in contrast to wild-type virus (A12-IC), does not spread from the initial site of infection after aerosol exposure of bovines . We have identified secondary cells from susceptible animals, i.e., bovine, ovine, and porcine animals, in which infection with A12-LLV2, in contrast to A12-IC infection, does not produce plaques; this result indicates that this virus cannot spread from the site of initial infection to neighboring cells . Nevertheless, A12-LLV2 can infect these cells, but cytopathic effects and virus yields are significantly reduced compared to those seen with A12-IC infection . Reverse transcription-PCR analysis demonstrates that both A12-LLV2 and A12-IC induce the production of alpha/beta interferon (IFN-alpha/beta) mRNA in host cells . However, only supernatants from A12-LLV2-infected cells have significant antiviral activity . The antiviral activity in supernatants from A12-LLV2-infected embryonic bovine kidney cells is IFN-alpha/beta specific, as assayed with mouse embryonic fibroblast cells with or without IFN-alpha/beta receptors . The results obtained with cell cultures demonstrate that the ability of A12-IC to form plaques is associated with the suppression of IFN-alpha/beta expression and suggest a role for this host factor in the inability of A12-LLV2 to spread and cause disease in susceptible animals.

Plant Physiol, 1999 Nov, 121(3), 805 - 812
Okadaic Acid Mimics Nitrogen-Stimulated Transcription of the NADH-Glutamate Synthase Gene in Rice Cell Cultures; Hirose N et al.; Okadaic acid (OKA), a potent and specific inhibitor of protein serine/threonine phosphatases 1 and 2A, induced the accumulation of NADH-glutamate synthase (GOGAT) mRNA within 4 h in rice (Oryza sativa L.) cell cultures . In contrast to the transient accumulation of NADH-GOGAT mRNA by NH(4)(+), OKA caused a continuous accumulation for at least 24 h . The induction of NADH-GOGAT mRNA by OKA was not inhibited in the presence of methionine sulfoximine, which inhibited the NH(4)(+)-induced accumulation of mRNA . These results suggest that the OKA-sensitive protein phosphatase is involved in the regulation of NADH-GOGAT gene expression and probably plays a role in the signal transduction pathway downstream from NH(4)(+), although a signal transduction pathway other than that of nitrogen sensing could be responsible . Nuclear run-on assays demonstrated that the accumulation of NADH-GOGAT mRNA induced by the supply of either NH(4)(+) or OKA was mainly regulated at the transcription level . OKA effects were synergistic to the NH(4)(+)-induced expression of the NADH-GOGAT gene . In the presence of K-252a, a protein kinase inhibitor, the accumulation of NADH-GOGAT mRNA induced by either NH(4)(+) or OKA was reduced . The possible roles of protein phosphatases in the regulation of NADH-GOGAT gene expression are discussed.

Hum Exp Toxicol, 1999 Oct, 18(10), 634 - 9
Oxygen reactive radicals production in cell culture by okadaic acid and their implication in protein synthesis inhibition; Matias WG et al.; Okadaic acid (OA), a diarrhetic shellfish toxin is a potent promoter of tumours in mouse skin and a specific inhibitor of protein phosphatases 1 and 2A . Recently it has been shown that OA inhibited protein synthesis in a cell-free system, with 50% inhibitory concentration of 6.3x10(-12) M but the mechanism whereby this inhibition is mediated was still unclear . In the present study, the effect of OA on protein synthesis in Vero cell cultures was investigated . Protein synthesis was inhibited by OA alone in Vero cells in a concentration-dependent manner (IC50=27 ng/ml i.e . 3 . 3x10(-8) M) . Since OA also induced lipid peroxidation and likely oxygen reactive radicals, it was interesting to know whether these radicals impair the protein synthesis process . Therefore, SOD+catalase known as scavenger of active oxygen radicals were added in the culture medium in the presence of OA and labelled leucine . These enzymes partially prevented the inhibition of protein synthesis induced by OA, indicating that the formation of high reactive oxygen free radicals could be one of the pathways this marine toxin induces its toxicity . Since the prevention by SOD+catalase was only partial (the IC50 increased from 27 ng/ml to 48 ng/ml i.e . 3.3x10(-8) M to 5.9x10(-8) M) it was speculated that the production of oxygen reactive radical scavengered by SOD+catalase is not the main mechanism whereby OA induces its cytotoxicity . Vitamins E and C completely prevent the lipid peroxidation induced by OA in cells, but failed to reduce the inhibition of protein synthesis to the same level, indicating that a more specific mechanism might be responsible for protein synthesis inhibition . That is the hyperphosphorylation of elongation factor EF-2 in the protein synthesis machinery . However our results pointed to lipid peroxidation being a precocious phenomenon following the OA exposure, since a concentration with enhanced MDA production was lower than that inducing significant cellular protein synthesis inhibition.

Eur J Immunol, 1999 Nov, 29(11), 3538 - 48
Myasthenia gravis: selective enrichment of antiacetylcholine receptor antibody production in untransformed human B cell cultures; Padberg F et al.; B cells producing antibodies against the acetylcholine receptor (AchR) play a central role in the pathogenesis of myasthenia gravis (MG) . Although anti-AchR autoantibodies have been studied extensively, not much is known about autoimmune B cells and their antigen-driven activation . This has mainly been due to difficulties in establishing and maintaining untransformed antigen-specific B cells in vitro . In this study, we show that highly enriched B cells from peripheral blood and thymus of MG patients can be maintained in culture over a period of 4 weeks when grown on the AchR-expressing rhabdomyosarcoma cell line TE671 together with an anti-CD40 stimulus and lymphokines . Anti-AchR antibody secretion could be detected in the majority of B cell cultures on TE671 cells up to 4 weeks . In contrast, B cells cultured on CDw32-transfected L cells binding anti-CD40 antibodies (the CD40 system) produced only small amounts of anti-AchR antibodies at single time points, whereas the overall IgG production was higher than on TE671 cells . The expression of the relevant autoantigen on the adherent cell line in addition to other growth stimuli could account for this difference and may provide a useful tool for investigating antigen-dependent B cell activation in MG and other B cell-mediated autoimmune conditions.

J Immunol Methods, 1999 Oct 29, 229(1-2), 81 - 95
Automatic image analysis for quantification of apoptosis in animal cell culture by annexin-V affinity assay; Plasier B et al.; Apoptosis is a form of cell death in which the dying cell plays an active part in its demise . At the morphological level, it is characterised by cell shrinkage rather than the swelling seen in necrotic cell death . In cell culture, apoptosis limits the yield of economically and medically important products, and can result in synthesis of imperfect molecules . Therefore, this process must be identified, monitored and fully understood, so that a means to regulate it can be developed . We have developed a new automatic image analysis assay for detecting apoptosis in animal cell culture on the basis of the annexin-V affinity assay . The results of this assay were compared with data generated by flow cytometry and manual scoring . All three methods were found to correspond well but image analysis like flow cytometry offers operator-independent results, and can be used as a tool for rapid monitoring of viable cell number, apoptosis and necrosis in animal cell culture . Furthermore, reduction in cell size was measured and was found to precede the appearance of phosphatidylserine on the cell surface.

Arch Environ Contam Toxicol, 2000 Jan, 38(1), 52 - 8
Biochemical responses of fish sac fry and a primary cell culture of fish hepatocytes exposed to polychlorinated naphthalenes; Pesonen M et al.; Chlorinated naphthalenes are planar halogenated aromatic compounds, which are widespread in the environment . Knowledge of their biochemical and toxicological actions in aquatic biota is, however, limited . The objective of this study was to assess the toxicity of highly chlorinated naphthalene congeners found in the aquatic environment on fish sac fry and to study their effects on xenobiotic metabolizing enzymes (CYP) using a short-term primary culture of fish hepatocytes and liver microsomes . A few days after hatching, rainbow trout sac fry were administered either Hallovax 1014, a mixture of 1,2,3,4,6,7-hexachloronaphthalene and 1,2,3,5,6, 7-hexachloronaphthalene (HxCN-mix), or 1,2,3,4,5,6, 7-heptachloronaphthalene (HpCN) (0.08, 0.8, and 4 microg/sac fry injected into the yolk sac) . The exposure was terminated 2 weeks later . The naphthalene preparations did not cause any clinical signs of toxicity or difference in mortality rates between the control and treated groups . Immunohistochemical analysis of CYP1A expression in the treated sac fry revealed that staining was most pronounced in the hepatocytes and thereafter in kidney tubular epithelial cells . Moderate CYP1A staining was also seen in the mucosal epithelium of pyloric caecae and mild staining in the epithelium of olfactory organ . Staining in control sac fry was weak or absent . Exposure of the primary cell culture of trout hepatocytes to a low doses (</=10 ng/ml) of the chlorinated naphthalenes increased significantly CYP1A-associated EROD activity and CYP1A mRNA content, HxCN-mix being the most potent and thereafter HpCN and Hallovax 1014 . The higher doses (50-100 ng/ml) of each naphthalene also inhibited EROD activity . However, the content of CYP1A mRNA or the intensity of the CYP1A protein band (58 kDa) recognized by anti-trout CYP1A peptide antibodies were not decreased with increasing polychlorinated naphthalene (PCN) concentration, indicating that the inhibition was not due to reduced protein synthesis . Furthermore, in vitro analyses of the inhibitory potential of PCNs on CYP1A activity with trout liver microsomes suggested that these naphthalene preparations may be CYP1A substrates and act as competitive inhibitors of CYP1A catalyst . Our results demonstrate that highly chlorinated naphthalenes are potent modulators of fish CYP1A enzyme and suggest that hepatocytes and tubular epithelial cells are the cell types that may be vulnerable to their metabolic products for cell injury in fish sac fry.

Neurosci Lett, 1999 Nov 5, 275(1), 53 - 6
Early hypoxia modulates the phenotype of dopaminergic cells in rat di- and mesencephalic cell cultures and induces a higher vulnerability of non-dopaminergic neurons to a second hypoxic exposure; Husemann B et al.; To investigate long-term effects of hypoxia on a cellular level, di- and mesencephalic cell cultures were exposed to hypoxia on in vitro day 2 (incubation in culture medium, pO2 = 10-20 mmHg, 24 h) and on in vitro day 13 (incubation in an electrolyte solution, pO2 = 10-20 mmHg, 8 h) . The numbers of neuron-specific enolase immuno-reactive (NSE-IR) and tyrosine hydroxylase immuno-reactive (TH-IR) neurons and the levels of dopamine, its main metabolites and the spontaneous and potassium-stimulated DA release were determined on DIV 15 . Hypoxia on DIV 2 did not affect the numbers of NSE-IR and TH-IR neurons, but increased the dopamine content and dopamine release by about 100% in both di-and mesencephalic cultures . In addition, this hypoxia increased the vulnerability of non-TH-IR neurons to the second hypoxic episode applied during more advanced stages of the culture development on DIV 13 . On the contrary, hypoxia exposure did not affect the vulnerability of TH-IR cells.

Clin Oral Implants Res, 1999 Oct, 10(5), 379 - 93
Cell culture tests for assessing the tolerance of soft tissue to variously modified titanium surfaces; Sauberlich S et al.; The aim of our research project was to achieve an improvement in the integration of enossal dental implants in the region of peri-implantary soft tissue . Improvement in the adhesion of the gingiva of the surface of enossal implants was to be achieved by modification of the titanium surface . The effect of different modifications on the biocompatibility of the modified titanium surfaces was tested: sulfur dioxide plasma treatment of titanium; acetylene plasma treatment of titanium followed by sulfur dioxide plasma etching; plasma nitration of titanium; replacement of titanium by glycidoxypropyltrimethoxy silane; coating titanium with poly{(ethene-co-vinyl acetate)-graft-vinyl chloride} and coating titanium with fibronectin . Determination of the chemical composition of the surface was carried out using X-ray photospectroscopy . The adsorption of fibronectin at the surface of the titanium was tested using an Enzyme Linked Immunosorbent Assay . In selected in vitro tests with human gingival fibroblasts, cell morphology was assessed using scanning electron microscopy and light microscopy . Cell proliferation and protein synthesis, as well as the activity of mitochondrial dehydrogenases were evaluated . By means of centrifugation and by determining initial cell adhesion, the adhesion of gingival fibroblasts was investigated . According to the kind of modification made to the titanium surfaces, it was possible to observe differences in the cellular behavior of gingiva fibroblasts on the differently modified surfaces of the implants . Coating the titanium using fibronectin produced optimization of cell growth and improvement in the adhesion of gingiva fibroblasts to the implant surface . In contrast, modification of the titanium with poly{(ethene-co-vinyl acetate)-graft-vinyl chloride} generally resulted in a deterioration of the biocompatibility of the surface . A marked correlation between the cellular compatibility of the modified titanium and the surface modification made did not become apparent . One reason for this is the large number of parameters determining the interaction between implant and tissue.

Arch Virol, 1999, 144(10), 1961 - 75
Antigenicity and pathogenicity characteristics of molecularly cloned chicken anaemia virus isolates obtained after multiple cell culture passages; Scott AN et al.; The Cux-1 isolate of chicken anaemia virus (CAV), which had received 310 (P310) cell culture passages, was substantially less pathogenic than virus that had been passaged 13 times (P13) . Molecularly cloned virus isolates, selected from the P310 and P13 virus populations using recombinant DNA cloning and transfection procedures, reacted differently with 4 CAV-specific monoclonal antibodies (MAbs), which had been raised to low-passage Cux-1 virus . In contrast to the strong immunofluorescence (IF) reactivities exhibited by all P13 cloned isolates tested, 80% and 57% of the P310 cloned isolates reacted weakly with MAbs 2A9 and 4H4, which are directed against conformational epitopes on the capsid protein, VP1 . Sequence analysis of the VP1 coding regions possessed by ten P310 and two P13 cloned isolates showed that 6 amino acid changes within VP1 had been selected by multiple-cell culture passage . One of these at position 89 in VP1 appeared to be crucial for determining reactivity with MAb 2A9 . Of nine P310 cloned isolates evaluated, 8 were substantially attenuated compared to the low-passage Cux-1 virus pool . It is concluded that the individual virus variants comprising the P310 virus pool differ with regards to their antigenicity and pathogenicity.

Mol Cell Biol Res Commun, 1999 Aug, 2(2), 131 - 7
Development of human and rabbit vaginal smooth muscle cell cultures: effects of vasoactive agents on intracellular levels of cyclic nucleotides; Traish A et al.; In this study, we subcultured and characterized human and rabbit vaginal smooth muscle cells and investigated the synthesis of second messenger cyclic nucleotides in response to vasodilators and determined the activity and kinetics of phosphodiesterase (PDE) type 5 (EC 3.1.4.35 3',5'-cyclic GMP phosphodiesterase) . Cultured vaginal cells exhibited growth characteristics typical of smooth muscle cells and immunostained with antibodies against alpha smooth muscle actin . The cells retained functional prostaglandin E and beta-adrenergic receptors as demonstrated by increased intracellular cAMP synthesis in response to PGE1, or isoproterenol . The response to these vasoactive substances was augmented with forskolin, suggesting stabilization of G-protein-activated adenylyl cyclases . Treatment with the nitric oxide donor, sodium nitroprusside, in the presence of sildenafil, a PDE type 5 inhibitor, enhanced intracellular cGMP synthesis and accumulation . Incubation of rabbit vaginal tissue with sildenafil, sodium nitroprusside, and PGE1 or forskolin produced a marked increase in intracellular cGMP . These observations were similar to findings with cultured cells and suggest that subcultured cells retain functional characteristics exhibited in intact tissue . The cells retained phosphodiesterase type 5 expression as shown by specific cGMP hydrolytic activity . Sildenafil and zaprinast inhibited cGMP hydrolysis competitively and bound with high affinity (Ki = 7 and 250 nM, respectively) . These observations suggest that cultured human and rabbit vaginal smooth muscle cells retained their metabolic functional integrity and this experimental system should prove useful in investigating the pathway of nitric oxide and PDE type 5 inhibitors in modulating vaginal smooth muscle tone.

Invest Ophthalmol Vis Sci, 1999 Nov, 40(12), 3012 - 6
Antioxidant pattern in uveal melanocytes and melanoma cell cultures; Blasi MA et al.; PURPOSE: To investigate the antioxidant status of cultured uveal melanocytes from patients with uveal melanoma and uveal melanoma cells to characterize some of the biochemical properties of these cells in respect to the normal cutaneous melanocytes . METHODS: The fatty acid pattern of membrane phospholipids, intracellular vitamin E level, and superoxide dismutase (SOD) and catalase activities were studied in uveal melanocytes (n = 10) and uveal melanoma cell (n = 10) cultures, by gas chromatography mass spectrometry or by spectrophotometer . RESULTS: Among the uveal melanocyte cultures, two groups were differentiated, according to catalase activity: group A with catalase values comparable to those of cutaneous ones and higher SOD activity and group B with catalase values 2 SD lower (P<0.001) and lower SOD activity . Vitamin E concentration was not significantly different between melanoma cells and melanocytes, whereas a significantly higher percentage of polyunsaturated fatty acids was found in melanoma cells and the B group of melanocytes (P = 0.022) . In uveal melanoma cells SOD activity was significantly lower than that detected in uveal melanocytes (P< 0.005) . CONCLUSIONS: These results show a different pattern of antioxidants in uveal melanocytes with respect to cutaneous ones, possibly related to the anatomic distribution . However, as in cutaneous melanocytes, two subgroups were identified on the basis of the antioxidant pattern that could be the expression of a constitutional increased susceptibility to oxidative stress in some subjects . Moreover, an imbalance of the antioxidants was observed in melanoma cells, possibly related to the disease status and progression.

Biochem Biophys Res Commun, 1999 Nov, 265(1), 233 - 9
TGF-beta enhances osteoclast differentiation in hematopoietic cell cultures stimulated with RANKL and M-CSF; Sells Galvin RJ et al.; TGF-beta has been shown to inhibit and stimulate osteoclastogenesis . The purpose of this study was to evaluate the effects of TGF-beta in hematopoietic cell cultures stimulated with RANKL and M-CSF . In cocultures of hematopoietic cells and BALC cells (a calvarial-derived cell line), TGF-beta inhibited tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cell formation . In contrast, TGF-beta enhanced TRAP-positive multinucleated cell formation up to 10-fold in hematopoietic cell cultures containing few osteoblastic/stromal cells . Likewise, TGF-beta increased the number of calcitonin receptor (CTR)-positive multinucleated and mononucleated cells in a concentration-dependent manner . An increase in cell size and multinuclearity was also observed in the presence of TGF-beta . The stimulatory effects of TGF-beta were dependent on the presence of M-CSF and RANKL . When differentiated on bovine cortical bone slices, these cells formed resorption lacunae . These results suggest that TGF-beta has a direct stimulatory effect on osteoclastogenesis in hematopoietic cells treated with RANKL and M-CSF .

Ann Urol (Paris), 1999, 33(5), 382 - 8
{Urothelium cell culture: what is the future? An experimental study in rabbits}; Bertschy C; Since the first human bladder reconstruction in 1989 using an ileal segment, many alternatives have been proposed to recreate a bladder reservoir as adapted as possible to physiological conditions . Since the development of urothelial cell culture alone and then in combination with matricial supports, various experimental trials have studied the possibility of using this neurothelium for surgical purposes . This experimental study in rabbits tested the compatibility of two different biosynthetic supports in an enterocystoplasty and the survival in in vitro urothelial cells grafted onto this support.

Can J Physiol Pharmacol, 1999 Aug, 77(8), 598 - 605
Slow wave and spike action potentials recorded in cell cultures from the muscularis externa of the guinea pig small intestine; Espinosa-Luna R et al.; Intracellular recordings were obtained to investigate whether slow wave and spike type action potentials are present in cell cultures of the muscularis externa from the guinea pig small intestine . The muscularis externa of the small intestine was dissociated by using specific purified enzymes and gentle mechanical dissociation . Cells were plated on cover slips and maintained in culture for up to 4 weeks . Dissociated cells obtained in this way reorganized themselves in a few days to form small cell clumps showing spontaneous movements . Intracellular recordings of these clumps displayed both spike and slow wave type action potentials . Spikes were observed on top of some slow waves and were abolished by the addition of nifedipine or the removal of extracellular calcium . Slow waves, however, were nifedipine insensitive and temperature sensitive, and were abolished by octanol (a gap junction blocker) and forskolin (an adenyl cyclase activator) . Slow waves were never observed in small clumps (<50 microm), suggesting that a critical mass of cells might be required for their generation . These observations demonstrated for the first time the presence of nifedipine-insensitive slow waves in cell cultures of the muscularis externa from the guinea pig small intestine . Cell cultures allow rigorous control of the immediate environment for the cells and this should facilitate future studies on the molecular and cellular mechanisms responsible for the slow waves in the gastrointestinal tract.

J Neurochem, 1999 Nov, 73(5), 1894 - 900
Serotonin N-acetyltransferase mRNA levels in photoreceptor-enriched chicken retinal cell cultures: elevation by cyclic AMP; Greve P et al.; Serotonin N-acetyltransferase (AA-NAT; arylalkylamine N-acetyltransferase; EC 2.3.1.87) is a key regulatory enzyme in the biosynthesis of melatonin . Previous studies have shown that the activity of this enzyme in the chicken retina is regulated by a cyclic AMP-dependent mechanism . In the present report, we investigated whether cyclic AMP can regulate the levels of AA-NAT mRNA in photoreceptor-enriched chick retinal cell cultures . AA-NAT mRNA levels were elevated by acute treatment with cyclic AMP protagonists, including forskolin; this response was blocked by H-89, a selective inhibitor of cyclic AMP-dependent protein kinase . Forskolin did not alter the rate of disappearance of AA-NAT mRNA in actinomycin D-treated cells, suggesting that cyclic AMP enhances transcription of the AA-NAT gene . Forskolin-induced elevation of AA-NAT mRNA levels was enhanced by cycloheximide, which decreased the degradation of the transcript in cells treated with actinomycin D . These studies indicate that the abundance of AA-NAT mRNA is regulated in part through a cyclic AMP-dependent mechanism.

Glia, 1999 Nov, 28(2), 114 - 27
Neuronal death in cytokine-activated primary human brain cell culture: role of tumor necrosis factor-alpha; Downen M et al.; We examined cytokine-mediated neuronal death in neuron-astrocyte cultures from second trimester human fetal cerebrum . In these cultures, high-output inducible nitric oxide synthase (NOS) and tumor necrosis factor-alpha (TNFalpha) are expressed in astrocytes after exposure to IL-1beta/IFNgamma . Neuronal cell death was evident at >/=48 h following cytokine stimulation . Neutralizing anti-TNFalpha antiserum inhibited ( approximately 48%) neurotoxicity in IL-1beta/IFNgamma-treated cultures, demonstrating a role for endogenously produced TNFalpha . Interestingly, the degree of neuroprotection conferred by superoxide dismutase or N-methyl D-aspartate (NMDA) receptor antagonists in these cultures was smaller and variable . Similarly, the effect of the NOS inhibitor, N(G)-monomethyl L-arginine (NMMA) on IL-1beta/IFNgamma-induced neuronal death was variable, showing no statistically significant effect when results from more than 30 independent cultures were averaged . Neurons die by apoptosis in cytokine-treated human fetal CNS cultures as shown by the characteristic nuclear morphology as well as positive labeling for TUNEL . Our results demonstrate a potent neurotoxicity mediated by the cytokine combination IL-1beta/IFNgamma in primary human neuron-astrocyte cultures and a crucial role for endogenous TNFalpha in mediating neurotoxicity in this system . These results firmly establish the neurotoxic potential of the inflammatory cytokines IL-1beta and TNFalpha in the human CNS .

Life Sci, 1999, 65(14), 1455 - 61
Paradoxical effect of neuroleptic drugs on prolactin secretion by rat pituitary cell cultures; Braghiroli L et al.; Several antipsychotic drugs reverse the dopamine-induced inhibition of prolactin release by rat pituitary cell cultures . Paradoxically, at high doses and without dopamine, antipsychotic drugs can also inhibit prolactin secretion . The mechanism underlying this phenomenon is unclear . Some evidence suggests that these drugs have an agonistic action . We sought to verify whether clozapine and fluphenazine, at doses higher than those reversing dopamine-induced inhibition of prolactin secretion in vitro, show this paradoxical effect and eventually a partial agonistic action . Both antipsychotics inhibited prolactin secretion, clozapine at doses starting from 10(-6) M and fluphenazine from 10(-7) M . Haloperidol reversed clozapine-induced prolactin inhibition but left fluphenazine-induced inhibition unchanged . These in vitro findings suggest that clozapine has a partial agonistic action on dopaminergic receptors but fluphenazine does not.

Brain Res, 1999 Oct 2, 843(1-2), 95 - 104
Cellular localization of dopamine-releasing protein (DARP) in rat C6 glioma and primary mesencephalic cell cultures; Smith S et al.; Dopamine-releasing protein (DARP) is a multisubunit protein shown to have dramatic effects on development, recovery, and function of the rat catecholaminergic (CA) system . This study details efforts to determine if glial cells are responsible for the production of DARP in the central nervous system (CNS) . Enzyme-linked immunosorbent assays (ELISA), Western blotting, and immunocytochemical techniques were employed to measure DARP levels and identify DARP immunoreactive proteins in rat C6 glioma cells and medium, respectively . ELISA analysis of serum-free C6 culture media revealed a maximal concentration of DARP by culture day 1 . However, ELISA analysis of C6 cultures grown in F-12K/serum medium revealed that maximal levels of DARP were detected on culture day 6 with a 108% increase in DARP immunoreactivity from culture day 1 . These values were determined using a polyclonal antibody generated against DARP-36aa (anti-DARP-36aa), a synthetic peptide with dopamine (DA) releasing activity, and anti-DARP B9-B10, a monoclonal antibody generated against partially purified DARP . Western blot analysis revealed that anti-DARP B9-B10 recognized proteins of approximately 60, 50, and 45 kDa in C6 cell homogenates while anti-DARP-36aa had immunoreactivity with the 60-kDa protein alone . Immunocytochemical studies demonstrated that anti-DARP-36aa and anti-DARP B9-B10 had strong immunoreactivity with proteins throughout the cytosol and in several processes of C6 cells . These results reveal that DARP is detected in glioma cells and secreted in a time-dependent fashion during culture . Primary rat mesencephalic cultures were also examined using immunocytochemistry . Incubation with DARP antibodies and antisera against glial fibrillary acidic protein (GFAP) revealed that DARP and GFAP immunoreactivity co-localized in primary mesencephalic cultures . However, the majority of DARP immunoreactivity was localized to cells without GFAP staining . These findings reveal that DARP is detected in astrocytes although the majority of DARP immunoreactivity is found in non-astrocyte type cells.

Neurobiol Dis, 1999 Oct, 6(5), 347 - 63
Neurons undergo apoptosis in animal and cell culture models of diabetes; Russell JW et al.; Recent clinical trials indicate that the severity of diabetic neuropathy is correlated with the level of patient glycemic control . In the current study, hyperglycemia induces apoptotic changes in dorsal root ganglion neurons and Schwann cells in vivo both in streptozotocin-treated diabetic rats and in rats made acutely hyperglycemic with infused glucose . Typical apoptotic nuclear and cytoplasmic changes are observed . In addition mitochondrial changes recently reported to occur as part of the apoptotic cascade, such as ballooning of mitochondria and disruption of the internal cristae, are seen in diabetic dorsal root ganglion neurons and Schwann cells . Similar changes have been reported in neurons in the presence of oxidative stress . In order to study the neurotoxic effects of high glucose we developed an in vitro model using rat dorsal root ganglion neurons . In dorsal root ganglion cultured in defined medium, addition of moderate glucose levels results in neurite degeneration and apoptosis . These changes are coupled with activation of caspase-3, dependent on the concentration of glucose . The apoptotic changes observed in vitro are similar to those observed in vivo . In contrast, addition of IGF-I, even at physiological concentrations, prevents activation of caspase-3 and neuronal apoptosis in vitro . We suggest that oxidative stress may promote the mitochondrial changes in diabetic animals and lead to activation of programmed cell death caspase pathways . These results imply a new pathogenetic mechanism for diabetic sensory neuropathy .

J Clin Microbiol, 1999 Nov, 37(11), 3722 - 4
Isolation of Rickettsia prowazekii from blood by shell vial cell culture; Birg ML et al.; A blood sample from a patient who returned from Algeria with a fever inoculated on human embryonic lung fibroblasts by the shell vial cell culture technique led to the recovery of Rickettsia prowazekii . The last clinical strain was isolated 30 years ago . Shell vial cell culture is a versatile method that could replace the classic animal and/or embryonated egg inoculation.

Brain Res Dev Brain Res, 1999 Sep 6, 116(2), 159 - 67
Brain endogenous insulin effects on neurite growth within fetal rat neuron cell cultures; Schechter R et al.; We have previously described insulin to be synthesized "de novo" within the fetal rat brain and that brain endogenous insulin {I(n)} promoted neurofilament distribution within fetal neurons . In this study, we investigated the role of I(n) in neuron axonal growth . Rat fetal brain stem cells from 16-day gestational age were cultured in an IFDM and treated with an insulin antibody . In addition, the cell cultures were also treated in defined medium with the addition of: 5 ng, 20 ng or 100 ng/ml of insulin or 100 ng/ml insulin-like growth factor 1 (IGF-1) . The neuron cell cultures were studied at 1 and 3 days of incubation . The presence of preproinsulin mRNA and insulin immunoreaction confirmed the "de novo" synthesis of insulin by the fetal neuron cell cultures . Axonal growth was similar by day 1 of the study in all the media, but in insulin medium containing 100 ng/ml of insulin the axonal length was significantly longer . By day 3 of incubation I(n) promoted axonal growth . Treating the neurons with an insulin antibody confirmed these findings, with a significant decrease in axonal length (p<0.05) . The treatment with different concentrations of exogenous insulin did not promote axonal growth beyond I(n) by day 3 of incubation . IGF-1 did not promote axonal growth by day 3 of incubation . In summary, I(n) may promote axonal growth during brain development.

Vet Microbiol, 1999 Sep 1, 69(1-2), 67 - 8
Quasispecies evolution of a hypervariable region of the feline calicivirus capsid gene in cell culture and persistently infected cats; Radford AD et al.; The study determines the sequence evolution of feline calicivirus both in cell culture and in persistently infected cats and relates this to changes in virus neutralisation.

J Med Chem, 1999 Oct 7, 42(20), 4122 - 8
Design and synthesis of lipophilic phosphoramidate d4T-MP prodrugs expressing high potency against HIV in cell culture: structural determinants for in vitro activity and QSAR; Siddiqui AQ et al.; A series of new substituted-aryl phosphoramidate derivatives of the anti-HIV drug d4T were synthesized as membrane-soluble nucleotide prodrugs, to extend and quantify the SAR observed for an earlier series of related derivatives . All of the compounds were found to be significantly more potent against HIV in cell culture than the nucleoside analogue d4T, and most were also found to be significantly more potent than the parent phosphoramidate . A Hansch type QSAR analysis was applied to the combined series of 21 compounds . The results of this analysis revealed anti-HIV activity to be principally dependent on lipophilicity in a quadratic manner, with terms representing substituent steric bulk and electronic effects having a minimal significance.

Biomaterials, 1999 Oct, 20(19), 1773 - 82
Biological evaluation of RGD peptidomimetics, designed for the covalent derivatization of cell culture substrata, as potential promotors of cellular adhesion; Marchand-Brynaert J et al.; Our aim was to replace the proteins and peptides, generally used for the biocompatibilization of polymer substrata, with synthetic molecules mimicking the RGD (Arg-Gly-Asp) active sequence . Based on the (L)-tyrosine template, RGD peptidomimetics were constructed; one molecule 3 was equipped with an anchorage arm that allowed its covalent grafting on a culture substratum made from poly(ethylene terephthalate) (PET) microporous membrane . The amount of fixed molecules was readily determined by XPS, using a fluorine tag incorporated in the peptidomimetic structure . The binding of peptidomimetics 1-3 to the vitronectin (VN) and fibronectin (FN) receptors could not be revealed in a test of inhibition of MSC 80 cells adhesion, by the synthetic compounds in solution placed in competition with the adhesive proteins (VN and FN) coating polystyrene plates . However, the cell-attachment activity of peptidomimetic 3 was shown by culturing CaCo2 cells, in the absence of serum, on the PET substratum grafted with 3 . The performance of this support was similar to that of PET grafted with the reference peptide RGDS (Arg-Gly-Asp-Ser), and only reduced by half comparatively to the PET grafted with FN.

Histol Histopathol, 1999 Oct, 14(4), 1231 - 40
Quantitative in situ hybridization for the evaluation of gene expression in asynchronous and synchronized cell cultures and in tissue sections; Barlati S et al.; We describe an image analysis (IA) system that has been applied for the quantitative evaluation of mRNAs evidenced by in situ hybridization (ISH) with radiolabelled probes in cultured cells and in tissue sections . The ISH-IA method was used for the evaluation of cultured cell morphological parameters such as cell and nucleous area (CA and NA, respectively) in parallel with the levels of mRNAs detected as hybridization grains areas (GA) . The evaluation of these parameters, together with the analysis of the levels of mRNAs (c-jun, cyclin A) specific for given cell cycle phases (i.e . G1 and S/G2), allowed the identification, in asynchronous cultures of human skin fibroblasts, of cells in G1 and S/G2 phases . The mRNA levels measured by ISH-AI were comparable with those detected by RT-PCR . This method was also applied for the analysis of fibronectin (FN) gene expression in control skin fibroblasts in relationship with the different phases of the cell cycle and in comparison with a tumor cell line (Sk-Hep1), heterogeneous either for morphometric parameters or for the levels of this transcript . Finally, the ISH-AI was applied for the semiquantitative evaluation of the expression, localization and alternative splicing pattern of FN mRNA in normal liver and in hepatocellular carcinoma (HCC) tissue sections.

AIDS Res Hum Retroviruses, 1999 Sep 20, 15(14), 1265 - 77
Effect of extracellular human immunodeficiency virus type 1 glycoprotein 120 on primary human vascular endothelial cell cultures; Huang MB et al.; During the course of an HIV-1 infection, free infectious and noninfectious virus particles, and free HIV-1 proteins, circulate within the host, exposing the host endothelium to these viral factors, even if the endothelium is not infected . This suggests that extracellular HIV-1 proteins could influence endothelial cell function, leading to pathogenesis . In light of this, we have used primary cultured human vascular endothelial cells (HUVECs) to screen for effects of the HIV-1 protein gp120 on endothelial cell function . The results of this study show that short exposure of HUVEC cultures to this protein causes significant levels of cytotoxicity . Further, using several different assays, we have shown that this cytotoxic effect on HUVECs appears to be due to induction of an apoptotic program . The biphasic nature of gp120 titration curves suggests that multiple cellular factors are mediating these gp120-induced effects . Competition studies appear to confirm this by showing that the apoptotic effect is mediated through two cell surface receptors on HUVECs, CCR5 and CXCR4 . Alternatively, competition studies examining CD4 receptors suggests that CD4 played no role in gp12O-induced effects on HUVECs.

Neurosci Lett, 1999 Sep 24, 273(1), 57 - 60
Astrocytes rather than neurones mediate interleukin-1beta dependent nitric oxide and superoxide radical release in primary hypothalamic rat cell cultures; Tolias CM et al.; The cellular sources of nitric oxide in the hypothalamus are thought to be 'NOergic' neurones . Using free radical electrochemical sensors we investigated nitric oxide and superoxide radical release in primary hypothalamic cell cultures . We present evidence that under interleukin-1beta (IL-1beta) stimulation hypothalamic astrocytes rather than neurones release nitric oxide . Under L-arginine deprivation and IL-1beta stimulation a concentration-dependent release of superoxide was also observed, which was inhibited in the presence of nitric oxide synthase inhibitor nitro-L-argininemethyl-ester . These findings support the hypothesis that the balance between nitric oxide and superoxide may be of vital importance in hypothalamic pathophysiology.

Life Sci, 1999, 65(13), PL167 - 70
Effect of estradiol metabolites on prostacyclin synthesis in human endothelial cell cultures; Seeger H et al.; Estradiol can stimulate prostacyclin production in the vessel wall, thereby eliciting vasodilatation . In the present work the effect of the estradiol metabolites estrone, 2-methoxyestrone, 2-methoxyestradiol, and 16alpha-hydroxyestrone were investigated to find out if they are also able to stimulate prostacyclin synthesis . All metabolites triggered an increase of prostacyclin synthesis in human endothelial cells starting at a concentration of 10(-9) M . The parent substance, 17beta-estradiol, accomplished this effect only starting at a concentration of 10(-8) M . These results indicate that estradiol metabolites may take part in the estradiol-induced vasodilatation in vivo.

Gynecol Oncol, 1999 Oct, 75(1), 72 - 7
Ovarian carcinoma cell cultures are resistant to TGF-beta1-mediated growth inhibition despite expression of functional receptors; Yamada SD et al.; OBJECTIVE: The purpose of this study was to determine the response of ovarian carcinoma cells to TGF-beta1 and to examine components of the TGF-beta signaling pathway . METHODS: Twenty-three primary ovarian cancer cell (CSOC) cultures established from solid ovarian carcinomas were treated with TGF-beta1 and assayed for growth response by MTT assay . Expression of TGF-beta receptor I (TbetaR-I) and receptor II (TbetaR-II), essential for effective signaling, was determined by Western analysis of CSOC cultures . TGF-beta1 ligand-induced phosphorylation of TbetaR-I was determined by immunoprecipitation of TbetaR-I followed by a protein kinase assay to assess TbetaR-I phosphorylation, an essential first step in TGF-beta signal transduction . Gelatin zymography performed on 5 CSOC cultures incubated with TGF-beta1 was used to determine TGF-beta's effect on matrix metalloproteinase production . Normal ovarian surface epithelial cells were used for comparison . RESULTS: Eighteen of twenty-three (78%) CSOC cultures demonstrated no significant growth inhibition in response to TGF-beta1 treatment . All cell cultures expressed TbetaR-I and TbetaR-II and exhibited TbetaR-I phosphorylation following TGF-beta1 treatment . CSOC cultures produced significantly higher levels of matrix metalloproteinase-2 (MMP-2) than normal ovarian surface epithelial cells; however, the level of MMP-2 expression was not regulated by TGF-beta1 . CONCLUSION: These results indicate that TGF-beta1 resistance and higher levels of MMP-2 production may be inherent properties of the ovarian cancer phenotype . The initial steps in the TGF-beta signaling pathway, receptor expression, ligand binding, and TbetaR-I phosphorylation, appear to be functional in primary ovarian cancer cell cultures . Therefore, the mechanism of growth resistance is downstream of TbetaR-I phosphorylation .

J Neurosci Res, 1999 Oct 15, 58(2), 308 - 17
Aging in a dish: age-dependent changes of neuronal survival, protein oxidation, and creatine kinase BB expression in long-term hippocampal cell culture; Aksenova MV et al.; Results from different experimental systems demonstrate that increased oxidative damage plays a role in normal aging and age-associated pathology . In the current study, long-term cultures of hippocampal neurons were examined as a model system . It was established that neuronal survival in long-term culture decreases according to the Gompertz law and that neuronal "aging in the dish" is associated with increased oxidative damage of cell proteins . The increase of protein carbonyl formation in aged neurons was demonstrated both by Western blot analysis for oxidized proteins and by in situ immunocytochemical method, which was developed to analyze protein oxidation in fixed cells . In aging neuronal cultures, a gradual increase in creatine kinase (CK) content but decreased activity of enzyme per immunoreactive protein was found, suggesting the accumulation of inactive CK molecules . The increase in CK content was not a result of generalized protein elevation, since analysis of beta-actin content showed a time-dependent loss, probably reflecting decreased number of cellular processes with aging . These findings, showing "aging in a dish," consistent with the notion that aging is associated with increased protein oxidation, provide a system for study of age-related neurodegenerative disorders associated with oxidative stress .

Adv Exp Med Biol, 1999, 457, 415 - 21
Cellular drug resistance in childhood acute myeloid leukemia . A mini-review with emphasis on cell culture assays; Kaspers GJ et al.; Cellular drug resistance is an important limiting factor in the success of chemotherapy in childhood acute myeloid leukemia (AML) . We summarize the results of the studies published sofar that have focussed on drug resistance in childhood AML, using cell culture assays . We also briefly report our own results of an ongoing study . Finally, potential applications of cellular drug resistance testing are discussed . It appears that cellular drug resistance differs between AML and acute lymphoblastic leukemia and between subgroups of AML patients, that AML cells of relapsed patients are more resistance to cytarabine than those of untreated patients, and that in vitro resistance to cytarabine and daunorubicin is related to a worse prognosis . However, more and larger studies are required to determine the exact role of cellular drug resistance testing in the treatment of childhood AML.

Toxicology, 1999 Aug 13, 136(1), 27 - 39
Extracellular calcium is required for the polychlorinated biphenyl-induced increase of intracellular free calcium levels in cerebellar granule cell culture; Mundy WR et al.; Recent studies from the laboratory indicate that polychlorinated biphenyl (PCB) congeners can alter signal transduction and calcium homeostasis in neuronal preparations . These effects were more pronounced for the ortho-substituted, non-coplanar congeners, although the mechanisms underlying these effects are not clear . In the present study the time-course and concentration-dependent effects of coplanar and non-coplanar PCBs on intracellular free calcium concentration ({Ca2+}i) in cerebellar granule cell cultures were compared using the fluorescent probe fura-2 . The ortho-substituted congeners 2,2'-dichlorobiphenyl (DCB) and 2,2',4,6,6'-pentachlorobiphenyl (PeCB) caused a gradual increase of {Ca2+}i while the non-ortho-substituted congeners 4,4'-DCB and 3,3',4,4',5-PeCB had no effect . The increase of {Ca2+}i produced by 2,2'-DCB was time- and concentration-dependent . Further studies examined possible mechanisms for this rise in {Ca2+}i . In contrast to the muscarinic agonist carbachol, the effects of 2,2'-DCB on {Ca2+}i were not blocked by thapsigargin and required the presence of extracellular calcium . The effects of ortho-substituted PCBs may depend on their ability to inhibit calcium sequestration as 2,2'-DCB significantly inhibited 45Ca2+-uptake by microsomes and mitochondria while 3,3',4,4',5-PeCB had no effect . In addition, 2,2'-DCB significantly increased the binding of {3H}inositol 1,4,5-trisphosphate to receptors on cerebellar microsomes, suggesting another possible mechanism by which ortho-substituted PCBs can mobilize {Ca2+}i . These results show that PCBs increase {Ca2+}i in vitro via a mechanism that requires extracelluar calcium, and support previous structure-activity studies indicating that ortho-substituted PCBs are more potent than non-ortho-substituted PCBs.

Bioorg Med Chem Lett, 1999 Sep 6, 9(17), 2555 - 60
Simple mono-derivatisation of the aryl moiety of D4A and DDA-based phosphoramidate prodrugs significantly enhances their anti-HIV potency in cell culture; Siddiqui AQ et al.; Simple mono-derivatisation of the aryl moiety of some phosphoramidate pronucleotide derivatives of d4A and ddA served to increase the lipophilicity of these membrane-soluble prodrugs . A concomitant and significant enhancement of potency against HIV-1 and HIV-2 in vitro was observed for the ddA- and d4A-based prodrugs compared to the original underivatised prodrugs.

Pharm Res, 1999 Sep, 16(9), 1380 - 5
Effects of pharmaceutical compounds on ciliary beating in human nasal epithelial cells: a comparative study of cell culture models; Agu RU et al.; PURPOSE: To test two in vitro human nasal epithelial cell culture systems for their ability to screen the effects of pharmaceutical compounds on ciliary beating . METHODS: Human nasal epithelial cells were cultured as monolayer and in a sequential monolayer-suspension culture with in vitro ciliogenesis . The influence of reference cilio-stimulatory compounds (glycocholate, isoprenaline), reference cilio-inhibitory compounds (chlorocresol, diphenhydramine) and pH on ciliary beating was investigated using computerized microscope photometry . RESULTS: Sodium glycocholate (0.5% w/v) maximally and reversibly increased CBF of the cells in both culture systems by 26 +/- 4% (monolayer) and 18 +/- 6% (suspension) . Similarly, isoprenaline (10(-3) M) maximally, but irreversibly increased CBF of the cells by 14 +/-3% (monolayer) and 17 +/- 4% (suspension) . Chlorocresol (0.005% w/ v) reversibly reduced the CBF of the cells by 50 +/- 6% (monolayer) and 34 +/- 4% (suspension); at a higher concentration (0.1% w/v) it resulted in instantaneous and irreversible ciliostasis . Diphenhydramine (0.1% w/v) reversibly reduced CBF in both culture systems by 45 +/- 13% (monolayer) and 69 +/- 5% (suspension); irreversible cilio-stasis occurred in less than 2 minutes in both culture systems upon cell exposure to diphenhydramine (1.0% w/v) . In the monolayer culture system, CBF was stable only within the physiological pH range of 6.5-8.0; ciliary beating in the suspension culture remained stable within a pH range of 4.0-10.0 . CONCLUSIONS: Both cell culture systems are suitable for screening the effects of pharmaceutical compounds on ciliary beating . Especially the sequential monolayer-suspension culture appears to be very promising as ciliary activity can be preserved for as long as 6 months.

Cell Physiol Biochem, 1999, 9(3), 150 - 72
Impact of culture conditions, culture media volumes, and glucose content on metabolic properties of renal epithelial cell cultures . Are renal cells in tissue culture hypoxic?
Gstraunthaler G, Seppi T, Pfaller W.
When renal proximal tubular cells are brought into tissue culture, they revert from oxidative metabolism and gluconeogenesis to high rates of glycolysis . Among the factors possibly responsible for this metabolic conversion, limited oxygen availability and/or substrate supply are discussed . In order to study the role of these factors on long-term cultures, the impact of growth conditions, culture media volume, and glucose content on carbohydrate metabolism of the continuous renal cell lines LLC-PK(1) (porcine kidney) and OK (opossum kidney) was investigated . The impact of culture media volumes and glucose content, respectively, was determined by overlaying confluent monolayer cultures of LLC-PK(1) and OK cells (i) with increasing volumes of culture medium and thus increasing amounts of glucose, and (ii) with increasing culture medium volumes at constant absolute amounts of glucose by adding glucose-free medium, in order to increase volume at a constant glucose supply . Alternatively, and in order to improve cell oxygenation, LLC-PK(1) cells were also cultured in roller bottles . Cell carbohydrate metabolism was assessed by measuring rates of glucose consumption and lactate production, respectively, and by determination of specific activities of the key glycolytic enzymes hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) . Mitochondrial phosphate-dependent glutaminase (PDG) was assayed as marker enzyme of oxidative metabolism of glutamine . In LLC-PK(1) and OK cells, rates of glucose consumption were independent of the initial glucose concentrations and/or the culture media volumes used . Glucose was quantitatively converted to lactate, which accumulated in a 1:2 molar ratio . Lactate in culture media reached a maximum content after 24 h, and was reutilized by the cell lines thereafter . Interestingly, the rates of lactate reuptake strictly depended on culture medium volume, indicating a volume-induced stimulation of oxidative lactate metabolism . Marked changes were found for the specific activities of glycolytic enzymes . In LLC-PK(1) cells, increased glucose supply caused increases in HK, PFK, PK and LDH activities, which were superimposed to the stimulatory effects of increased media volumes . Enzyme activity showed a biphasic response, indicating that both glucose supply and culture media volumes covering the cell monolayer are factors determining glycolytic rates of LLC-PK(1) renal cells . Conversely, in OK cells glycolytic enzyme activities decreased with increasing culture media volumes at constant glucose levels . As expected, under conditions of enhanced oxygenation of LLC-PK(1) cells in roller bottle culture, glycolytic enzyme activities decreased, whereas PDG activity increased, which was paralleled by increased rates of ammonia generation . Thus, changes in nutrient supply and oxygenation of renal epithelial cell cultures by altered culture media volumes dramatically influence metabolic rates and levels of enzyme activities, respectively.

Cell Physiol Biochem, 1999, 9(3), 139 - 49
Cell culture conditions determine apolipoprotein CIII secretion and regulation by fibrates in human hepatoma HepG2 cells; Clavey V et al.; Fibrates are widely used drugs which lower triglycerides and increase HDL concentrations in serum . Recent findings from our laboratory have shown that fibrates repress apolipoprotein (apo) CIII gene expression, an effect that explains partially the triglyceride-lowering activity of these drugs . The goal of the present study was to compare the effect of various fibrates on apo CIII gene expression in the human hepatoblastoma cell line HepG2 . First, we demonstrate that the level of apo CIII secretion by HepG2 cells is controlled by serum factors whereas apo CIII mRNA levels are not and even increase under conditions when apo CIII secretion dramatically decreases . Twelve different fetal calf serum batches were tested during this study and apo CIII secretion in cell medium could only be detected with three of them . The effect of serum on apolipoprotein secretion was more pronounced for apo CIII whereas other apolipoproteins (apo E, apo B, apo AII and apo AI) were affected to a lesser extent . Under serum conditions allowing apo CIII secretion, treatment with the peroxisome-proliferator activated receptor (PPAR)alpha activators fenofibrate, gemfibrozil and Wy-14643 result in a marked lowering of apo CIII secretion and gene expression, this effect being most pronounced with Wy-14643 . Comparison of the activity of a PPARgamma-specific ligand, the antidiabetic thiazolidinedione, BRL-49653 and a PPARalpha ligand Wy-14643 showed a marked decrease of apo CIII secretion and gene expression after activation of PPARalpha but not PPARgamma . In conclusion, fibrates down-regulate apo CIII gene expression in human HepG2 cells, most likely via PPARalpha but not via PPARgamma . However, these effects are only observed in HepG2 cells cultured under appropriate conditions.

Dev Biol Stand, 1999, 98, 177 - 81; discussion 197
Cell culture influenza vaccine: an Australian perspective; Chang L et al.; The Australia Influenza Vaccine Committee makes independent decisions concerning the influenza vaccine formulation for Australia . Large-scale cell culture using MDCK cells would improve response time for vaccine production in the face of a new pandemic . There must be a consensus with respect to the use of MDCK or other cells before the next pandemic . It is unrealistic to expect any national regulatory authority to determine what safety requirements should be met before approving a cell substrate for vaccine production during an influenza pandemic . Regulatory issues seen as obstacles to the approval of MDCK cells as an accepted cell substrate for influenza vaccine production are identified.

Dev Biol Stand, 1999, 98, 171 - 5; discussion 197
Regulatory perspective in the United States on cell cultures for production of inactivated influenza virus vaccines; Levandowski RA; The United States Code of Federal Regulations requires that all influenza virus vaccines produced for use in the United States adhere to specific regulatory standards including the demonstration of safety and efficacy . For vaccines produced in cell lines, rigorous characterization for manufacturing is particularly important . Influenza vaccines produced by the passage of viruses in mammalian cell lines will require careful evaluation to ensure the removal or inactivation of potential adventitious agents.

Dev Biol Stand, 1999, 98, 93 - 100; discussion 111
Influvac: a safe Madin Darby Canine Kidney (MDCK) cell culture-based influenza vaccine; Brands R et al.; Influenza vaccine production technology based on large scale cell culture technology has been developed . From the characterization of the continuous cell line MDCK as well as drug safety studies we conclude that this cell line and the cell culture system are suitable for biological production . The Down Stream Process (DSP) of the virus-containing harvest fluids guarantees sufficient inactivation of influenza viruses and adequate removal or inactivation of putative adventitious or endogenous viruses, mycoplasma or bacteria . Our data indicate that the tissue culture-based production technology is feasible.

Biol Reprod, 1999 Oct, 61(4), 1090 - 8
Expression and regulation of relaxin-like factor gene transcripts in the bovine ovary: differentiation-dependent expression in theca cell cultures; Bathgate R et al.; The relaxin-like factor (RLF) was recently discovered as a new member of the insulin-insulin-like growth factor-relaxin family of growth factors and hormones predominantly in the Leydig cells of the testis . In cattle, in contrast to other species, the RLF gene is also expressed to a high level in the ovary, where its expression pattern in the corpus luteum of the late cycle and pregnancy is similar to that of relaxin in the pig . The RLF gene was also transcribed to a high level in the theca cells of estrogen-rich, large antral follicles . Long-term primary cultures of bovine theca cells showed that expression was insulin dependent . After an initial decline in specific mRNA concentrations, there was a switch to a transcript with a longer poly(A) tail at about Day 6 of culture, which continued to increase to very high levels by Day 15 of culture . Addition of fetal calf serum to cultures caused a rapid and irreversible down-regulation of the RLF gene . Also, LH caused a decline in specific gene expression in long-term primary theca cell cultures . As in the Leydig cells of the testis, the pattern of RLF gene expression appears to reflect the differentiation state of the ovarian theca-luteal cell lineage, and should prove useful for mapping the fate of these cells under differing stimulation regimes.

Artif Organs, 1999 Sep, 23(9), 881 - 4
Partially degradable film/fabric composites: textile scaffolds for liver cell culture; Karamuk E et al.; In this study, a composite scaffold combining textile superstructures and biomimetic glycopolymers is introduced, which may allow engineering of organotypic liver tissue in vitro . Woven poly(ethylene therephtalat) (PET) fabrics were coated on one side with a thin biodegradable polymer film (poly{D-L-lactic-co-glycolic acid} PLGA), in order to obtain a polar structure . The composite structure ensured the stability of the membrane during in vitro degradation, independently of mesh size . Matrix porosity increased when a polymer blend matrix was used . For hepatocyte culturing studies, the scaffolds were additionally coated with an artificial glycopolymer (poly{N-p-vinylbenzyl-D-lactoamide}, PVLA) in order to improve cell attachment . It was observed that formation of aggregates depends on the scaffold geometry as well as on the pretreatment and medium conditions . After 4 days in culture, the pores of the fabric were filled with aggregates illustrating the possibility of immobilizing hepatocyte aggregates in well-defined spatial configurations on textile structures.

Artif Organs, 1999 Sep, 23(9), 809 - 16
Recombinant human erythropoietin stimulates production of interleukin 2 by whole blood cell cultures of hemodialysis patients; Bryl E et al.; The impairment of function of human T lymphocytes, leading to an inappropriate cytokine production, is partially responsible for defective immunological response in hemodialysis patients (HD) . Recent data suggest that recombinant human erythropoietin (rhEPO) may exert immunological effects . The aim of this study was to find out whether rhEPO treatment of the HD patients may have an effect on the interleukin 2 (IL-2) production by their whole blood cell cultures . The study was carried out in 10 HD patients receiving rhEPO for 6 months . Compared with the levels seen before the treatment, the concentration of IL-2 increased in the phytohemagglutinin-stimulated whole blood cell cultures of 7 of 10 patients under study . Addition of rhEPO in vitro to the whole blood cell cultures of the HD patients before implementation of erythropoietin confirmed that rhEPO is able to directly stimulate IL-2 production . Our studies show that the therapy with rhEPO affects IL-2 secretion.

J Biomed Mater Res, 1999 Dec 5, 47(3), 424 - 33
Osteoclastic responses to various calcium phosphates in cell cultures; Doi Y et al.; Disks made of hydroxyapatite, beta-tricalcium phosphate, carbonate apatite, tetracalcium phosphate, alpha-tricalcium phosphate, dicalcium phosphate dihydrate, and octacalcium phosphate were incubated in osteoclastic cell cultures for 2 days . The first five salts were sintered and the last two were compressed before incubation . Osteoclasts resorbed only the sintered carbonate apatite disks . However, osteoclasts were able to resorb octacalcium phosphate disks that were preincubated for 1 day in medium without cells, indicating that surface conditioning was important for osteoclastic resorption of this calcium phosphate . Although resorption did not occur, medium calcium and phosphorus changed to an appreciable extent after a 2-day incubation of beta-tricalcium phosphate, tetracalcium phosphate, alpha-tricalcium phosphate, and dicalcium phosphate dihydrate . These changes in the medium calcium and phosphate concentrations could explain why osteoclasts appeared to have lost their activity on these calcium phosphate disks and were not capable of resorbing them . With hydroxyapatite disks no changes were observed in the medium calcium and phosphorus before and after incubation . Moreover, the osteoclasts appeared to be essentially the same as with the sintered carbonate apatite disks and with bone slices used as a control . Nevertheless, no pits or lacunae were observed on the hydroxyapatite disks, indicating that sintered carbonate apatite should be superior to sintered hydroxyapatite as a bioresorbable bone substitute .

Hum Mol Genet, 1999 Oct, 8(11), 1975 - 84
Cis and trans effects of the myotonic dystrophy (DM) mutation in a cell culture model; Amack JD et al.; The mutation causing myotonic dystrophy (DM) has been identified as a CTG expansion in the 3'-untranslated region (3'-UTR) of the DM protein kinase gene ( DMPK ), but the mechanism(s) of pathogenesis remain unknown . Studies using DM patient materials have often produced confusing results . Therefore, to study the effects of the DM mutation in a controlled environment, we have established a cell culture model system using C2C12 mouse myoblasts . By expressing chimeric reporter constructs containing a reporter gene fused to a human DMPK 3'-UTR, we identified both cis and trans effects that are mediated by the DM mutation . Our data show that a mutant DMPK 3'-UTR, with as few as 57 CTGs, had a negative cis effect on protein expression and resulted in the aggregation of reporter transcripts into discrete nuclear foci . We determined by deletion analysis that an expanded (CTG) (n) tract alone was sufficient to mediate these cis effects . Furthermore, in contrast to the normal DMPK 3'-UTR mRNA, a mutant DMPK 3'-UTR mRNA with (CUG)(200)selectively inhibited myogenic differentiation of C2C12 myoblasts . Genetic analysis and the Cre- loxP system were used to clearly demonstrate that the myoblast fusion defect could be rescued by eliminating the expression of the mutant DMPK 3'-UTR transcript . Characterization of spontaneous deletion events mapped the inhibitory effect to the (CTG) (n) expansion and/or the 3' end of the DMPK 3'-UTR . These results provide evidence that the DM mutation acts in cis to reduce protein production (consistent with DMPK haploinsufficiency) and in trans as a 'riboregulator' to inhibit myogenesis.

J Immunol Methods, 1999 Jul 30, 227(1-2), 53 - 63
The use of Teflon cell culture bags to expand functionally active CD8+ cytotoxic T lymphocytes; Garland RJ et al.; We have investigated the ability of Teflon cell culture (TCC) bags, compared to conventional tissue culture flasks and plates, to support the expansion of human CD8+ T cells in response to an allogeneic stimulus . TCC bags, which are compatible with good manufacturing practice (GMP), facilitated CD8+ T cell growth as well as conventional culture vessels and resulted in cytotoxic T cells which were able to kill allogeneic targets . Growth characteristics were compared by investigating the number, immunophenotype and cell cycle properties of the cells generated . The kinetics of cell growth were not significantly different over the first 14 days of culture in each vessel type, with the cell counts being highest at day 10 in all cases . However, the TCC bags resulted in a significantly higher proportion of cells with the morphology of typical lymphocytes than tissue culture flasks after 14 and 18 days in culture . There were no significant differences in the percentage of typical lymphocytes expanded in TCC bags compared to those expanded in plates . Expanded CD8+ cells maintained their initial level of expression of CD3, CD11a, CD18 and T cell receptor (alphabeta heterodimer, TCR (alphabeta)) but increased expression of CD45RO, CD95 and of activation markers HLA-DR and CD25 in each culture vessel . Studies of cell cycle parameters showed that each vessel supported CD8+ T cell stimulation, as demonstrated by significantly higher levels of S phase than fresh PBMN cells . The cells generated in TCC bags were able to kill allogeneic targets and also possessed natural killer (NK) cell activity . Thus, TCC bags are able to support the expansion of CD8+ T lymphocytes as well as flasks or tissue culture plates and are applicable to lymphocyte expansion for use in immunotherapy.

Mikrobiol Z, 1999 May-Jun, 61(3), 37 - 45
{Virus reproduction in cell cultures synchronized by macromolecular platinum complexes}; Hyrin VM et al.; A new method of application of platinum macromolecular complex poly inverted question markhexacis{chloroamine aquaplatinum (II)} inverted question mark-mu-deoxyribonucleate (MCP) as a cell cycle synchronization inductor has been developed . The process of some RNA-genome viruses reproduction in cell cultures synchronized by MCP was studied . It was shown that the amount of virus protection in such cultures under special conditions can be increased 100-350 times without increasing the cell substrate amount . The obtained results may be used in biotechnology to increase production of virus antigens and vaccines.

Plant Cell Physiol, 1999 Jun, 40(6), 651 - 5
One type of chalcone synthase gene expressed during embryogenesis regulates the flavonoid accumulation in citrus cell cultures; Moriguchi T et al.; To elucidate the relationship between the expression of chalcone synthase (CHS) genes and the production of flavonoid in citrus cell cultures, two cDNA clones encoding CHS were isolated (CitCHS1 and CitCHS2) from the citrus . The accumulation of CitCHS2 mRNA was notably induced by embryogenesis but CitCHS1 mRNA was not . There was no detectable accumulation of flavonoid in the undifferentiated calli, but flavonoid accumulated after the morphological changes to embryoids . These results indicate that two CHS genes differentially expressed during citrus somatic embryogenesis and CitCHS2 may regulate the accumulation of flavonoid in citrus cell cultures.

J Infect Dis, 1999 Oct, 180(4), 976 - 86
Hepatocytes are permissive for human cytomegalovirus infection in human liver cell culture and In vivo; Sinzger C et al.; The cytopathic potential of human cytomegalovirus (HCMV) in human liver cells was analyzed in cell culture and in tissue sections from patients with HCMV hepatitis . Liver cell cultures, consisting of hepatocytes, bile duct epithelial cells, and stromal cells were infected by various HCMV strains . Cytopathic effects, viral gene expression, and virus production were detected . Infected cell types were identified by immunocytochemical double labeling . Hepatocytes were the predominant target cells of HCMV infection in liver tissues and in cell culture . Late-stage infected cultured hepatocytes produced infectious progeny virus, and infectious virus was propagated from liver tissue specimens . HCMV infection in cultured liver cells closely resembled in vivo infection of the liver with regard to the target cell spectrum and the permissive course of infection . It is concluded that HCMV can cause direct liver parenchyma damage by efficient cytolytic infection of hepatocytes.

Mutat Res, 1999 Jul 21, 444(1), 217 - 25
Evaluation of the genotoxicity of 2,4-dichlorophenoxyacetic acid and its derivatives in mammalian cell cultures; Gollapudi BB et al.; 2,4-dichlorophenoxyacetic acid and its derivatives (collectively known as 2,4-D) are herbicides used to control a wide variety of broadleaf and woody plants . The genetic toxicity of an ester (2,4-D 2-butoxyethylester) and two salts (2,4-D isopropylamine and 2,4-D triisopropanolamine) was investigated in cultured mammalian cells . The end points used were the induction of chromosomal aberrations in primary cultures of rat lymphocytes and forward mutations at the HGPRT locus of Chinese hamster ovary cells . There was no evidence of genotoxicity for the test materials in the experimental systems used . These results were consistent with the general lack of genotoxic potential for 2,4-D in a number of other test systems.

Anticancer Drugs, 1999 Jun, 10(5), 445 - 52
Selective cell kill of the combination of gemcitabine and cisplatin in multilayered postconfluent tumor cell cultures; Padron JM et al.; Both gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and cisplatin (cis-diammine-dichloroplatinum) have significant anticancer activity against ovarian, head and neck, and non-small cell lung cancer (NSCLC) . dFdC can be incorporated into DNA and RNA, and inhibit DNA repair, while cisplatin can form Pt-DNA adducts . We previously observed schedule-dependent synergism of the combination of dFdC and cisplatin in monolayer cell cultures . We now evaluated whether the combination would also enable selective cell kill in multilayered postconfluent cell cultures, since each compound showed variable activity in multilayered cells . The combination was tested in multilayered cultures from cell lines with a different histological origin: the human head and neck squamous cell carcinoma cell line UMSCC-22B (22B), the human NSCLC cell line H322, and ADDP, a cisplatin-resistant variant of the human ovarian cancer cell line A2780 . Sensitivity of the multilayered cells was dependent on exposure duration and sequence of the drug combinations, which were added in a constant molar ratio (dFdC:cisplatin 1:100), with a total exposure time of 96 h . The type of interaction was related to the degree of resistance of the cell lines to either dFdC or cisplatin . Thus, the very sensitive 22B cells only showed an additive effect when cells were preincubated for 24 h with dFdC prior to exposure to the combination . In contrast, in the resistant ADDP and H322 cells, synergism was the most common profile (three out of four schedules tested) . This is of special relevance when we take into account that these drugs only show cytostatic effects when administered alone, whereas the combination produced cytotoxic cell killing . In conclusion, combining dFdC with cisplatin can be at least additive, but synergistic in multilayered postconfluent cells resistant to dFdC and cisplatin.

Clin Diagn Lab Immunol, 1999 Sep, 6(5), 729 - 33
Cell culture of sporadic hepatitis E virus in China; Huang R et al.; The isolation and identification of the 87A strain of epidemic hepatitis E virus (HEV) by means of cell culturing have been described previously . This paper reports the successful isolation of a sporadic HEV strain (G93-2) in human lung carcinoma cell (A549) cultures . The etiology, molecular and biological properties, and serological relationship of this new strain to other, epidemic HEV strains are described . The propagation of both sporadic and epidemic HEV strains in a cell culture system will facilitate vaccine research.

FEBS Lett, 1999 Aug 27, 457(2), 200 - 4
Induction of myelin gene expression in Schwann cell cultures by an interleukin-6 receptor-interleukin-6 chimera; Haggiag S et al.; Expression of myelin basic protein (MBP) and Po gene products is induced during the final postnatal maturation of Schwann cells and reinduced during nerve regeneration . We show that a chimeric protein containing interleukin-6 fused to its soluble receptor (IL6RIL6 chimera) induces MBP and Po RNAs and proteins in cultures of dorsal root ganglia (DRG) from 14 day old mouse embryos . Activation of gp130 signaling by IL6RIL6 appears comparable to cyclic AMP elevating agents to induce the myelin gene products in DRG and in pure Schwann cell cultures.

J Neurovirol, 1999 Aug, 5(4), 384 - 91
Restricted replication of herpes simplex virus in satellite glial cell cultures clonally derived from adult mice; Wilkinson R et al.; To determine the possible influence of satellite glial cells on restricting the spread of herpes simplex virus in the peripheral nervous system, HSV replication was studied in clonally derived cultures of satellite glial cells from adult animals . Satellite cells were purified by exploiting their close anatomical association with primary sensory neurons . Dissociated neurons from dorsal root ganglia were micro-manipulated to remove all but one of the attached satellite cells and cultured in the presence of the mitogenic stimulators bovine pituitary extract and cholera toxin . Following a lag phase of 20-30 days some of the individual satellite cells began to proliferate . Initial cultures demonstrated bipolar morphology similar to cultured Schwann cells, some of which differentiated into large astrocytic whorl-like cells on subsequent passage . Immunocytochemical and molecular studies demonstrated that these cells, designated Sat.1, express glial fibrillary acidic protein, confirming their glial origin and by electron microscopy they were shown to be phagocytic . Under single step viral growth conditions Sat.1 cells were restrictive for HSV replication, producing in the order of 1000 times less infectious virus than Vero cells, a standard permissive cell line . These results suggest that satellite cells, which tightly encase sensory neurons, play a role in restricting interneural spread of HSV within the peripheral nervous system.

Folia Med (Plovdiv), 1999, 41(1), 43 - 5
In vitro study on cytotoxic effect of some chemicals on serum McCoy and serum-free McCoy-Plovdiv cell culture systems; Docheva D et al.; The cytotoxicity of test agents on serum-free McCoy cultures has not been studied at all . The cytotoxic effect of EDTA, methanol, DMSO, and cycloheximide on serum-free McCoy-Plovdiv cell culture (SF) was detected visually on inverted microscope and quantitatively by tests for viability (NR) and total protein (KBP) . The IC50 values for the tested chemicals were calculated . SF showed the lowest IC50 values for cycloheximide, DMSO and EDTA and the highest for methanol according to both tests . EDTA, methanol, DMSO and cycloheximide had dose-effect relationship in the cell test systems after treatment . The data indicate that McCoy-Plovdiv cell line is a suitable serum-free cell system for in vitro cytotoxic studies.

Histochem Cell Biol, 1999 Jul, 112(1), 41 - 9
Characterisation of caveolins from cartilage: expression of caveolin-1, -2 and -3 in chondrocytes and in alginate cell culture of the rat tibia; Schwab W et al.; This study was performed to determine if rat articular chondrocytes express caveolin, the structural protein of caveolae, and to determine differences in the distribution of the caveolin subtypes 1, 2 and 3 in knee joints of newborn and adult rats . All three subtypes of caveolin were detected in adult cartilage by immunocytochemical staining . In newborn rats, only caveolin-1 was found in the hyaline cartilage . Caveolin-1, -2 and -3 messenger RNA and protein were also detected in chondrocyte cell cultures . Ultrastructural investigations of cell culture and cartilage tissue revealed the presence of caveolae at the plasma membrane of chondrocytes . These findings represent the first report on the different expression of caveolin isoforms, in particular the expression of the muscle cell-specific caveolin-3 in chondrocytes . There is evidence that caveolin-2 and -3 are upregulated during growth and development of articular cartilage, suggesting a role for caveolins in chondrocyte differentiation.

Proc Soc Exp Biol Med, 1999 Sep, 221(4), 386 - 90
5-hydroxytryptamine evokes endothelial nitric oxide synthase activation in bovine aortic endothelial cell cultures; McDuffie JE et al.; Activation of endothelial nitric oxide synthase (eNOS) results in the production of nitric oxide (NO) that mediates the vasorelaxing properties of endothelial cells . The goal of this project was to address the possibility that 5-hydroxytryptamine (5-HT) stimulates eNOS activity in bovine aortic endothelial cell (BAEC) cultures . Here, we tested the hypothesis that 5-HT receptors mediate eNOS activation by measuring agonist-stimulated {3H}L-citrulline ({3H}L-Cit) formation in BAEC cultures . We found that 5-HT stimulated the conversion of {3H}L-arginine ({3H}L-Arg) to {3H}L-Cit, indicating eNOS activation . The high affinity 5-HT1B receptor agonist, 5-nonyloxytryptamine (5-NOT)-stimulated {3H}L-Cit turnover responses were concentration-(0.01 nM to 100 microM) and time-dependent . Maximal responses were observed within 10 min following agonist exposures . These responses were effectively blocked by the 5-HT1B receptor antagonist, isamoltane, the 5-HT1B/5-HT2 receptor antagonist, methiothepin, and the eNOS selective antagonists (0.01-10 microM): L-Nomega -monomethyl-L-arginine (L-NMMA) and L-N omega-iminoethyl-L-ornithine (L-NIO) . Pretreatment of BAEC cultures with pertussis toxin (PTX; 1-100 ng/ml) for 16 hr resulted in significant inhibition of the agonist-stimulated eNOS activity, indicating the involvement of Gi proteins . These findings lend evidence of a 5-HT1B receptor/eNOS pathway, accounting in part for the activation of eNOS by 5-HT . Further investigation is needed to determine the role of other vascular 5-HT receptors in the stimulation of eNOS activity.

J Med Virol, 1999 Oct, 59(2), 215 - 20
Comparison of two nested PCR, cell culture, and antigen detection for the diagnosis of upper respiratory tract infections due to influenza viruses; Magnard C et al.; Influenza surveillance requires sensitive and rapid diagnostic methods . Different diagnostic procedures have been evaluated on a selected set of nasal swabs sample collected from patients presenting with acute respiratory infection . One hundred fifty-four samples collected during the peak of the influenza epidemic recorded during winter of 1997-1998 in the south of France were processed for influenza detection using antigen detection (ELISA-immunocapture assay), two different nested RT-PCR assays (targeting M and HA genes), and cell culture . Among 154 samples, 93 (60.4%) were positive for influenza detection . Forty specimens (26%) were positive by ELISA, 77 (50%) by culture, 88 (57.1%) using the multiplex HA-PCR and 76 (49.4%) using the M-PCR . Multiplex HA-PCR was thus the most sensitive test . The PCR assay offers an alternative to culture for influenza detection . Nevertheless, culture is efficient for influenza diagnosis and is the only technique that allows the reference centres to collect viral strains and characterise fully new variants .

Acta Anat (Basel), 1992, 145(4), 307 - 20
Experiments on the induction of antibody dependent macrophage-mediated cellular cytotoxicity in mixed brain cell cultures; Korn J; These examinations were based on the discussion whether in demyelinating diseases anti-lipid antibody associated with brain macrophages could have a cytotoxic effect on oligodendrocytes . We used mixed brain cell cultures of newborn rats where, among others, both oligodendrocytes and vacuolated macrophage-like cells were found . On these macrophage-like cells, the presence of Fc-receptors was proven . Besides Fc-receptor-dependent phagocytosis, these cells showed an Fc-receptor-independent type of phagocytosis . The Fc-receptor-bearing cells moved within the culture and adhered to glass fibers . In the cytoplasm of these cells, unspecific esterase, acid phosphatase and peroxidase could be visualized . The vacuolated cells showed strong autofluorescence, expressed a surface marker found on all types of rat leukocytes and were marked by Griffonia simplicifolia lectin . These results definitely characterized the vacuolised cells as macrophages . We saw globular and pleomorphic macrophages . After incubation of anti-GC serum in a highly diluted solution, significantly more macrophages bound to oligodendrocytes than in the controls . In these cases, we found target cell lysis . It could be shown in vitro that anti-GC serum together with macrophages of neonatal brains can induce a cytotoxic effect on oligodendrocytes.

Bone Marrow Transplant, 1999 Jul, 24(2), 179 - 89
Natural killer (NK) cells prevent virus production in cell culture; Baraz L et al.; Natural killer (NK) cells (CD3-/CD16+/CD56+ lymphocytes) play an important role in early immune defense against viral infection, a fact which is of prime significance for heavily immunosuppressed patients after bone marrow transplantation . In this study we demonstrate that NK cells preferentially lyse human colon adenocarcinoma (Colo-205) tumor cells infected with herpes simplex virus type 1 (HSV-1) and vaccinia virus (VV) and autologous T cells infected with VV . This phenomenon was assessed by the viral infectious center (IC) method and compared with the results obtained by means of the standard 51Cr-release assay . Using the IC assay, we found that NK cells lyse virus infected cells at an early stage of infection, thereby preventing viral dissemination to neighboring cells . 51Cr-release assay verified by propidium iodide (PI) penetration showed that the early effects of NK mediated anti-viral activity are not the result of membrane damage . The effect of NK cells on HSV-1 infected Colo-205 cells appears to be independent of the level of expression of major histocompatibility complex (MHC) class I molecules while the killing of autologous VV-infected T cells correlates with a reduction in MHC class I expression . Our results suggest that additional factors besides MHC play a role in the regulation of NK cell-mediated lysis of virus infected cells . This may be of clinical importance in patients who are heavily immunosuppressed after bone marrow transplantation.

Clin Infect Dis, 1999 May, 28(5), 1100 - 3
Antibody response after a four-site intradermal booster vaccination with cell-culture rabies vaccine; Tantawichien T et al.; The current World Health Organization recommendation for booster vaccination of previously immunized individuals with potential exposure to rabies is two doses of vaccine intramuscularly or intradermally on days 0 and 3 . We report responses to two types of postexposure treatment of healthy individuals who had received preexposure rabies vaccination 1 year previously . Group A individuals received four intradermal doses (one-fifth of the diluent volume of vaccine per dose) on day 0, and group B individuals received two intramuscular doses on days 0 and 3 . Immunogenicity of the two booster regimens was assessed by titrating the amount of neutralizing antibody (Nab) . We found that the booster doses of vaccine produced remarkable responses in all subjects . Nab titers of > or = 0.5 IU/mL (acceptable antibody level for protection against rabies) were detected in all subjects on day 14, and they were shown to be consistently high 1 year after the booster vaccination . We also found that the Nab titers for group A were significantly higher (two- to eightfold) than those for group B on days 5, 14, 150, and 360 after the initial booster vaccination (P < .05) . Our study shows that the four-site intradermal booster regimen with use of one-fifth of the diluent volume of cell-culture rabies vaccine on day 0 is associated with a significantly higher antibody response than is the conventional booster regimen for subsequent postexposure rabies treatment of individuals who have received preexposure rabies vaccination with cell-culture rabies vaccine 1 year previously.

FEBS Lett, 1999 Jul 30, 456(1), 41 - 4
Short-term block of Na+/K+-ATPase in neuro-glial cell cultures of cerebellum induces glutamate dependent damage of granule cells; Stelmashook EV et al.; Granule cells in a dissociated neuro-glial cell culture of cerebellum when exposed to ouabain (10(-3) M) for 25 min apparently swell, increase their {Ca2+}i with obvious depolarization of the mitochondrial membrane . In 3 h after ouabain was omitted from the solution, 62 +/- 3% of granule cells had pycnotic nuclei . The supplement of a solution with competitive specific antagonist of NMDA receptors, L-2-amino-7-phosphonoheptanoate (10(-4) M, APH) together with ouabain prevented cells from swelling, mitochondrial deenergization, neuronal death and increase of {Ca2+}i . These data suggest that cellular Na+/K+-ATPase inactivation in neuro-glial cell cultures of cerebellum leads to glutamate (Glu) accumulation, hyperstimulation of glutamate receptors, higher Ca2+ and Na+ influxes into the cells through the channels activated by Glu . This process leads to cell swelling, mitochondrial deenergization and death of granule cells . Possibly, the decrease of Na+/K+-ATPase activity in brain cells can lead to the onset of at least some chronic neurological disorders.

Zhonghua Yan Ke Za Zhi, 1997 Sep, 33(5), 357 - 9
{Cell culture of human lamina cribrosa in vitro}; Luo X et al.; OBJECTIVE: To investigate the physiology and pathology of the biosynthesis of extracellular matrix (ECM) by lamina cribrosa cells (LCC) and to establish a model of cultured LCC . METHODS: The LCC of 10 human dead fetal eyes were cultured with enzyme digestion procedures in vitro . The cultured cells were identified by immunohistochemistry assays and electron microscopy . RESULTS: The cultured cells were flat, polygonal in shape, and their cytoplasm contained abundant perinuclear granules . The nuclei were relatively transparent and nucleoli were clear, there were basement membrane-like material and ECM surrounding the cell membrane . The immunofluorescent staining was positive for collagen type I, III, IV, fibronectin and laminin, negative for glial fibrillar acid protein and factor-VIII . The characteristics mentioned above were coincident with the morphology of LCC . This cell line has been cultured to its fourth generation . CONCLUSION: We have successfully cultured a cell line of LCC, and it may be utilized in the study of pathogenesis of glaucoma.

J Nutr Sci Vitaminol (Tokyo), 1999 Apr, 45(2), 203 - 12
Formation of 8,11,14-octadecatrienoic acid (18:3 n-4) from naturally occurring unique fatty acid, 9,12-hexadecadienoic acid (16:2 n-4), in animal cell cultures; Nakano N et al.; 9,12-Hexadecadienoic acid (16:2 n-4), present in small amounts in fish oils as a naturally occurring unique fatty acid, was incorporated into the phospholipids in rat liver BRL-3A cells to a similar extent as linoleic acid (18:2 n-6) . 11,14-Octadecadienoic acid (18:2 n-4) and 8,11,14-octadecatrienoic acid (18:3 n-4) were detected in the cellular lipids of BRL-3A cells when incubated in a medium supplemented with 16:2 n-4 methyl ester . The cellular levels of these acids increased in parallel with 16:2 n-4 methyl ester added to the medium . These compounds were probably formed through conversion from 16:2 n-4 to 16:3 n-4 by delta 6 desaturation, and then 18:3 n-4 was produced by elongation, and part of the surplus 16:2 n-4, not desaturated to 16:3 n-4, elongated to 18:2 n-4 . These results suggested that 16:2 n-4 was desaturated by delta 6 desaturase in vitro . It was also shown that 16:2 n-4 inhibited arachidonic acid synthesis from exogenous linoleic acid in BRL-3A cells as efficiently as alpha-linolenic acid (18:3 n-3).

Int J Cancer, 1999 Sep 24, 83(1), 135 - 40
Inverse regulation of neuronal cellular adhesion molecule (NCAM) by IFN-gamma in melanoma cell cultures established from CNS lesions; Geertsen R et al.; In advanced stages of malignant melanoma (MM), metastases to the CNS are frequently observed . Few results are available on trophic factors and immunological features involved in the process of invasion and adhesion of circulating metastatic cells into the CNS . A direct comparison of remote metastases found in different locations of the same patient might help to identify such properties . For this purpose, we screened a panel of MM cell cultures, which had been established from patients with surgically removed MM lesions of the CNS, for expression and regulation of immunorelevant molecules . The results were compared with standard controls and cultures established from non-CNS metastatic lesions of the same patients . No significant differences were observed for expression of HLA-I, HLA-II, ICAM-1 and the melanoma-associated antigens Mage-3, MelanA and tyrosinase . Constitutive expression of the neuronal cell adhesion molecule (NCAM) was found in all CNS-derived samples and in fewer than 50% of non-CNS derived cultures . IFN-gamma was found to have a weak up-regulating effect in all non-CNS-derived cultures, except normal melanocytes . However, in 6/7 CNS-derived cultures, pre-treatment with IFN-gamma reduced expression of NCAM to 28% to 77% of the level in untreated cultures . The presence and regulation of NCAM differs between MM cells derived from CNS metastases and non-CNS-derived melanocytic cells . Thus, NCAM might be a candidate immunoregulating molecule involved in the formation of CNS metastases of MM .

Brain Res, 1999 Jul 17, 835(1), 74 - 9
Instability of the CAG repeat in immortalized fibroblast cell cultures from Huntington's disease transgenic mice; Manley K et al.; Huntington's Disease transgenic mice were used for an exploration into the stability of a trinucleotide repeat . The brain shows heterogeneous somatic instability that increases quantitatively with age . To test somatic CAG-repeat alterations during long-term culture, DNA was extracted from transgenic tissue, primary fibroblasts, and SV40-immortalized fibroblasts at intervals of approximately 100 cell doublings . In fibroblasts derived from an adult mouse, there was an initial short truncation of the repeat, followed by an emerging population of cells showing continuous slow expansion . After 15 months in continuous culture (approximately 600 cell doublings following transformation) the major CAG peak has increased from 155 to approximately 170 triplets . This in vitro system can now be used to assay factors that affect instability .

J Cell Sci, 1999 Sep, 112 ( Pt 17), 2823 - 32
Matrix expression and proliferation of primary gingival fibroblasts in a three-dimensional cell culture model; Hillmann G et al.; The growth of cultured primary human gingival fibroblasts and the three-dimensional arrangement of the extracellular matrix in a polyester carrier system was investigated using various histological techniques . The results were compared with monolayer cultures . Collagen types I, III, V, and VI were investigated by conventional and fluorescence microscopy, scanning and transmission electron microscopy, and confocal laser scanning microscopy . Human gingival fibroblasts were obtained from tissue biopsies of five donors and were cultivated up to 5 weeks under three-dimensional culture conditions . The cells displayed an elongated, spindle-like or stellate morphology resembling the in vivo situation . Collagen type I revealed thick fiber bundles, and collagens type III and V were distributed as fine fibrils or small bundles throughout the culture system . Frequently, the fibers were oriented parallel to the long axis of the cells . Type VI collagen formed thin fibers and revealed a reticular pattern . In histological sections the cultured cells exhibited a morphology clearly different from that of cells cultured in monolayers . Their shape and spatial distribution resembled that of cells in tissue biopsies more closely . The culture system presented here promotes a dynamic model for performing studies for instance on the interactions of cultured cells with extracellular matrix molecules, on the pathogenesis of inflammatory processes or on the interactions with biomaterials, thus providing qualitative and quantitative information.

J Neuroendocrinol, 1999 Jul, 11(7), 491 - 502
Expression of functional growth hormone secretagogue receptors in human pituitary adenomas: polymerase chain reaction, triple in-situ hybridization and cell culture studies; Barlier A et al.; We examined the expression of functional growth hormone secretagogue receptors (GHS-R) in a series of 30 human pituitary adenomas-six secreting GH, three GH-PRL, six prolactin (PRL), five adrenocorticotrophic hormone (ACTH), one thyroid stimulating hormone (TSH), four gonadotroph and five non-secreting adenomas . By reverse transcriptase polymerase chain reaction (RT-PCR), the coexpression of the two GHS-R isoforms (Ia and Ib) was found in all the GH-, GH-PRL- and PRL-secreting adenomas, and only in two out of three corticotroph, two out of four gonadotroph and one out of five non-secreting tumours . They were absent in the TSH-secreting adenoma . The PCR products of GHS-R Ia and Ib were identical in size to those from two normal pituitaries . PCR cloning and sequencing of isoforms performed in two somatotroph adenomas revealed only two single, silent base mutations . Triple in-situ hybridization showed colocalization of GHS-R mRNA with messengers of GH and PRL, conjointly or separately, in individual cells of somatotroph, mammosomatotroph, and lactotroph adenomas . The presence of GHS-R mRNA in cells expressing PRL mRNA is emphasized . In cultured cells from six somatotroph and two mammosomatotroph adenomas, the powerful GHS MK-0677 stimulated GH release in a dose-dependent manner, with maximal effect at 6 h . Contrarily, when GHRH was applied, only three somatotrophs and two mamosomatotrophs were stimulated . In the two mammosomatotrophs, the PRL response to MK-0677 and to GHRH was similar to the GH response . An homologous desensitization of the GHS-R and the GHRH receptor was observed 24 h after a first stimulation by a single dose of the corresponding agonist . Heterologous desensitization was not observed . Interestingly, MK-0677 also stimulated, in a dose-dependent way, the hormone release of cells from all tested lactotroph and corticotroph adenomas . The existence of a functional expression of GHS-R in somatotroph, mammosomatotroph, lactotroph and corticotroph adenomas rises the question of the role played by GHS-R in pituitary adenomas, particularly those not engaged in GH secretion.

Arch Esp Urol, 1999 Mar, 52(2), 133 - 9
{Development of cell cultures in basic research on renal carcinoma}; Moreno Sierra J et al.; OBJECTIVE: The development of in vitro cell tissue culture techniques has provided most of the current knowledge on the physiology of both normal and tumor cells . The results of cell culture studies using nephrectomy specimens are presented . METHODS: Surgical specimens from 7 patients (3 males, 42.9%; 4 females, 57.1%) aged 43-79 years (mean 62.14), who had undergone nephrectomy for renal adenocarcinoma between April 1997 and February 1998 were utilized . The tumor cell samples were obtained during surgery, after the kidney had been excised . The samples were cut, washed in a balanced saline solution and enzymatically dissociated in a trypsin solution (0.1% Hank's solution) for 30 minutes at 37 degrees, centrifuged, placed in Multiwell plates, covered with 0.1 mg/ml polylysine (ICN) or 3 mg/ml collagen S (Boehringer) and cultured in RPMI 1640 (Sigma) supplemented with 5% fetal calf serum (reagent), which was changed every 2-3 days for one month . After the culture had developed, samples were fixed in 70% ethanol and stained with hematoxylin-eosin for cell identification . Cell types were identified by cytokeratin analysis . RESULTS: The histological types were: clear cell renal adenocarcinoma in three cases (42.9%), renal oncocytoma in two (28.6%), mixed cells renal adenocarcinoma in one (14.3%) and papillary carcinoma in one case (14.3%) . Polylysine and collagen were found to be good substrates for normal and tumor cell culture . Polylysine was found to be a better substrate for epithelial cells; there were less epithelial and more mesangial cells in the collagen substate . The positive cytokeratin expression (a marker for intermediate filaments) corroborates the well-known epithelial origin of renal cell carcinoma . Epithelial cells from normal kidney grew well in the culture medium . Mean cell survival was 30.83 days (range 15-50) . To evaluate the viability of the culture, positivity for a neutral red stain was used as marker for good metabolic activity after one month . All cultures that survived for more than one month (5 of 7) stained positively . CONCLUSIONS: The study and development of human cell lines is useful to analyze the metabolic aspects of renal carcinoma . Cell cultures permit conducting genetic and molecular studies and investigating the radio and chemosensitivity of renal carcinoma in vitro.

Lijec Vjesn, 1999 Apr-May, 121(4-5), 137 - 43
{Skin cell culture: utilization in plastic surgery and laboratory studies}; Boranic M et al.; Skin is the largest organ of the human body (8-10 kg, 1.5-2.0 m2, 10(11) cells of epidermal, mesenchymal and neural origin) . Although endowed with remarkable regeneration ability, the recovery after major injuries viz . burns requires appropriate surgical treatment, temporary coverage of defects and supportive measures . Large defects are covered with viable transplants of autologous or allogeneic skin, frozen or lyophilized human and animal skin, bioartificial tissues made of synthetic or biodegradable materials, sheets of keratinocytes cultured in vitro . The use of autotransplants is limited by the size of preserved skin areas as well as by general condition of the patient . Allotransplants collected from cadavers or volunteers are rejected after 1 or 2 weeks and thus afford only temporary coverage . Grafts of human or animal skin devitalized by lyophilization or freezing in glycerol accommodate connective tissue and blood vessels ingrowing from the graft bed but eventually dissolve . Artificial skin consists of collagen, chondroitin or similar fiber network (substituting the dermis) covered by semipermeable silicon foil (substituting the epidermis) . After healing in, the silicon foil is peeled off and replaced by skin autotransplants or autologous keratinocytes grown and expanded in vitro . The technique for massive production of human keratinocytes, invented some twenty years ago, has been applied for clinical purposes by several specialized centers . During the culture period of approximately three weeks the keratinocyte population may enlarge five to ten thousandfold . Keratinocytes obtained from a 1.5 cm2 piece of skin (half of a postal stamp) may thus yield progeny sufficient for the coverage of 1.5 m2, which is almost the total body surface . The period required for culturing autologous keratinocytes is bridged by temporary transplants and vigorous supportive treatment of the patient . Cultured keratinocytes display all essential features of keratinocytes in situ . They divide and differentiate, express membrane structures required for intercellular communication and reception of signals regulating cell division and differentiation, secrete cytokines . In addition to clinical application, the culture of human keratinocytes is a convenient and useful model for studies of cellular biology . This review is illustrated by first examples of keratinocyte cultures grown in our laboratory.

Philos Trans R Soc Lond B Biol Sci, 1999 Jun 29, 354(1386), 1047 - 55
Evidence for both the nucleus and cytoplasm as subcellular sites of pathogenesis in Huntington's disease in cell culture and in transgenic mice expressing mutant huntingtin; Hackam AS et al.; A unifying feature of the CAG expansion diseases is the formation of intracellular aggregates composed of the mutant polyglutamine-expanded protein . Despite the presence of aggregates in affected patients, the precise relationship between aggregates and disease pathogenesis is unresolved . Results from in vivo and in vitro studies of mutant huntingtin have led to the hypothesis that nuclear localization of aggregates is critical for the pathology of Huntington's disease (HD) . We tested this hypothesis using a 293T cell culture model system by comparing the frequency and toxicity of cytoplasmic and nuclear huntingtin aggregates . Insertion of nuclear import or export sequences into huntingtin fragments containing 548 or 151 amino acids was used to reverse the normal localization of these proteins . Changing the subcellular localization of the fragments did not influence their total aggregate frequency . There were also no significant differences in toxicity associated with the presence of nuclear compared with cytoplasmic aggregates . These studies, together with findings in transgenic mice, suggest two phases for the pathogenesis of HD, with the initial toxicity in the cytoplasm followed by proteolytic processing of huntingtin, nuclear translocation with increased nuclear concentration of N-terminal fragments, seeding of aggregates and resultant apoptotic death . These findings support the nucleus and cytosol as subcellular sites for pathogenesis in HD.

Clin Infect Dis, 1999 Jul, 29(1), 193 - 5
Isolation of eastern equine encephalitis virus in A549 and MRC-5 cell cultures; Sotomayor EA et al.; Eastern equine encephalitis (EEE) has been diagnosed either serologically or by virus isolation . Until now, the recovery of EEE virus has been delegated to reference laboratories with the expertise and resources needed to amplify the virus in a susceptible vertebrate host and/or to isolate and identify the virus in cell culture . We report a case in which EEE virus was recovered directly from a patient's cerebrospinal fluid in A549 and MRC-5 cell cultures . Many clinical virology laboratories routinely use these cells to recover adenovirus, herpes simplex virus, and enterovirus . To the best of our knowledge, this is the first report of isolation of EEE virus in A549 cell culture . This report demonstrates the possibility of recovery of EEE virus in cell culture without the necessity of bioamplification or maintaining unusual cell lines.

Endocrinology, 1999 Aug, 140(8), 3835 - 42
Identification of link protein during follicle development and cumulus cell cultures in rats; Kobayashi H et al.; Cumulus oocyte complex (COC) expansion is induced through hyaluronic acid production and accumulation of proteins of the inter-alpha-trypsin inhibitor family in the gonadotropin-stimulated cumulus cells . Link protein, a glycoprotein found in cartilage, interacts specifically with hyaluronic acid and stabilizes the binding of proteoglycan monomers to hyaluronic acid to form aggregates . The aim of this study was to investigate the expression of immunoreactive link protein during follicle development in rats and in cumulus cells in culture by immunohistochemistry and Western blot as well as by specific enzyme-linked immunosorbent assay . Immunohistochemical analysis revealed that the extracellular matrix of cumulus cells that were morphologically at a stage of COC expansion were markedly stained for link protein, whereas granulosa cells from immature follicles were not stained . Cumulus cells deposited link protein into the extracellular matrix in an in vitro culture system . The staining intensity was negated by the treatment with hyaluronidase, suggesting that the link protein is bound to hyaluronic acid . We have identified a 42-kDa immunoreactive link protein in rat ovary during the preovulatory period and in COC extracts . Addition of FSH to the medium of cumulus cells in culture supplemented with 10% FBS and oocyte-conditioned medium resulted in an increased rate of link protein synthesis . This work suggests that the cumulus cells synthesize the link protein that may stabilize the binding of inter-alpha-trypsin inhibitor or dermatan sulfate proteoglycan to hyaluronic acid to make up hyaluronic acid-rich matrix aggregate.

Ophthalmologe, 1999 Jun, 96(6), 375 - 81
{Quantifying the damage to conjunctival and corneal cell cultures caused by uv light using CASY (Cell Analysis System) . A method for reducing animal experiments}; Schmut O et al.; BACKGROUND: By depletion of stratospheric ozone, enhanced levels of UV radiation reach the surface of the earth . Exposure of the anterior parts of the eye to UV radiation leads to irritation of the conjunctival and corneal cells . METHOD: By the CASY (cell analysis) system the influence of UV radiation on cultures of conjunctival and corneal cells was observed by determination of the cell counts, cell diameter, and the cell volume . RESULTS: By comparing these parameters with the control, damage of the conjunctival and corneal cells by UV radiation can be determined within 2 s of exposure of the cells in quartz glass vials to the UV light . CONCLUSION: By the CASY system the dramatic influence of UV radiation on cells of the anterior parts of the eye can be determined . This system enables objective statements on the cytotoxicity of radiation, chemical substances and drugs on cell cultures without the use of radioactive methods, complicated determination of cell metabolism or staining methods which are difficult to standardize . Also, studies on animals can be reduced by the CASY system.

Fertil Steril, 1999 Jul, 72(1), 71 - 6
Induction of bone formation in rat osteoprogenitor cell culture by sera of climacteric women before and after hormone replacement therapy; Rojansky N et al.; OBJECTIVE: To evaluate the effect of hormone replacement therapy (HRT) on growth and differentiation of cultured osteoprogenitor cells . DESIGN: Prospective clinical study . SETTING: Outpatients in a menopause clinic . PATIENT(S): Women with climacteric symptoms . INTERVENTION(S): Daily oral conjugated estrogen, 0.625 mg, and medroxyprogesterone acetate, 2.5 mg, for 7-12 months . Bone density measurement before HRT and blood sampling before and after HRT . MAIN OUTCOME MEASURE(S): Sera of climacteric women were added to the culture of rat osteoprogenitor cells, and indices of cell proliferation and differentiation (alkaline phosphatase activity and mineralization) were measured before and after HRT . RESULT(S): Sera after HRT significantly decreased cell counts but not alkaline phosphatase activity or mineralization as compared with sera before HRT . However, mineralization induced in the bioassay by both sera showed a positive correlation (r = 0.56) with E2 levels before treatment and a negative correlation (r = -0.6181) with time in menopause of serum donors . The change in mineralization showed a significant correlation with hip bone mineral density z scores (r = -0.67) but not with spine z scores (r = -0.1915), whereas the change in cell count correlated with spine bone mineral density z scores (r = 0.49) only . CONCLUSION(S): Changes in serum-induced cell proliferation and mineralization may be helpful in studying the response to HRT in climacteric women . Serum-induced mineralization is more efficient in diagnosing osteopenia than in monitoring HRT effects.

Ther Apher, 1999 Aug, 3(3), 257 - 63
In vitro cell culture systems as the basis for an extracorporeal blood purification strategy in multiorgan failure treatment; Mitteregger R et al.; Multiorgan failure (MOF) based on septic processes is very common but prognostically an extremely severe disease that has to be treated exclusively under intensive care conditions . Extracorporeal blood purification (ECBP) using specific and efficient systems such as the microspheres based detoxification system (MDS) (Artif Organs 1996;20:420) could improve significantly the situation of MOF in terms of the efficient removal of endotoxins as well as key mediators such as tumor necrosis factor alpha (TNF alpha) . The purpose of the study was to test the effectiveness of endotoxin and cytokine removal to blunt cellular response . In terms of the in vitro principle methodology, isolated peripheral blood mononuclear cells (PBMC) were incubated with endotoxins and a selective endotoxin adsorbent, which was added at various times (immediately or 30, 60, 120, 240, or 360 min) after the onset of incubation . TNF alpha release of monocytes was measured following a standard procedure after 20 h . Human TNF alpha was incubated with cultured human endothelial cells with and without a specific TNF alpha adsorbent (polyclonal antibodies coated on polystyrene particles) . The results showed that after the initial addition of endotoxins, the activation of monocytes can be stopped within 120 min by addition of endotoxin adsorbents . In addition, specific TNF alpha adsorbents are able to prevent intercellular adhesion molecule 1 (ICAM-1) expression of endothelial cells, therefore avoiding activation of endothelial cells . In conclusion, cell culture models are suitable to simulate cell interaction in MOF . Specific adsorbents are able to reduce or block pathophysiologically relevant cell interactions, and the time frame for effective ECBP seems to be very short, and therefore, efficiency must be high.

Appl Environ Microbiol, 1999 Aug, 65(8), 3427 - 32
Detection of infectious Cryptosporidium parvum oocysts in surface and filter backwash water samples by immunomagnetic separation and integrated cell culture-PCR; Di Giovanni GD et al.; A new strategy for the detection of infectious Cryptosporidium parvum oocysts in water samples, which combines immunomagnetic separation (IMS) for recovery of oocysts with in vitro cell culturing and PCR (CC-PCR), was field tested with a total of 122 raw source water samples and 121 filter backwash water grab samples obtained from 25 sites in the United States . In addition, samples were processed by Percoll-sucrose flotation and oocysts were detected by an immunofluorescence assay (IFA) as a baseline method . Samples of different water quality were seeded with viable C . parvum to evaluate oocyst recovery efficiencies and the performance of the CC-PCR protocol . Mean method oocyst recoveries, including concentration of seeded 10-liter samples, from raw water were 26.1% for IMS and 16.6% for flotation, while recoveries from seeded filter backwash water were 9.1 and 5.8%, respectively . There was full agreement between IFA oocyst counts of IMS-purified seeded samples and CC-PCR results . In natural samples, CC-PCR detected infectious C . parvum in 4.9% (6) of the raw water samples and 7.4% (9) of the filter backwash water samples, while IFA detected oocysts in 13.1% (16) of the raw water samples and 5.8% (7) of the filter backwash water samples . All CC-PCR products were confirmed by cloning and DNA sequence analysis and were greater than 98% homologous to the C . parvum KSU-1 hsp70 gene product . DNA sequence analysis also revealed reproducible nucleotide substitutions among the hsp70 fragments, suggesting that several different strains of infectious C . parvum were detected.

J Mol Cell Cardiol, 1999 Aug, 31(8), 1419 - 27
Preservation of contractile characteristics of human myocardium in multi-day cell culture; Janssen PM et al.; Functional studies of different human cell types have been successfully conducted under in vitro conditions . Despite many efforts, it has not been possible to develop a human myocardial preparation in which contractile function can be studied over several days . We hypothesize that by mimicking the in vivo situation in an in vitro environment we can preserve viability and function of human myocardial preparations over several days . From explanted hearts of patients undergoing cardiac transplantation, multicellular preparations were dissected and mounted in a sterile muscle chamber . Muscles were stimulated at 0.5 or 1 Hz at 1.75 mmol/l Ca(2+), a pH of 7.4 and at 37 degrees C, and kept contracting isometrically for 2-6 days . This study shows for the first time that contractile function of human myocardial tissue can be preserved over several days; active force development had not significantly changed after 48 h (10.6+/-1.2 at t=0 v 11.4+/-2.8 mN/mm(2)at t=48, n=10), nor had diastolic force (1.0+/-0.1 v 0.9+/-0.1 mN/mm(2)) . After at least 48 h, the contractile response to stimulation with 1 micromol/l isoproterenol was clearly present: developed force increased to 631+/-146% of control values, while half-relaxation time declined to 57+/-6% (n=7) . In addition, both pharmacological responses and regulatory physiological behavior, such as post-rest potentiation and force-frequency relationships, are preserved . This technique allows the study of the regulation of contractile function of human myocardium in vitro and may be used to link changes in protein expression to consequent changes in myocardial contraction .

Histochem J, 1999 Mar, 31(3), 175 - 80
Effect of follicular cells on calcitonin gene expression in thyroid parafollicular cells in cell culture; Zabel M et al.; Expression of calcitonin (CT) gene in thyroid parafollicular cells involves alternate formation of CT mRNA or CGRP mRNA . High amounts of CT mRNA are formed only in thyroid gland and formation of CGRP mRNA dominates in the remaining organs . Apart from paracrine and endocrine factors, mRNA formation on the CT gene seems to be affected also by direct contacts with other cells present in the thyroid gland, in which parafollicular cells are located next to follicular cells . The present study aimed at examining whether thyroid follicular cells affect formation of mRNAs for CT and CGRP in parafollicular cells . The studies were performed in cell cultures . A parafollicular cell line (TT cells) and a follicular cell line (F6BTY cells) served as the experimental model . For comparison, co-cultures with fibroblasts, 3T3 cells, and malignant melanoma, MM cells, were also examined . CT gene expression was examined at the level of mRNAs (in situ hybridization and morphometric studies) and at the level of hormones (immunocytochemistry, morphometric studies and radioimmunological estimation of hormone levels in the medium) . The immunocytochemical and hybridocytochemical studies, in line with morphometry studies, demonstrated that F6BTY and 3T3 cells were capable of affecting mRNA production for CT and CGRP and that they changed the ratio of CGRP/CT secretion by TT cells, as a sequel of contact between the two cell types and due to mediation of secreted substances . On the other hand, the malignant melanoma MM cells showed no effect on the secretion ratio . Our study seems to indicate that control of mRNA formation from CT gene may involve not only humoral factors but also direct contacts with other cells, which may explain differences in expression of the gene between cells localized in different organs.

Methods Find Exp Clin Pharmacol, 1999 Jun, 21(5), 331 - 8
Effects of Cerebrolysin on in vitro primary microglial and astrocyte rat cell cultures; Lombardi VR et al.; In recent years the potential use of neurotrophic factors in the prevention and/or treatment of neurodegenerative diseases has received much attention . To determine whether Cerebrolysin, a porcine brain-derived peptide preparation, was able to modulate in vitro lipopolysaccharide (LPS)-induced microglial activation and to test the direct effect of Cerebrolysin on astrocyte morphology, survival and proliferation, rat glial and astrocyte cell culture experiments were carried out . The morphology of microglia, ameboid/activated and flat/resting, was examined under contrast microscopy and cell counts obtained . In addition, the release of interleukin (IL)-1 beta and brain-derived neurotrophic factor (BDNF) was measured from cell culture supernatant using an enzyme-linked-immunoassay (ELISA) . The results obtained in this study clearly suggest a protective effect of Cerebrolysin as revealed by downregulation of microglial activation after LPS treatment as well as by the control of IL-1 beta expression . No significant differences were observed on astrocyte morphology, survival or the production and/or release of BDNF . In conclusion, these in vitro studies indicate that Cerebrolysin might exert a neuroimmunotrophic function which can in turn reduce the extent of inflammation and accelerate neuronal death under pathological conditions such as human neurodegenerative disorders.

Ann N Y Acad Sci, 1999 Jun 18, 875, 353 - 63
Three-dimensional in vitro cell culture leads to a marked upregulation of cell function in human hepatocyte cell lines--an important tool for the development of a bioartificial liver machine; Selden C et al.; Upregulating hepatocyte function in proliferating human liver cell lines could provide cells for a bio-artificial liver . Ideally, a means of mimicking the biological extracellular matrix with a relatively inert, bio-compatible matrix is required . Alginate encapsulation of primary hepatocytes is biocompatible . This study aimed to characterize cells grown in a 3D configuration in alginate . A human-derived liver cell line encapsulated in 1% alginate was assessed for synthetic and detoxification functions . Secreted proteins measured (e.g., albumin, fibrinogen, alpha-1-antitrypsin etc.) were increased in alginate compared with monolayers . Cytochrome P450 1A1 activity increased three- to fourfold, whilst urea synthesis, undetectable in monolayer cultures, was synthesized by cells in alginate at levels approaching in vivo production . TEM revealed good ultrastructure reminiscent of normal hepatocytes . Alginate promotes 3D colonies of proliferating cells with upregulated liver functions . Rapid recovery of function of cryopreserved cells (< 18 h) provides added advantages for this system to support the biological component of an artificial liver for patients with fulminant hepatic failure.

Toxicol Lett, 1999 Jun 30, 107(1-3), 81 - 7
Induction of mitotic cell division distrubances and mitotic arrest by pyrethroids in V79 cell cultures; Hadnagy W et al.; Five pyrethroids (fenvalerate, deltamethrin, cypermethrin, permethrin, cyfluthrin) differing in their chemical purity were investigated on their cytotoxic effects, especially on their ability to induce mitotic cell division disturbances using Chinese hamster lung cells of line V79 . The colony forming ability (CFA) resulted in distinct differences of the cytotoxic effect of the tested pyrethroids, whereby permethrin was found to be most toxic . With the exception of fenvalerate all tested pyrethroids gave rise to inhibition of cell cycle progression as shown by G2/M-arrest of synchronized V79 cells by flow cytometry as well as by the increase of the mitotic index as evaluated by light microscopy . The mitotic arresting activity could be attributed to the occurrence of abnormal mitotic figures such as initial and full C-metaphases . The results however indicate, that pyrethroids per se do not contribute to the cytotoxic effects but that other factors such as chemical impurities, source as well as manufacturing process and isomer composition may be responsible for the observed cytotoxic effects.

Neuroscience, 1999, 92(2), 699 - 704
Hypoxia during early developmental period induces long-term changes in the dopamine content and release in a mesencephalic cell culture; Gross J et al.; The present study was conducted to elucidate the long-term effects of exposure to hypoxia of dopaminergic neurons during the early developmental period . Primary mesencephalic cell cultures prepared from fetal rats and containing 0.5-2% of dopaminergic neurons were exposed to hypoxia between in vitro days 1 and 6, the putative critical developmental period . Changes in the content, release and uptake of dopamine were found to depend on the degree of hypoxia and on the duration of exposure . Following moderate hypoxia (7 h, 5% O2) on two consecutive days between in vitro days 1 and 3, the cultures showed a small increase in the dopamine levels, by 16% . After severe hypoxia (0% O2/95% N2 for 24 h), during the same time window, the cellular dopamine content was elevated by 100% . Moreover, severe hypoxia produced long-lasting modulations of the dopaminergic system . On in vitro day 14, cells exhibited increased levels of 3,4-dihydroxyphenylacetic acid and homovanillic acid (by 34% and 55%, respectively), and elevations of both the spontaneous and potassium-stimulated dopamine release by 70% . The dopamine transport and metabolism of cells exposed to hypoxia between in vitro days 4 and 6 remained unchanged with regard to long-term effects . The present study provides strong evidence for the induction of long-term changes in dopaminergic cells due to hypoxia during the critical developmental period in mesencephalic culture . The developmental period capable of inducing long-lasting changes in dopamine metabolism is restricted to in vitro days 1-3.

J Clin Microbiol, 1999 Aug, 37(8), 2518 - 24
Invasion and intracellular development of the human granulocytic ehrlichiosis agent in tick cell culture; Munderloh UG et al.; Human granulocytotropic ehrlichias are tick-borne bacterial pathogens that cause an acute, life-threatening illness, human granulocytic ehrlichiosis (HGE) . Ehrlichias within neutrophil granulocytes that invade tick bite sites are likely ingested by the vector, to be transmitted to another mammalian host during the tick's next blood meal . Thus, the cycle of replication and development in the vector is prerequisite to mammalian infection, and yet these events have not been described . We report tick cell culture isolation of two strains of the HGE agent directly from an infected horse and a dog and have also established a human isolate from HL60 culture in tick cells, proving that the blood stages of the HGE agent are infectious for tick cells, as are those replicating in the human cell line HL60 . This required changes to the culture system, including a new tick cell line . In tick cell layers, the HGE agent induced foci of infection that caused necrotic plaques and eventual destruction of the culture . Using the human isolate and electron microscopy, we monitored adhesion, internalization, and replication in vector tick cells . Both electron-lucent and -dense forms adhered to and entered cells by a mechanism reminiscent of phagocytosis . Ehrlichial cell division was initiated soon after, resulting in endosomes filled with numerous ehrlichias . During early development, pale ehrlichias with a tight cell wall dominated, but by day 2, individual bacteria condensed into dark forms with a rippled membrane . These may become compacted into clumps where individual organisms are barely discernible . Whether these are part of an ehrlichia life cycle or are degenerating is unknown.

Dev Biol Stand, 1999, 99, 153 - 6
Elimination of serum from cell culture medium; Lubiniecki AS; Growth of continuous cell lines for preparing biopharmaceuticals in the absence of animal serum has been attempted by many organizations to improve process and product quality, prevent exposure to adventitious agents, and reduce costs . Literature surveys suggest that substantial academic studies on serum-free medium have been pursued for many decades, with varying levels of success for different cell types and cell lines in terms of achieving cell growth while retaining cell function . Industrial research proceeded for at least three decades . Recent work with CHO cells and with some hybridomas has been successful in providing the basis for serially propagating cells on a large scale in suspension in the total absence of serum, while preserving the ability to prepare biopharmaceuticals . In some cases, this can be achieved not only without serum, but also without the use of other animal-derived proteins.

Dev Biol Stand, 1999, 99, 3 - 8
Benefits and risks due to animal serum used in cell culture production; Wessman SJ et al.; Infection with bovine viral diarrhoea virus (BVDV) and other viruses is frequent in the bovine population . In utero infection leads to virus and antibody contamination of foetal and other serum used in cell culture production . The use of contaminated cells for vaccine production may result in contaminated vaccines, which may lead to seroconversion or disease in the vaccinated animal . Contaminated serum or cell cultures may also interfere with the diagnosis of viral infections . Methods for the detection of BVDV and other viruses in serum, cell cultures, seed viruses and vaccines at the CVB-L, and the frequency of detection are described . Reasons for continued use of serum in cell culture production, and the risks of using serum, are discussed.

Clin Chim Acta, 1999 May, 283(1-2), 21 - 32
Thiol and redox reactive agents exert different effects on glutathione metabolism in HeLa cell cultures; Hultberg B et al.; Glutathione protects cells against oxidative damage, free radical damage and other types of toxicity . The aim of the present study was to investigate the impact on glutathione metabolism exerted by different thiol or redox reactive agents . Intracellular concentrations of glutathione in HeLa cell cultures were lowered after addition of agents mainly exerting oxidative stress (homocysteine and hydrogen peroxide), whereas thiol reactive oxidative agents (mercury ions, copper ions and hydroquinone) in concentrations not affecting cell growth seemed to stimulate the production of glutathione . Possibly, the thiol reactive agents decrease the concentration of glutathione, thereby stimulating further synthesis of glutathione, since glutathione synthesis is subject to feedback regulation by glutathione on gamma-glutamylcysteine synthase . Redox changes after addition of thiol and redox reactive agents were also investigated . Copper ions lowered the concentrations of reduced forms of all extracellular thiols and of intracellular reduced cysteine in HeLa cell cultures . The addition of mercury ions, hydroquinone, homocysteine or hydrogen peroxide did not change the proportions between reduced and total thiol concentrations . After addition of buthionine sulfoxime, the total concentrations of intra- and extracellular glutathione were markedly decreased and the ratio between reduced and total glutathione concentrations was lowered . However, both cysteine and homocysteine exhibited normal ratios between the concentrations of reduced and total thiols in the presence of buthionine sulfoxime . This finding could be due to other cellular antioxidants, such as thioredoxin, ascorbic acid or tocopherols, maintaining redox status of these thiols.

Biochem Pharmacol, 1999 Jul 1, 58(1), 95 - 107
Cell cycle-dependent distribution and specific inhibitory effect of vectorized antisense oligonucleotides in cell culture; Helin V et al.; Factors limiting the use of antisense phosphodiester oligodeoxynucleotides (ODNs) as therapeutic agents are inefficient cellular uptake and intracellular transport to RNA target . To overcome these obstacles, ODN carriers have been developed, but the intracellular fate of ODNs is controversial and strongly depends on the means of vectorization . Polyamidoamine dendrimers are non-linear polycationic cascade polymers that are able to bind ODNs electrostatically . These complexes have been demonstrated to protect phosphodiester ODNs from nuclease degradation and also to increase their cellular uptake and pharmacological effectiveness . We studied the intracellular distribution of a fluorescein isothiocyanate-labeled ODN vectorized by a dendrimer vector and found that intracellular ODN distribution was dependent on the phase of the cell cycle, with a nuclear localization predominantly in the G2/M phase . In addition, in order to evaluate the relevance of ODN vectors in enhancing the inhibition of the targeted genes' expression, we developed a rapid screening system which measures the transient expression of two reporter genes, one used as target, the other as control and vice versa . This system was validated through investigating the effect of the dendrimer vector on ODN biological activity . Antisense sequence-specific inhibition of more than 70% of one reporter gene was obtained with a chimeric ODN containing four phosphorothioate groups, two at each end.

Glia, 1999 Jul, 27(1), 53 - 61
Regulation of gelatinases in microglia and astrocyte cell cultures by plant lectins; Liuzzi GM et al.; The effects of 25 recently discovered plant lectins on cell proliferation and enzyme release were compared to those of previously known lectins on rat microglia and astrocyte cell cultures . A dose-dependent proliferation of microglial cells, but not of astrocytes, was induced by seven lectins, whereas five lectins showed dose-dependent cytotoxicity on both microglia and astrocyte cell cultures . The activity of gelatinase B (MMP-9) was strongly increased in microglial cells by the aforementioned seven lectins, by concanavalin A, and by phytohemagglutinin (PHA-E4), whereas gelatinase A (MMP-2) remained at constitutive levels . The five cytotoxic lectins decreased the activity of gelatinase B in microglia and of gelatinase A in astrocytes, in a dose-dependent manner . The lectin wheat germ agglutinin induced a decrease in gelatinase B activity in microglia, but stimulated gelatinase A and B activity in astrocytes . These results indicate that lectins possess neuromodulatory effects that may motivate the study of their effects on central nervous system (CNS) function in vivo . This, in turn, may lead to better insight into whether lectin or lectin-like molecules can interact with glial cells, and whether they have a role in acute toxicity and in multifactorial diseases in which environmental factors may play a role.

Neurosci Lett, 1999 Jun 11, 268(1), 1 - 4
Insulin effects on extracellular signal regulated kinase cascade in fetal rat astrocyte cell cultures; Schechter R et al.; Insulin within the nervous system promotes cell differentiation and survival . In addition extracellular signal regulated kinase (ERK) promotes cell differentiation and survival . We studied the effects of insulin on astrocyte ERK-1 and 2, in fetal enriched astrocyte cell cultures incubated in an insulin free-defined medium (G3 medium) . After 2 days in G3 medium the astrocytes were stimulated with 5 ng/ml of insulin for 2 min, and insulin effects on insulin receptor, insulin receptor substrate-1 (IRS-1) and ERK-1 and 2 were studied using Western blots . Insulin inhibited the phosphorylation of the insulin receptor and IRS-1 and decreased the activity of the ERK-1, and inhibited the activation of the ERK-2 . Thus, insulin is involved in ERK regulation in fetal astrocytes.

J Biomed Mater Res, 1999 Jan, 44(1), 44 - 52
Signal transduction and cytoskeletal reorganization are required for cell detachment from cell culture surfaces grafted with a temperature-responsive polymer; Yamato M et al.; We have developed a new cell culture substrate grafted with a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm) using an electron beam irradiation method . These surfaces are hydrophobic in culture at 37 degrees C due to the hydration/dehydration changes intrinsic to PIPAAm at 32 degrees C, and they become highly hydrophilic below 32 degrees C . At 37 degrees C grafted and ungrafted surfaces showed no difference with regard to attachment, spreading, growth, confluent cell density, and morphology of bovine aortic endothelial cells . Stress fibers, peripheral bands, and focal contacts were established in similar ways . After the medium temperature was decreased to 20 degrees C, spread cells lost their flattened morphology, acquiring a rounded cell appearance similar to that of cells immediately after plating . After mild agitation cells floated free from the dish surface without trypsin treatment . Neither cell morphological changes nor cell detachment occurred on ungrafted surfaces . An ATP synthesis inhibitor, sodium azide, and a tyrosine kinase inhibitor, genistein, suppressed cell morphological changes and cell detachment while a protein synthesis inhibitor, cycloheximide, slightly enhanced cell detachment . An actin filament stabilizer, phalloidin, and its depolymerizer, cytochalasin D, also inhibited cell detachment . These findings suggest that cell detachment on grafted surfaces is mediated by intracellular signal transduction and reorganization of the cytoskeleton . While trypsinization causes damage to the cell membrane surface and extracellular matrix proteins, this alternative low temperature treatment is exceptionally noninvasive . The temperature-responsive cell culture surface also should prove useful for investigating the molecular machinery involved in cell-surface detachment .

Avian Dis, 1999 Apr-Jun, 43(2), 182 - 8
Genome analysis of Marek's disease virus strain CVI-988: effect of cell culture passage on the inverted repeat regions; van Iddekinge BJ et al.; The genomes of different derivatives of Marek's disease virus (MDV) strain CVI-988, a low oncogenic isolate of a serotype 1 MDV, were analyzed by restriction enzyme analyses to detect whether alterations occurred after passages in cell culture . DNA molecules of strain 988 isolated directly from blood cells contained mainly two copies of the 132-bp repeat sequence previously reported within BamH1-H and -D fragment as previously reported for more virulent MDV strains . Although a minority of virus particles showed repeat amplification was already at the fifth passage level, amplification mainly occurred between passages 17 and 34 in cell culture . In addition, a 400-bp deletion was detected within the BamH1-A fragment of two derivatives of CVI-988, 988C and 988C/R6.

Proc Natl Acad Sci U S A, 1999 Jul 6, 96(14), 8074 - 9
Exon shuffling mimicked in cell culture; van Rijk AA et al.; Undesired side products of DNA transfections are usually discarded . However, here, we show that such products may provide insight into mutational events that are also a major driving force in protein evolution . While studying the small heat-shock protein alphaA-crystallin, we transfected the hamster alphaA-crystallin gene into a mouse muscle cell line . One of the stable transfected cell lines expressed, in addition to the expected normal alphaA- and alternatively spliced alphaAins-crystallins, two slightly larger, immunologically cross-reacting proteins . These proteins were found to be encoded by a mutant alphaA-crystallin gene with a large intragenic duplication, arisen by illegitimate recombination at two CCCAT homologies, approximately 1.8 kilobases apart in the normal hamster alphaA-crystallin gene . As a consequence, a tandem-duplicated exon 3 sequence is present in the mature mRNA of this gene, resulting in a 41-residue repeat in the translated proteins . Cells expressing the elongated alphaA-crystallins have normal growth characteristics and the usual diffuse cytoplasmic distribution of immunoreactive alphaA-crystallin . Size-exclusion chromatography of cell extracts indicated that the mutant proteins are readily incorporated into the normal large water-soluble alphaA-crystallin complexes, showing that the insert does not disturb the integrity of these complexes . This viable alphaA-crystallin mutant thus mimics the origins and effects of exon duplication, which is a common consequence of exon shuffling in mammalian genome evolution.

Am J Pathol, 1999 Jul, 155(1), 123 - 33
A cell culture system for the study of amyloid pathogenesis . Amyloid formation by peritoneal macrophages cultured with recombinant serum amyloid A; Kluve-Beckerman B et al.; A murine macrophage culture system that is both easy to employ and amenable to manipulation has been developed to study the cellular processes involved in AA amyloid formation . Amyloid deposition, as identified by Congo red-positive, green birefringent material, is achieved by providing cultures with recombinant serum amyloid A2 (rSAA2), a defined, readily produced, and highly amyloidogenic protein . In contrast to fibril formation, which can occur in vitro with very high concentrations of SAA and low pH, amyloid deposition in culture is dependent on metabolically active macrophages maintained in neutral pH medium containing rSAA2 at a concentration typical of that seen in acute phase serum . Although amyloid-enhancing factor is not required, its addition to culture medium results in larger and more numerous amyloid deposits . Amyloid formation in culture is accompanied by C-terminal processing of SAA and the generation of an 8.5-kd fragment analogous to amyloid A protein produced in vivo . Consistent with the possibility that impaired catabolism of SAA plays a role in AA amyloid pathogenesis, treatment of macrophages with pepstatin, an aspartic protease inhibitor, results in increased amyloid deposition . Finally, the amyloidogenicity exhibited by SAA proteins in macrophage cultures parallels that seen in vivo, eg, SAA2 is highly amyloidogenic, whereas CE/J SAA is nonamyloidogenic . The macrophage culture model presented here offers a new approach to the study of AA amyloid pathogenesis.

Am J Pathol, 1999 Jul, 155(1), 67 - 70
Mitochondrial DNA depletion syndrome is expressed in amniotic fluid cell cultures; Blake JC et al.; Mitochondrial DNA depletion syndrome is an autosomal inherited disease associated with grossly reduced cellular levels of mitochondrial DNA in infancy . Most patients are born after a full and uncomplicated pregnancy, are normal at birth, but develop symptoms in the early neonatal period . These observations have led to the suggestion that the patients have a defect affecting the control of mitochondrial DNA copy number after birth . Using immunocytochemical techniques, we demonstrated that the disease is already expressed in amniotic fluid cells . Detection of mitochondrial DNA depletion in these fetal cells indicates that the defect may already be expressed early in embryological development.

Vet Microbiol, 1999 Jun 1, 67(1), 1 - 12
Identification of group I porcine enteroviruses by monoclonal antibodies in cell culture; Dauber M; A panel of monoclonal antibodies (mAb) against porcine enteroviruses (PEV) was established . One of these mAbs reacts group-specifically with PEV of serotype group I in the indirect immunofluorescence assay (IIF) . This mAb is very well suited for diagnosis of PEV infections . However, the mAb neither neutralizes virus nor does it react with virus particles in immuno electron microscopy (IEM) . Another mAb is PEV-1 specific in IIF, neutralizes virus, and is suited for IEM . Both mAbs presumably recognize conformation-dependent epitopes of the virus.

Appl Microbiol Biotechnol, 1999 May, 51(5), 579 - 85
Metabolic responses to different glucose and glutamine levels in baby hamster kidney cell culture; Cruz HJ et al.; In this work, a BHK21 clone producing a recombinant antibody/cytokine fusion protein was used to study the dependence of cell metabolism on the glucose and glutamine levels in the culture medium . Results obtained indicate that both glucose and glutamine consumptions show a Michaelis-Menten dependence on glucose and glutamine concentrations respectively . A similar dependence is also observed for lactate and ammonia productions . The estimated value of the Michaelis constant for the dependence of lactate production on glucose (KLacGlc) was 1.4 +/- 0.1 mM and for the dependence of ammonia production on glutamine (KAmmGln) was 0.25 +/- 0.11 mM and 0.10 +/- 0.03 mM, at glucose concentrations of 0.28 mM and 5.6 mM respectively . At very low glucose concentrations, the glucose to lactate yield decreased markedly, showing a metabolic shift towards lower lactate production . This metabolic shift was also confirmed by the significant increase in the specific oxygen consumption rate also observed at low glucose concentrations . Although it was highly dependent on glucose concentration, the oxygen consumption also increased with the increase in glutamine concentration . At very low glutamine concentrations, the glutamine to ammonia yield increased, showing a more efficient glutamine metabolism.

Dig Dis Sci, 1999 Jun, 44(6), 1208 - 15
Decreased corticosensitivity in quiescent Crohn's disease: an ex vivo study using whole blood cell cultures; Franchimont D et al.; Corticosensitivity influences the degree and the duration of an inflammatory reaction by altering target cell responses to endogenous and/or exogenous glucocorticoids . Indeed, different clinical responses to glucocorticoids have been observed among patients with Crohn's disease, suggesting different degrees of corticosensitivity in these subjects . The purpose of this study was to compare the corticosensitivity of patients with quiescent Crohn's disease to that of healthy subjects (HS) . Nineteen patients with quiescent Crohn's disease and 14 HS were studied; all patients were steroid-free for at least six months; 7 of the 19 were corticosteroid-dependent (CSD) and treated with nonglucocorticoid immunosuppressants at the time of the study . Corticosensitivity was measured by the inhibition of LPS-induced cytokine secretion in whole blood cell cultures treated with increasing concentrations (10(-9) to 10(-6) M) of dexamethasone . Tumor-necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-1 beta (IL-1 beta) were measured using specific immunoassays . Crohn's disease patients had a markedly decreased dexamethasone-mediated inhibition of TNF-alpha (P < 0.01), IL-6 (P < 0.001), and IL-1 beta (P < 0.01) compared to healthy subjects, with a shift of the dexamethasone dose-response curve to the right . No significant differences in the basal and LPS-stimulated secretion of the three cytokines were observed between CSD and non-CSD patients, and both subgroups of patients had similar degrees of dexamethasone-mediated cytokine inhibition . We conclude that patients with Crohn's disease have a significant decrease in the corticosensitivity of their leukocytes . This may be related to a specific genetic/constitutional background and/or could be acquired, due to inflammation-related endocrine and/or immune factors.

Radiat Res, 1999 Jul, 152(1), 76 - 82
Measurement of dose rate at the interface of cell culture medium and glass dishes by means of ESR dosimetry using thin films of alanine; Furre T et al.; Previous studies on human cervical cancer cells (NHIK 3025) have indicated that the cells, when X-irradiated in suspension, appeared to be more radiosensitive than when they were irradiated attached to glass dishes . However, this result depends on dosimetry, which is difficult in the situation where cells are attached to glass dishes due to backscattering electrons at the glass-liquid interface . Recently developed dosimetry that is based on detection of radiation-induced stable radicals in alanine and uses ESR spectroscopy offers a possibility for more relevant dosimetry at the glass-liquid interface than the previous estimates of doses based on Fricke dosimetry . Thin alanine films (>/=10 microm) were used to measure dose at the interface by irradiating the films while they were placed tightly against the bottom of dishes and covered with 1 mm of wax simulating the medium above cells . Fricke dosimetry was also performed, with different depths of Fricke solution in the dishes, to elucidate the contribution to the dose delivered by backscattering electrons at the glass-liquid interface . A dose rate of 1.9 Gy/min was measured with a thin layer (0.2-0.3 mm) of Fricke solution in petri dishes made of glass . However, this estimate appears to be too high, due to a contribution to dose by short-ranged electrons generated when the X rays passed through a steel lid 4.5 cm above the dishes . Dosimetry using alanine films resulted in dose rates of 1.15 and 0.87 Gy/min at the interfaces of glass-liquid and plastic- liquid, respectively . Hence there is a significant contribution to dose from backscattering electrons on dishes made of glass . The reason for our previous observation of a difference in radiosensitivity between cells irradiated in suspension and cells irradiated attached to glass appears to be a lack of accurate dosimetry at the glass-liquid interface.

Eur J Pharm Sci, 1999 Jul, 8(3), 167 - 75
Mucus as a barrier to the permeability of hydrophilic and lipophilic compounds in the absence and presence of sodium taurocholate micellar systems using cell culture models; Meaney C et al.; A mucus secreting CaCo-2-Ht29GlucH cell co-culture model was characterised and used to examine the influence of mucus as a barrier to the transport of hydrophilic and lipophilic compounds in the absence and presence of sodium taurocholate micellar (NaTC) systems . TEER measurements and permeability studies using the hydrophilic markers (mannitol, polyethylene glycols (PEGS) 900 and 4000) indicated that the paracellular permeability of the co-culture model was greater than that of the CaCo-2 model . At pH 7.4, no difference in the transport of a model lipophilic drug, dextropropoxyphene, was observed between the two models . However, at pH 4.5, when the drug was highly ionised the transport was significantly lower across the co-culture monolayers . NaTC micellar systems appeared to affect the different cell culture models in the order CaCo-2>CaCo-2-Ht29GlucH>Ht29GlucH . Following removal of the mucus layer by incubation with the mucolytic agent, N-acetyl-l-cysteine, the absorption enhancing potential of NaTC micellar systems was increased in the co-culture model.

Toxicology, 1999 Apr 15, 133(2-3), 85 - 92
Impairment of TNF-alpha expression and secretion in primary rat liver cell cultures by acetaminophen treatment; Nastevska C et al.; Tumor necrosis factor-alpha (TNF-alpha) is assumed to act as a mediator in toxic liver injury, aggravating the primary damage to the parenchymal liver cell, but also stimulating liver regeneration . Reports on the effect of acetaminophen in vivo on TNF-alpha transcript concentrations and serum TNF-alpha concentrations, under different experimental, or clinical conditions have yielded controversial results . We used primary rat hepatocyte and Kupffer cell cultures to test the direct action of subtoxic and toxic concentrations of acetaminophen on TNF-alpha expression and release . We observed a dose-dependent decrease of TNF-alpha mRNA in the hepatocytes, and of TNF-alpha release into the medium of hepatocyte cultures . The data also indicate an impairment of TNF-alpha release in Kupffer cell cultures after treatment with nontoxic, as well as with toxic, acetaminophen concentrations . The results suggest that inhibition of TNF-alpha expression and release in the liver is a consequence of acetaminophen exposure . It is at present unknown how this effect modulates the course of acetaminophen intoxication.

Arh Hig Rada Toksikol, 1998 Sep, 49(3), 219 - 29
Application of anterior pituitary cell culture in toxicological research; Zechner-Krpan V et al.; High density plating procedure was used to evaluate the effect of atrazine on anterior pituitary cells of rats in monolayer culture . Collagenase-dispersed pituitary cells plated in suspension with medium-199 and 10% foetal calf serum attached quantitatively to plastic surfaces within 24 hours . Electron microscopy showed subpopulations of different cell types . After prolonged cultivation, most cells established small colonies with extensive contacts among them . Cell-to-cell formation of aggregates was significant and the colonies manifested morphological changes . The cells retained their enzymatic activity, converting testosterone into 5 alpha-dihydrotestosterone by enzyme 5 alpha-reductase . Immunohistochemical techniques facilitated differentiation of gonadotrophs producing follicle stimulating hormone (FSH) and luteinising hormone (LH) . Atrazine in concentrations of 5 to 50 micrograms/ml of medium was associated with a significant reduction in the number of viable cells within 72 hours . The results suggest that the pituitary cell culture may prove useful in toxicological testing of various toxic compounds and reduce or replace in vivo animal experiments.

Exp Eye Res, 1999 Jul, 69(1), 97 - 107
Maintenance of retinoid metabolism in human retinal pigment epithelium cell culture; von Recum HA et al.; If transplantation of cultured retinal pigment epithelium (RPE) or iris pigment epithelium (IPE) is to be successful in the treatment of ocular disease, it is imperative to demonstrate that these cells can perform all of their necessary metabolic functions . Unfortunately, a critical function of the RPE, retinoid metabolism, is often lost rapidly in culture . We have examined whether or not nonspecific proteolytic enzymes commonly used in cell isolation and serial passaging may be responsible for this loss of function, and we have investigated novel isolation and passaging techniques which can alleviate this loss of retinoid metabolism.RPE cells were obtained from human donor eyes by enzymatic and nonenzymatic methods . Cells were cultured either on control tissue culture inserts or on inserts coated with a layer of thermally responsive poly(N -isopropylacrylamide-co-cinnamoylcarbamidemethylstyrene) . Upon confluence, cells were detached either by trypsinization or by lowering dish temperature . Retinoid metabolism of cells was assessed after isolation and culture by incubating membrane fractions with3H-all- trans -retinol . Retinoid metabolism was also measured in freshly isolated IPE, corneal endothelium (CE), an RPE cell line (D407), and two hepatocyte cell lines (Hepa 6 and HepG2).Membrane fractions from cells isolated nonenzymatically or using collagenase/hyaluronidase formed 11- cis -retinol, retinal isomers and retinyl esters . Retinoid metabolism of RPE cells freshly isolated by trypsinization showed no 11- cis -retinal and little 11- cis -retinol formation . Nondamaged cells cultured on thermally responsive surfaces detached in sheets upon temperature change . They showed metabolism similar to that of cells freshly isolated by nonenzymatic means . After trypsinization, confluent cultures dissociated into individual cells, but these cells showed poor retinoid metabolism, including no detectable retinyl esters or 11- cis -retinoid isomers . IPE, CE and Hepa 6 did not show any retinoid metabolism . D407 and HepG2 produced retinals, but not the 11- cis isomer.RPE cells isolated using trypsin lose the ability to form critical intermediates in the visual cycle . Collagenase/hyaluronidase or nonenzymatic cell isolation techniques enable these functions to be maintained . After cell culture, thermally responsive surfaces allow nonenzymatic cell detachment and excellent maintenance of retinoid metabolism .

Nucleic Acids Res, 1999 Jul 1, 27(13), 2737 - 44
Enzymatic and antisense effects of a specific anti-Ki-ras ribozyme in vitro and in cell culture; Giannini CD et al.; Due to their mode of action, ribozymes show antisense effects in addition to their specific cleavage activity . In the present study we investigated whether a hammerhead ribozyme is capable of cleaving mutated Ki-ras mRNA in a pancreatic carcinoma cell line and whether antisense effects contribute to the activity of the ribozyme . A 2{prime}-O-allyl modified hammerhead ribozyme was designed to cleave specifically the mutated form of the Ki- ras mRNA (GUU motif in codon 12) . The activity was monitored by RT-PCR on Ki- ras RNA expression by determination of the relative amount of wild type to mutant Ki-ras mRNA, by 5-bromo-2{prime}-deoxy-uridine incorporation on cell proliferation and by colony formation in soft agar on malignancy in the human pancreatic adenocarcinoma cell line CFPAC-1, which is heterozygous for the Ki-ras mutation . A catalytically inactive ribozyme was used as control to differentiate between antisense and cleavage activity and a ribozyme with random guide sequences as negative control . The catalytically active anti-Ki-ras ribozyme was at least 2-fold more potent in decreasing cellular Ki-ras mRNA levels, inhibiting cell proliferation and colony formation in soft agar than the catalytically inactive ribozyme . The catalytically active anti-Ki-ras ribozyme, but not the catalytically inactive or random ribozyme, increased the ratio of wild type to mutated Ki-ras mRNA in CFPAC-1 cells . In conclusion, both cleavage activity and antisense effects contribute to the activity of the catalytically active anti-Ki-ras hammerhead ribozyme . Specific ribozymes might be useful in the treatment of pancreatic carcinomas containing an oncogenic GTT mutation in codon 12 of the Ki-ras gene.

Int J Pharm, 1999 Apr 15, 180(2), 225 - 34
Temporal dependence of ectopeptidase expression in alveolar epithelial cell culture: implications for study of peptide absorption; Forbes B et al.; There is little data available regarding the extent of peptide metabolism encountered following inhalation to the lung . We have studied the activity of five ectopeptidases in primary rat alveolar epithelial cells, A549 cells and pulmonary macrophages . Peptidase activity was assayed in the plasma membrane fractions (PMF) of primary type II alveolar epithelial cells (ATII cells) after 2 days in culture and after 7 days in culture when they had formed monolayers of type I-like cells (ATI-like cells) . Dipeptidyl peptidase IV (DPP) activity fell from 36.65 mU/mg protein to 16.29 mU/mg protein between day 2 and day 7 in culture, aminopeptidase N (AMN) activity increased from 16.16 mU/mg protein to 23.99 mU/mg protein, angiotensin-converting enzyme (ACE) activity was lost (4.29 mU/mg protein at day 2, not detected at day 7), and carboxypeptidase M (CPM) activity was acquired (not detected at day 2, 5.20 mU/mg protein at day 7) . The profile of exopeptidase expression in A549 cells was similar to that of primary rat alveolar cells at day 7 in culture (DPP 24.24 mU/mg protein, AMN 47.74 mU/mg protein, CPM 4.28 mU/mg protein, ACE not detected) . Macrophages expressed high levels of aminopeptidases (DPP 46.85 mU/mg protein, AMN 28.28 mU/mg protein) but carboxypeptidase activity was not detected . Low neutral endopeptidase 24.11 (NEP) activity was found in all cell types studied (0.96-2.41 mU/mg protein) . The qualitative and quantitative changes in the peptidase activity of primary cultured rat alveolar cells between day 2 and day 7 in culture has implications for the use of alveolar cell monolayers as drug absorption models to investigate peptide absorption from the lung . Ectopeptidase activity in cultured alveolar cells can be used to infer the peptide-metabolising capacity of the surface of the alveolar epithelium . The aminopeptidase activity in particular, if representative of enzyme activity in vivo, would offer a significant metabolic barrier to systemic delivery of peptides via the lung . Copyright

Calcif Tissue Int, 1999 Jul, 65(1), 59 - 65
Aluminum accelerates osteoblastic differentiation but is cytotoxic in long-term rat calvaria cell cultures; Bellows CG et al.; We have examined the effects of aluminum (Al) on osteoprogenitor proliferation and differentiation, cell survival, and bone formation in long-term rat calvaria (RC) cell cultures . RC cells were grown in alpha minimal essential medium containing 10% fetal bovine serum, 50 microg/ml ascorbic acid, and 10 mM beta-glycerophosphate with or without Al added to final concentrations of 1 microM-1 mM . Al caused a dose-dependent increase in the number of bone nodules present at early times (day 11) but had no significant effect on nodule numbers at later times (day 17) . Time course experiments showed that Al increased nodule number beginning from day 7 . Alkaline phosphatase activity, assessed at four stages during the differentiation sequence of RC cell cultures (from 4 to 13 days) was stimulated by Al at all times . However, Al decreased colony formation, inhibited cell growth in late log phase, and decreased saturation density of the treated cultures . Al concentrations of 30 microM and above resulted in degeneration of the cell layer and an increasing fibrillar appearance of the matrix present in between or adjacent to nodules when cultures were maintained for more than 15 days . The presence of Al significantly decreased the viability of cells obtained from 13-17 days cultures, as determined by plating efficiency and trypan blue exclusion . We frequently observed cellular toxicity (in 8 of 10 experiments) in cultures containing 300 microM Al, and by days 17-19, cells, nodules, and matrix were disintegrating in these cultures . We conclude that Al accelerates the rate of osteoprogenitor cell differentiation and the formation of bone nodules while concomitantly inhibiting nodule mineralization . However, concentrations that accelerate differentiation appear to be cytotoxic in long-term cultures.

J Am Soc Mass Spectrom, 1999 Jun, 10(6), 502 - 11
Investigation of cell culture media infected with viruses by pyrolysis mass spectrometry: implications for bioaerosol detection; Madonna AJ et al.; Mass spectrometry coupled with a pyrolysis inlet system was used to investigate media from cell cultures infected with viruses . Cell culture media is an intricate mixture of numerous chemical constituents and cells that collectively produce complicated mass spectra . Cholesterol and free fatty acids were identified and attributed to lipid sources in the media (blood serum supplement and plasma membranes of host cells) . These lipid moieties could be utilized as signature markers for rapidly detecting the cell culture media . Viruses are intracellular parasites and are dependent upon host cells in order to exist . Therefore, it is highly probable that significant quantities of media needed to grow and maintain viable host cells would be present if a viral agent were disseminated as an aerosol into the environment . Cholesterol was also detected from a purified virus sample, further substantiating its use as a target compound for detection . Implications of this research for detection of viral bioaerosols, using a field-portable pyrolysis mass spectrometer, is described.

Anticancer Res, 1999 Mar-Apr, 19(2A), 1245 - 8
Falsification of tetrazolium dye (MTT) based cytotoxicity assay results due to mycoplasma contamination of cell cultures; Denecke J et al.; Mycoplasma contamination of cell cultures is a frequently observed problem . Due to the inconspicuous growth in cell cultures, periodical screening procedures represent the only protection . Many influences of mycoplasma on cell culture parameters have been described . We addressed the question of whether mycoplasma contamination affects the most frequently used cytotoxicity assay, the tetrazolium based MTT assay . We contaminated C6 glioma cells with mycoplasma and performed MTT assays with doxorubicin, vincristine, etoposide and cisplatinum under various conditions . Contaminated cells demonstrated significant different results when tested with the MTT assay than mycoplasma free controls . Differences were not detectable when cells were counted as toxicity assay . Due to an additional reduction of tetrazolium by mycoplasmas, contaminated cells appeared up to 15 fold resistant to doxorubicin, vincristine and etoposide, but not to cisplatinum . Differences decreased with decreasing drug doses and decreasing plated cell count . Our findings confirm the compelling need for periodical mycoplasma screening, especially when tetrazolium based cytotoxicity assay (MTT) are used.

Med Biol Eng Comput, 1998 Sep, 36(5), 654 - 8
Cell culture approach to biocompatibility evaluation of unconventionally prepared hydroxyapatite; Kundu PK et al.; Hydroxyapatite (HA), Ca10(PO4)6(OH)2 was produced by microwave irradiation of calcium nitrate (CaNO3.4H2O) and di-ammonium phosphate in aqueous solution . The HA formation was confirmed by X-ray diffraction analysis . HA prepared by this unconventional route was subjected to biocompatibility assay by a cell-culture method using the hybridoma cell line AE9D6 in both conventional Dulbecco's modification of Eagle's medium (DMEM) and simulated body fluid (SBF), both supplemented with 5% fetal calf serum . HA synthesised through this unconventional method showed the presence of tricalcium phosphate which can be reduced only after heat treatment at 1150 degrees C . The HA conformed to the X-ray data index file for hydroxyapatite . Biocompatibility assays showed reproducible growth and secretion patterns of cells both in DMEM as well as in SBF, thereby indicating the effectiveness of this method for the production of biocompatible HA.

Br J Anaesth, 1999 Feb, 82(2), 295 - 8
Comparison of basic methods in clinical studies and in vitro tissue and cell culture studies reported in three anaesthesia journals; Watters MP et al.; Tissue and cell culture (in vitro) studies reported in the 1997 issues of the British Journal of Anaesthesia, Anesthesia and Analgesia, and Anesthesiology were compared with groups of clinical studies selected at random from the same issues . Comparisons were of some basic aspects of study design and reporting that might lead to bias . The aspects examined were sample size, randomization and reporting of exclusions and withdrawals . Two groups of 53 articles were compared: sample size was smaller in in vitro than in clinical studies (median 6 vs 19); randomization was reported in five in vitro studies and in 37 studies; and failures were reported in two in vitro studies and in 43 clinical studies . This hinders interpretation of reported tissue and cell culture studies . Where possible, tissue and cell culture studies should be conducted, reported and assessed for publication to standards equivalent to those for clinical studies.

J Clin Microbiol, 1999 Jul, 37(7), 2137 - 41
Borrelia burgdorferi in tick cell culture modulates expression of outer surface proteins A and C in response to temperature; Obonyo M et al.; The Lyme disease spirochete Borrelia burgdorferi sensu stricto downregulates outer surface protein A (OspA) and upregulates outer surface protein C (OspC) during tick feeding . The switching of these proteins correlates with increased spirochetal infectivity for the mammal . We examined the effect of temperature on differential expression of OspA and OspC by B . burgdorferi cocultivated with a cell line isolated from the vector tick Ixodes scapularis . The effect of incubation at 31, 34, or 37 degrees C on expression of OspA and OspC by B . burgdorferi JMNT and N40 was analyzed by indirect fluorescent-antibody microscopy, polyacrylamide gel electrophoresis, and immunoblotting . The amount of OspA relative to the amount of flagellin was highest in spirochetes cocultivated with tick cells at 31 degrees C and declined with increasing temperature in both strains . OspC production was enhanced in spirochetes cocultivated with tick cells at 37 degrees C . Spirochetes grown axenically in BSK-H medium also produced more OspC at 37 degrees C, but OspA content was not appreciably affected by temperature . Our findings indicate that temperature, along with cultivation in a tick cell culture system, plays a role in the differential expression of OspA and enhances differential expression of OspC by spirochetes.

Math Biosci, 1999 Jun, 159(1), 47 - 78
A stochastic model of temporally regulated generation of oligodendrocytes in cell culture; Boucher K et al.; The results of our previous analyses suggest that O-2A progenitor cells become competent for differentiation in vitro after they complete a certain number of critical mitotic cycles . The number of critical cycles varies from clone to clone and should be thought of as a random variable . We propose an approach to the analysis of oligodendrocyte generation in vitro based on a stochastic model allowing for an arbitrary distribution of this random variable with a finite support . When applied to experimental data on clonal growth and differentiation of purified O-2A progenitor cells obtained from optic nerves of 1 and 7 day-old rats, the model provides a good quantitative description not only of the first two moments (mean and variance) of the number of O-2A progenitor cells and oligodendrocytes at different times after the start of experiment, but of the corresponding distributions as well . As our estimates show, there are scarcely any O-2A progenitor cells that divide in vitro more than twice before they acquire the competence for differentiation . Those O-2A cells that have undergone the critical divisions differentiate into an oligodendrocyte in each of the subsequent mitotic cycles with a certain probability . We give estimates of this probability for O-2A cells under different growth conditions . Our analysis suggests that the effect of thyroid hormone is twofold: it reduces the mean duration of the mitotic cycle for progenitor cells, and it increases the probability of their transformation into oligodendrocytes.

Blood, 1999 Jun 15, 93(12), 4425 - 35
The expression of human blood group antigens during erythropoiesis in a cell culture system; Southcott MJ et al.; Phenotypic analysis of hematopoietic stem and progenitor cells has been an invaluable tool in defining the biology of stem cell populations . We use here flow cytometry to examine the expression of human erythroid-specific surface markers during the maturation of early committed erythroid cells derived from cord blood in vitro . The temporal order of the expression of erythroid specific markers was as follows: Kell glycoprotein (gp), Rh gp, Landsteiner Wiener (LW) gp, glycophorin A (GPA), Band 3, Lutheran (Lu) gp, and Duffy (Fy) gp . The time at which some of these markers appeared suggests possible roles for some of these erythroid-specific polypeptides during the differentiation of these committed progenitors . The early appearance of Kell gp raises the possibility that it may have an important role in the early stages of hematopoiesis or cell lineage determination . Kell gp may also be a useful marker for the diagnosis of erythroleukemia . The late expression of Lu gp suggests it may be involved in the migration of erythroid precursors from the marrow . Fy gp is also expressed late consistent with a role as a scavenger receptor for cytokines in the bone marrow and circulation . Rh c antigen appeared before Rh D antigen, and it is suggested that this may reflect a reorganization of the developing erythroid cell membrane involving the Rh polypeptides and other components, including GPA and Band 3.

J Physiol, 1999 Jun 15, 517 ( Pt 3), 721 - 30
Target-dependent regulation of acetylcholine secretion at developing motoneurons in Xenopus cell cultures; Liou JC et al.; 1 . Myocyte-dependent regulation of acetylcholine (ACh) quantal secretion from developing motoneurons was studied in day-3 Xenopus nerve-muscle co-cultures . Spontaneous synaptic currents (SSCs) were measured in manipulated synapses by using whole-cell voltage-clamped myocytes . Changes in SSC amplitude were assumed to reflect changes in the ACh content of secreted quantal packets . Compared with natural synapses, motoneurons without any contact with a myocyte (naive neurons) released ACh in smaller quantal packets . 2 . Bipolar cultured motoneurons, which were in contact with a myocyte with one axon branch (contact-end) but remained free at another axon branch (free-end), were further used to examine quantal ACh secretion . The ACh quantal size recorded at free-end terminals was similar to that of naive neurons and was smaller than that at the contact-end, indicating that myocyte contact exerts differential regulation on quantal secretion in the same neuron . 3 . Some of the neurons that formed a natural synapse with a myocyte continued to grow forward and ACh quantal secretion from the free growth cone was examined . The ACh quantal size recorded at free growth cones was inversely proportional to the distance to the natural synapse, implying localized regulation of quantal secretion by the myocyte . 4 . Chronic treatment of day-1 cultures with veratridine and d-tubocurarine, respectively, increased and decreased the neurotrophic action of myocytes when assayed on day 3 . 5 . Taken together, these findings suggest that the myocyte is an important postsynaptic target in the regulation of quantal secretion and that the trophic action is spatially restricted to the neighbourhood of the neuromuscular junction.

Hum Reprod, 1999 Jun, 14(6), 1522 - 7
Androgen regulation of glycosidase secretion in epithelial cell cultures from human epididymis; Castellon EA et al.; The human epididymis and its secretions actively promote sperm fertilizing capacity and provide protection for spermatozoa against harmful influences . Among epididymal secretions, glycosidases have been recently studied and associated with molecular changes on the sperm surface . In the present work, we studied the influence of different concentrations of testosterone, dihydrotestosterone and cyproterone acetate on the secretion of alpha-glucosidase, N-acetyl-glucosaminidase, beta-glucuronidase and alpha-mannosidase by isolated and cultured epithelial cells from human caput, corpus and cauda epididymides . Cell cultures were obtained from aggregates of isolated tubule fragments plated on extracellular matrix-covered multi-well plates . Activities of the glycosidases were measured in conditioned culture media and were higher in the distal regions of the epididymis . Testosterone and dihydrotestosterone significantly increase the enzyme secretion in a concentration-dependent manner . This increase was higher in corpus and/or cauda than in caput epididymis . Cyproterone acetate caused a dose-dependent decrease in glycosidase secretion in cultures from all epididymal regions . It is concluded that the secretion of epididymal glycosidases is regulated by androgen, being stimulated by dihydrotestosterone and testosterone and inhibited by the androgen antagonist cyproterone acetate.

J Med Chem, 1999 Jun 3, 42(11), 1901 - 7
Lamellarin alpha 20-sulfate, an inhibitor of HIV-1 integrase active against HIV-1 virus in cell culture; Reddy MV et al.; HIV-1 integrase is an attractive target for anti-retroviral chemotherapy, but to date no clinically useful inhibitors have been developed . We have screened diverse marine natural products for compounds active against integrase in vitro and found a series of ascidian alkaloids, the lamellarins, that show selective inhibition . A new member of the family named lamellarin alpha 20-sulfate (1), the structure of which was determined from spectroscopic data, displayed the most favorable therapeutic index . The site of action of lamellarin alpha 20-sulfate on the integrase protein was mapped by testing activity against deletion mutants of integrase . Inhibition of isolated catalytic domain was detectable though weaker than inhibition of full length integrase; possibly lamellarin alpha 20-sulfate binds a site composed of multiple integrase domains . Lamellarin alpha 20-sulfate also inhibited integration in vitro by authentic HIV-1 replication intermediates isolated from infected cells . Lamellarin alpha 20-sulfate was tested against wild type HIV using the MAGI indicator cell assay and found to inhibit early steps of HIV replication . To clarify the inhibitor target, we tested inhibition against an HIV-based retroviral vector bearing a different viral envelope . Inhibition was observed, indicating that the HIV envelope cannot be the sole target of lamellarin alpha 20-sulfate in cell culture . In addition, these single round tests rule out action against viral assembly or budding . These findings provide a new class of compounds for potential development of clinically useful integrase inhibitors.

Neurosci Lett, 1999 May 7, 266(2), 89 - 92
Hyperammonemia: regulation of argininosuccinate synthetase and argininosuccinate lyase genes in aggregating cell cultures of fetal rat brain; Braissant O et al.; Hyperammonemia in the brain leads to poorly understood alterations of nitric oxide (NO) synthesis . Arginine, the substrate of nitric oxide synthases, might be recycled from the citrulline produced with NO by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL) . The regulation of AS and AL genes during hyperammonemia is unknown in the brain . We used brain cell aggregates cultured from dissociated telencephalic cortex of rat embryos to analyze the regulation of AS and AL genes in hyperammonemia . Using RNase protection assay and non-radioactive in situ hybridization on aggregate cryosections, we show that both AS and AL genes are induced in astrocytes but not in neurons of aggregates exposed to 5 mM NH4Cl . Our work suggests that the hyperammonemic brain might increase its recycling of citrulline to arginine.

Kidney Blood Press Res, 1999, 22(1-2), 81 - 97
Progression of diabetic nephropathy . Insights from cell culture studies and animal models; Phillips A et al.; Nephropathy in patients with type I and II diabetes mellitus is a rapidly increasing problem worldwide . Studies using both glomerular and tubular cells have delineated some of the consequences induced by acute hyperglycemia . In vitro studies have clearly demonstrated that exposure of cultured renal cells, such as glomerular mesangial cells and proximal tubular epithelial cells, to elevated glucose concentrations, may alter cell proliferation and/or extracellular matrix turnover . The latter is effected both directly and indirectly by the alteration of cytokine generation . Furthermore, these in vitro studies have allowed detailed examination of the mechanisms by which exposure of these cells to high ambient glucose concentrations may alter cell function . Extension of these studies to the experimental in vivo situation has confirmed most of the in vitro findings . Important insights gained from models of type I diabetes (i.e . streptocotocin-induced diabetes) as well as type II diabetes (i.e . Goto-Kakizaki (GK) rats and obese Zucker rats) include: (1) The demonstration that increased glomerular cell proliferation and renal matrix accumulation, driven by TGF-beta and/or PDGF, occur in streptocotocin-induced diabetes, yet that nephropathy in these rats does not progress to renal failure . (2) The demonstration that prolonged mild type II diabetes does induce morphological changes characteristic of pre-clinical diabetic nephropathy in GK-rats but does not result in albuminuria or progressive renal disease . (3) The demonstration that the association of type II diabetes with hyperlipidemia in obese Zucker rats results in early podocyte damage and subsequent progression to glomerulosclerosis, tubulointerstitial damage, and renal insufficiency . Identification of the mediators involved in the above processes and in particular of the conditions that will determine progression of subclinical morphological changes to overt nephropathy and renal failure will likely result in future novel therapeutic approaches to diabetic nephropathy.

Vet Parasitol, 1999 Apr 12, 82(3), 205 - 10
Determination of the activity of pyrimethamine, trimethoprim, sulfonamides, and combinations of pyrimethamine and sulfonamides against Sarcocystis neurona in cell cultures; Lindsay DS et al.; Equine protozoal myeloencephalitis (EPM) is a neurologic syndrome in horses from the Americas and is usually caused by infection with the apicomplexan parasite, Sarcocystis neurona . The activities of pyrimethamine, trimethoprim, sulfachloropyridazine, sulfadiazine, sulfadimethoxine, sulfamethoxazole, sulfamethazine, and sulfathiazole were examined against developing S . neurona merozoites in bovine turbinate cell cultures . A microtiter plate host cell lesion based assay was used to determine the effects of agents on developing merozoites . A cell culture flask assay was used to determine if selective concentrations of the agents killed or only inhibited development of S . neurona . Pyrimethamine was coccidiocidal at 1.0 microg/ml and trimethoprim was coccidiocidal at 5.0 microg/ml . None of the sulfonamides had activity when used alone at 50.0 or 100.0 microg/ml . Combinations of sulfonamides (5.0 or 10.0 microg/ml) with 0.1 microg/ml pyrimethamine demonstrated improved activity.

J Med Virol, 1999 Jun, 58(2), 178 - 81
Specific detection of echoviruses 22 and 23 in cell culture supernatants by RT-PCR; Oberste MS et al.; Reverse transcription-polymerase chain reaction (RT-PCR) methods are available for the rapid detection of enteroviruses in clinical specimens or virus isolates . Pan-enterovirus PCR primers, however, fail to amplify echovirus (E) type 22 or 23 because of their extreme sequence divergence from the other enteroviruses . We have developed an RT-PCR method to detect specifically E22 and E23 RNA directly in tissue culture supernatants without a viral RNA purification step . The E22/E23 primers successfully amplified 20 of 20 clinical isolates of E22 and 4 of 4 E23 isolates representing viruses isolated in 15 states over a 19-year period, as well as E22 and E23 prototype strains isolated in the 1950s . The primers did not amplify any of the other 64 enterovirus prototype strains.

J Biomed Mater Res, 1999, 48(2), 108 - 10
Cell culture test of TCP/CPLA composite; Kikuchi M et al.; Thermoplastic polymers filled with a ceramic powder (here described as a "composite") of beta-tricalcium phosphate (TCP) and copoly-L-lactide (CPLA) was prepared by a heat-kneading method . The TCP/CPLA composite obtained had a three-point bending strength of 51.26 +/- 634 MPa, a Young's modulus of 5.18 +/- 1.11 GPa, and a fracture strength of 52.64 +/- 2.975 MPa, which is sufficient for usage as an artificial bone implant material . According to cell culture tests using MC3T3-E1 cells, the TCP/CPLA composite showed no cytotoxicity and the cells were in close contact with the surfaces of the composite without any observable morphological change . These results suggest that a composite consisting of osteoconductive TCP and biodegradable CPLA is suitable as a bioactive artificial bone material.

Exp Toxicol Pathol, 1999 May, 51(3), 229 - 34
Cell cultures from cryopreserved renal biopsies and other tissue samples; Sommer M et al.; Answering questions regarding research or clinical aspects, histological and histochemical examinations of tissue specimens are playing an increasing role . The same is true for cell cultures obtained from organ specimens . In most cases, tissue samples are obtained only once and have to be examined immediately . This is often impracticable and therefore, it is necessary to store tissue samples or cells from established cell cultures so as to be able to continue examinations at a later time . Very rare reports exist on the preservation of tissue for performing cell culture examinations and they exclusively refer to tumour tissue and bone marrow, but not to normal organ tissue and biopsy samples . Therefore, in this study cell cultures from several organs have been prepared immediately after obtaining the tissue and compared with those established after cryopreservation several months later . The tissue specimens were obtained from rats (kidney, skin, heart) and from humans (kidney, placenta) or were biopsy specimens from the kidney and skin . From all these cryopreparations, typical cells were cultured . There was no significant difference in the mean population doubling time (MPD) and regarding morphological cell criteria between cell cultures obtained from fresh tissue samples after biopsy and those prepared several months after cryopreservation . There was nearly the same ratio between most cell types present in the tissue . From these results we can conclude that in the examined organs cells from cryopreserved tissue can be cultured even months or more than one year later . At least, these results make it possible to answer new questions and repeat different experiments at any time.

Int Endod J, 1997 Mar, 30(2), 141 - 4
Multilayer and monolayer cell cultures in a cytotoxicity assay of root canal sealers; Vajrabhaya L et al.; The aim of this study was to evaluate the response of a multilayer compared with a monolayer cell culture using six root canal sealers . Both monolayer and multilayer of MU-mu-1 (Mahidol University mouse cell line 1) were cultured in separate 96-well plates . Following incubation at 37 degrees C in 5% CO2 for 4 h in the presence of each sealer, cells were stained with 0.4% Sulphorhodamine-B, viable dye staining and the absorbance at 540 nm determined and calculated as cell viability . There was no statistical difference in the percentage viability for each sealer in both the multilayer and the monolayer cell culture (P > 0.01) . Apexit and AH-26 were less toxic (P < 0.01) than MU (Mahidol University) sealer, ROCANAL 3, ROCANAL 2, and Endomethasone which demonstrated the same toxicity (P > 0.01).

J Biol Chem, 1999 May 21, 274(21), 15110 - 4
Cholesterol-dependent generation of a seeding amyloid beta-protein in cell culture; Mizuno T et al.; Deposition of aggregated amyloid beta-protein (Abeta), a proteolytic cleavage product of the amyloid precursor protein (Abeta ), is a critical step in the development of Alzheimer's disease(Abeta++) . However, we are far from understanding the molecular mechanisms underlying the initiation of Abeta polymerization in vivo . Here, we report that a seeding Abeta, which catalyzes the fibrillogenesis of soluble Abeta, is generated from the apically missorted amyloid precursor protein in cultured epithelial cells . Furthermore, the generation of this Abeta depends exclusively on the presence of cholesterol in the cells . Taken together with mass spectrometric analysis of this novel Abeta and our recent study (3), it is suggested that a conformationally altered form of Abeta, which acts as a "seed" for amyloid fibril formation, is generated in intracellular cholesterol-rich microdomains.

Chem Res Toxicol, 1999 May, 12(5), 437 - 41
Metabolic activation of 4H-cyclopenta{def}chrysene in human mammary carcinoma MCF-7 cell cultures; Agarwal R et al.; The tumor initiating activities of 4H-cyclopenta{def}chrysene (C{def}C) and its two putative reactive metabolites, trans-1, 2-dihydroxy-anti-3,3a-epoxy-1,2,3, 3a-tetrahydro-4H-cyclopenta{def}chrysene (C{def}C-3,3a-DE) and trans-6,7-dihydroxy-anti-8,9-epoxy-6,7,8, 9-tetrahydro-4H-cyclopenta{def}chrysene (C{def}C-8,9-DE), were evaluated previously in mice {Amin, S., et al . (1995) Carcinogenesis 16, 2813-2817} . C{def}C-3,3a-DE was the more active inducer of lung tumors and elicited twice as many tumors as C{def}C-8,9-DE . In this study, the route of metabolism of C{def}C to DNA-reactive metabolites in the human mammary carcinoma cell line (MCF-7) was investigated using the 32P-postlabeling assay . The results show that metabolic activation to DNA-binding species proceeds through the formation of both trans-1,2-dihydrodiol and trans-6,7-dihydrodiol metabolites of C{def}C . At a 1 microM dose, adducts from the methylene-bridged (C{def}C-3,3a-DE) and bay region (C{def}C-8,9-DE) dihydrodiol epoxides were detected in comparable amounts . In contrast, the majority of the postlabeled adducts recovered from cells exposed to a 10 microM dose were derived from the bay region dihydrodiol epoxide, C{def}C-8,9-DE . Using markers from reactions of the dihydrodiol epoxides with deoxyguanosine 3'-phosphate and deoxyadenosine 3'-phosphate, it was shown that the major radioactive spots formed with both anti-C{def}C-3,3a-DE and anti-C{def}C-8,9-DE chromatographed with deoxyguanosine adduct markers . Thus, the human cells used in these studies can activate C{def}C to carcinogenic metabolites.

J Anim Sci, 1999 Apr, 77(4), 942 - 7
Alkaloid binding and activation of D2 dopamine receptors in cell culture; Larson BT et al.; Ergot and pyrrolizidine alkaloids, either extracted from endophyte-infected tall fescue, synthesized, or purchased commercially, were evaluated in cultured cells to estimate their binding to the D2 dopamine receptor and subsequent effects on cyclic AMP production in GH4ZR7 cells, transfected with a rat D2 dopamine receptor . Ergopeptide alkaloid (alpha-ergocryptine, bromocryptine, ergotamine tartrate, and ergovaline) inhibition of the binding of the D2-specific radioligand, {3H}YM-09151-2, exhibited inhibition constants (K(I)) in the nanomolar range, whereas dopamine was less potent (micromolar) . The lysergic acid amides (ergine and ergonovine) were 1/100th as potent as the ergopeptide alkaloids . Ergovaline and ergotamine tartrate were equally effective in inhibiting vasoactive intestinal peptide (VIP)-stimulated cyclic AMP production, with consistent nanomolar effective concentration (EC50) values . The remaining ergopeptide alkaloids (alpha-ergocryptine and bromocryptine), lysergic acid amides (ergonovine and ergine), and dopamine were 1/100th as potent . Two representative pyrrolizidines, N-formylloline and N-acetylloline, exhibited no binding activity at the D2 dopamine receptor or effects on the cyclic AMP system within the concentration ranges of nanomolar to millimolar . Our results indicate that the commercially available ergot alkaloids ergotamine tartrate and ergonovine may be used interchangeably in the D2 dopamine receptor system to simulate the effects of extracted ergovaline and ergine and to examine responses in receptor binding and the inhibition of cyclic AMP.

New Microbiol, 1999 Apr, 22(2), 77 - 83
Evidence of hepatitis E virus replication on cell cultures; Divizia M et al.; Several human and animal cell lines have been used to grow hepatitis E virus . The strain SAR-55 was adapted only on PLF/PLC/5 cell line without any visible cytopathic effect . The growth of the SAR-55 was monitored by examining the positive and the negative strands of HEV-RNA . Stool samples, obtained from hospitalised acute hepatitis patients at the Fever Hospital of Alexandria (Egypt), were used to confirm the susceptibility of PLF/PLC/5 cells . After more than one-week's cultivation, three stool samples out of 17 IgM anti-HEV positive and 1 from 52 IgG anti-HEV positive patients showed a specific RT-PCR amplification product . The nucleotide sequences of the methyltransferase region of the genome in the isolates revealed the maximum homology with Burma strain with several point mutations.

J Endocrinol, 1999 May, 161(2), 231 - 6
SC-19220, a prostaglandin E2 antagonist, inhibits osteoclast formation by 1,25-dihydroxyvitamin D3 in cell cultures; Inoue H et al.; 1,25 Dihydroxy vitamin D3 (1,25(OH)2D3), prostaglandin (PG) E2 and parathyroid hormone (PTH) induce osteoclast formation in cell cultures . Previously, we have shown that SC-19220, an antagonist of the EP1 subtype of PGE receptors, inhibited tartrate-resistant acid phosphatase (TRAP)-positive cell formation by PGE2 and PTH in adherent cell cultures taken from neonatal rats . Since 1,25(OH)2D3 has been shown to induce osteoclast formation through PGE2 synthesis, in this study we have examined the effect of SC-19220 on osteoclast formation induced by 1,25(OH)2D3 in cell cultures by measuring bone resorption as well as TRAP-positive cell formation . SC-19220 inhibited osteoclast formation by 1,25(OH)2D3 as well as by PGE2 in cell cultures . The addition of SC-19220 to the later half but not to the earlier half of the culture inhibited 1,25(OH)2D3-induced formation . In the culture in which hydroxyurea was added in the later half period, SC-19220 inhibited osteoclast formation by 1, 25(OH)2D3 . Under these conditions, 17-phenyl PGE1, an EP1 agonist, induced osteoclast formation . Thus, SC-19220 inhibits certain reactions in the later processes of osteoclast formation induced by 1,25(OH)2D3 . In addition, SC-19220 also inhibited osteoclast formation induced by interleukin (IL)-11 and IL-6 as well as by PTH . It is suggested that the SC-19220 inhibiting reactions are shared by all the inducers including 1,25(OH)2D3 and are essential for osteoclast formation.

Biophys J, 1999 May, 76(5), 2649 - 63
Subcellular Ca2+ distribution with varying Ca2+ load in neonatal cardiac cell culture; Winka LL et al.; Recent work in our laboratory has investigated and modeled subcellular calcium compartmentation and Ca2+ movement under steady-state control conditions . This experimental study is directed to the further description and quantitation of cellular calcium compartmentation patterns and movements as correlated with contraction in neonatal rat cardiac myocytes in culture under a variety of calcium loading conditions . Compartmental contents were assessed after incubations in various {Ca2+}o, 0 Na+/1 mM Ca2+, and 10 microM ouabain/1.0 mM Ca2+ test solutions . The cellular components investigated include sarcolemmal bound, sarcoplasmic reticulum (SR), and mitochondrial calcium . The results indicate that 1) sarcolemmal calcium binding is insensitive to changes in {Ca2+}o in the range tested (0.25-6.0 mM) while highly sensitive to changes in {Na+}i; 2) SR is sensitive to both changes in {Ca2+}o and {Na+}i and exhibits a maximum loading capacity of approximately 750 micromol Ca2+/kg dw; 3) in the {Ca2+}o range between 0.25 and 2.0 mM, contractile amplitude is proportional to SR content; 4) the mitochondria comprise a high-capacity calcium-containing compartment that is sensitive to changes in {Ca2+}o but does not reach saturation under the conditions tested (0.25-8.0 mM {Ca2+}o); 5) SR calcium is divided into at least two functionally discrete pools, one of which is available for release to the myofilaments during a normal ICa-triggered contraction and other of which is caffeine releasable but unavailable for release to the myofilaments during a normal triggered release; and 6) mitochondrial calcium serves as a reservoir of calcium capable of replenishing and/or augmenting SR stores with anywhere from 10% to 50% of mitochondrial calcium cycling through SR calcium compartments.

Free Radic Biol Med, 1999 Apr, 26(7-8), 936 - 43
EPR characterization of free radical intermediates formed during ultrasound exposure of cell culture media; Misik V et al.; Free radicals and/or hydrogen peroxide produced by exposure of cells to ultrasound are potentially cytotoxic and mutagenic . The formation and type of free radical species can be substantially modulated by the chemical composition of the media in which the ultrasound exposures of cells are carried out . In the current study, we examined the free radical intermediates formed during ultrasound exposure of a typical cell culture medium (RPMI-1640); the dominant free radicals that were identified by spin trapping were derived from the hydrophobic amino acids Trp, Leu, and Phe, and were formed by hydrogen abstraction from these amino acids . Compared to exposures in phosphate-buffered saline, the yield of *OH radicals and H2O2 was significantly reduced in the cell culture medium, glucose (the main organic component in the medium), and the hydrophobic amino acids (Trp, Phe, Tyr, Leu, Val, Met) being chiefly responsible for this effect . In contrast, other nonhydrophobic amino acids did not contribute significantly to the *OH or H2O2 decrease . These findings are consistent with the accumulation of hydrophobic solutes at the liquid-gas interface of the collapsing cavitation bubbles resulting in increased efficiency of radical scavenging.

J Neural Transm, 1999, 106(2), 111 - 22
Hypoxia induces differential changes of dopamine metabolism in mature and immature mesencephalic and diencephalic cell cultures; Gao J et al.; Perinatal hypoxia is known as a high risk factor for the development of long-lasting abnormalities in dopaminergic system . The early developmental alterations of dopamine (DA) metabolism induced by hypoxia could contribute to these abnormalities . To understand the hypoxia-induced changes of intra- and extracellular dopamine levels and its main metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in immature dopaminergic neurons, we compared these changes in rat mesencephalic and diencephalic cell cultures on day in vitro (DIV) 2 (immature cells), DIV 8 and DIV 13 (mature cells) . Cell cultures were exposed to an oxygen-free gas mixture in a Billups chamber for 2-4 hours . Mature cell cultures responded to hypoxia with an increase of DA levels in the cells and in the medium during the first 45 min (by an average of 57 and 114% respectively) . Thereafter, DA levels decreased, and returned to the baseline within the next 30 min . The cellular DA levels continued to decrease up to 15% of the baseline during 255 min hypoxia whereas the extracellular DA content stabilized at the prehypoxic levels . Immature cell cultures (DIV 2) in contrast to mature ones, were unable to maintain normal extracellular DA levels during hypoxia and showed a decrease of the cellular and extracellular levels to 50% of the prehypoxic levels . DOPAC and HVA changes mimick, however, at a lower level, the pattern of DA changes during the exposure to hypoxia . In principle, in the diencephalic cell culture similar effects of hypoxia exposure on the investigated parameters were found (studied during 0-120 min) . The present study demonstrates that mature and immature dopaminergic cells differ in the regulation of the extra- and intracellular DA levels during hypoxia . In immature cells the low synthetic capacity of tyrosine hydroxylase and the deficient capacities of the transport and storage processes result in decreased extracellular DA levels . This could be an important factor for the long-term modulation of the expression of tyrosine hydroxylase and subsequent long-term behavioral and/or neurological abnormalities induced by perinatal hypoxia.

J Neurosci Methods, 1998 Jul 1, 82(1), 97 - 103
An improved approach to freeze-fracture morphology of monolayer cell cultures; Bugnard E et al.; Much work is currently done on cell cultures to elucidate membrane processes associated with different cell functions . We describe here a modified freeze-fracture method to obtain systematically large fractured areas of the plasma membrane from monolayer cell culture in situ . Cells are grown until confluence on a Thermanox coverslip overlaid with poly-L-ornithine . After chemical fixation, the culture is flattened overnight by sandwiching it between the Thermanox coverslip, a Falcon membrane and a glass coverslip, under a 5 g weight . After freeze-fracture, vast pictures of the protoplasmic leaflets are obtained in a reproducible manner . Our approach was applied to cultures which were stimulated to release acetylcholine; it has been found very appropriate for studying modifications affecting intramembrane particles and vesicles openings in the plasmalemma . Accurate quantifications were performed and correlations were established between the membrane changes and the data revealed by thin sections . The present sandwich method can be applied to a variety of cell preparations, allowing for quantitative study of structure-function relationships.

J Invertebr Pathol, 1999 May, 73(3), 303 - 8
Serial passage of a Helicoverpa armigera nucleopolyhedrovirus in Helicoverpa zea cell cultures; Chakraborty S et al.; The serial passaging of baculoviruses in cell lines numerous times can result in a variety of mutations or defective viral populations becoming predominant in the cultures . The generation of these mutants during cell culture passage, also known as "the passage effect," can seriously hinder the use of in vitro methods for large-scale production of baculoviruses for use as biopesticides . In an effort to develop a large-scale in vitro method of producing Helicoverpa armigera singly enveloped nucleopolyhedrovirus (HaSNPV), it was essential to determine whether or not the passage effect was evident when this virus is serially passaged in cell cultures . An isolate of HaSNPV was serially passaged in Helicoverpa zea cell cultures up to 10 times . The production of occlusion bodies decreased with increasing passage number and there was evidence of defective viruses becoming predominant in cultures after 5 passages . The number of virions present within cross sections of passage 3 occlusion bodies was 1.5 times higher than those from passage 10 occlusion bodies when quantified using electron microscopy . A laboratory bioassay showed that potencies of passage 3 isolates against H . armigera larvae were 8 times higher than potencies of passage 10 isolates . This study indicated that changes typical of the passage effect were evident when HaSNPV was serially passaged in H . zea cell cultures up to 10 times .

Neuropharmacology, 1999 Mar, 38(3), 395 - 402
Effects of PKA and PKC modulators on high affinity glutamate uptake in primary neuronal cell cultures from rat cerebral cortex; Lortet S et al.; In this study, the effects of various agents known to alter protein phosphorylation, via protein kinase C or A, on high affinity glutamate uptake were investigated in primary neuronal cell cultures of rat cerebral cortex . Incubating the culture dishes with chelerythrine or H89 (N-{2-(p-bromocinnamylamino)ethyl}-5-isoquinolinesulfonamide), which inhibit PKC and PKA, respectively, dramatically decreased the glutamate uptake in a dose-dependent manner . Saturation kinetic analysis showed that chelerythrine and H89 decreased the Vmax (chelerythrine: -61%, P < 0.06; -59%, P < 0.05) without affecting the Km of the transport process as compared to the control values . These inhibitory effects were counteracted by the corresponding protein kinase activators, i.e . PMA (phorbol-12-myristate 13-acetate) in the case of PKC and forskolin in the case of PKA, although these protein kinase activators alone did not significantly affect the glutamate uptake . These results provide evidence that, in primary cultures of neuronal cells, the high affinity glutamate uptake may be regulated by both PKA and PKC-mediated phosphorylation processes.

Neuropharmacology, 1999 Feb, 38(2), 273 - 8
Endomorphin-1 potentiates HIV-1 expression in human brain cell cultures: implication of an atypical mu-opioid receptor; Peterson PK et al.; Endogneous delta and kappa opioid peptides possess a variety of immunomodulatory properties, and kappa-opioid receptor ligands recently were shown to suppress the expression of human immunodeficiency virus type 1 (HIV-1) in microglial cells, the resident macrophages of the brain . To determine whether the newly discovered endogenous mu-opioid receptor ligands endomorphin-1 and -2 would affect HIV-1 replication, these peptides were added to acutely infected brain cell cultures . Endomorphin-1 potentiated viral expression, in a bell-shaped dose-response manner with maximal enhancement approximately equal to 35% at 10(-10) M, in both mixed glial/neuronal cell and purified microglial cell cultures . Endomorphin-1's amplifying effect was blocked by pretreatment of brain cells with either the mu-opioid receptor selective antagonist beta-funaltrexamine or the G protein inhibitor pertussis toxin . However, the classical mu receptor agonists morphine and DAMGO (Tyr-d-Ala-Gly-N-Me-Phe-Gly-ol) had no effect on viral expression or on endomorphin-1's amplifying effect . Taken together, these findings suggest that in this in vitro model of HIV-1 brain infection, endomorphin-1 potentiates viral expression via activation of an atypical mu-selective opioid receptor . They also provide evidence, for the first time, that an endogenous mu-opioid peptide has neuroimmunomodulatory activity.

J Neurochem, 1999 May, 72(5), 1843 - 52
Analysis of the neuroprotective effects of various nitric oxide donor compounds in murine mixed cortical cell culture; Vidwans AS et al.; Nitric oxide (NO) has been implicated in both the pathogenesis of and protection from NMDA receptor-mediated neuronal injury . This apparent paradox has been attributed to alternate redox states of nitrogen monoxide, whereby, depending on the redox milieu, nitrogen monoxide can be neuroprotective via nitrosation chemistry or react with superoxide to form secondary toxic species . In our murine mixed cortical cell culture system, the NONOate-type NO donors diethylamine/NO complex sodium (Dea/NO), (Z)-{N-(3-ammoniopropyl)-N-(n-propyl)amino}diazen-1-ium++ +-1,2-diolate (Papa/NO), and spermine/NO complex sodium (Sper/NO), as well as the S-nitrosothiols S-nitroso-L-glutathione (GSNO) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) (NO+ equivalents), decreased NMDA-induced neuronal injury in a concentration-dependent manner . 8-Bromo-cyclic GMP did not mimic the inhibitory effects of the donors, suggesting that the neuroprotection was not the result of NO-stimulated neuronal cyclic GMP production . Furthermore, neuronal injury induced by exposure of cultures to H2O2 was not altered by the presence of Dea/NO, indicating the absence of a direct antioxidant effect . NONOates did, however, reduce NMDA-stimulated uptake of 45Ca2+, whereas high potassium-induced 45Ca2+ accumulation, a measurement of entry via voltage-gated calcium channels, was unaffected . The parallel reduction of 45Ca2+ accumulation and NMDA neurotoxicity by NONOates mimicked that seen with an NMDA receptor antagonist . Electrochemical measurements of NO via an NO-sensitive electrode demonstrated that neuroprotective concentrations of all donors produced appreciable amounts of NO over the 5-min time frame . Determination of the formation of NO+ equivalents, as assessed by N-nitrosation of 2,3-diaminonaphthylene, revealed little or no observable N-nitrosation by Sper/NO, GSNO, and SNAP with significant N-nitrosation observed by Papa/NO and Dea/NO . However, addition of ascorbate (400 microM) effectively prevented the nitrosation of 2,3-diaminonaphthylene produced by Dea/NO and Papa/NO without altering their neuroprotective properties or their effects on 45Ca2+ accumulation . Present results indicate that the intrinsic NO/NO+ characteristics of NO donor compounds may not be a good predictor of their ability to inhibit NMDA receptor-mediated neurotoxicity at the cellular level.

Avian Dis, 1999 Jan-Mar, 43(1), 16 - 21
Comparative evaluation of cell culture-adapted and chicken embryo-adapted fowl pox vaccine strains; Baxi MK et al.; Two types of vaccines, chicken embryo adapted (VacCE) and cell culture adapted (VacCC), were tested for their efficacy to elicite the immune response in birds vaccinated at 2 and 8 wk of age . The cell-mediated immune response studied by blastogenesis assay showed that birds vaccinated at the second week of age by both VacCE and VacCC vaccines had significant increase in T-lymphocyte count at 21 days postvaccination (PV) and 7 days postchallenge (PC), whereas in birds vaccinated at 8 wk of age, a significant increase was seen at 21 days PV and 7 days PC with the VacCC vaccine . The rise in passive hemagglutination titers was observed up to 21 days PV and 7 days PC in birds vaccinated at 2 wk of age . However, only the birds vaccinated with VacCC at 8 wk of age showed rise in titers at days 21 PV and 7 PC . Birds were challenged 90 days PV by scarification on the thigh region, and the birds vaccinated with VacCC showed 90% and 70% protection when vaccinated at 2 and 8 wk, respectively . The birds vaccinated with VacCE showed only 60% and 20% protection at the corresponding levels, respectively.

FEBS Lett, 1999 Mar 26, 447(2-3), 264 - 8
The extensin multigene family responds differentially to superoxide or hydrogen peroxide in tomato cell cultures; Wisniewski JP et al.; Changes in extensin gene expression were examined in cultured tomato cells following treatments leading to the production of activated oxygen species . Digitonin, a steroid glycoalkaloid compound, has been shown to trigger a rapid and transient production of superoxide anion, O2-* . 6 h after application of 50 or 100 microM of digitonin, the accumulation of four extensin transcripts (1.5, 2.6, 4.0 and 6.1 kb) was observed . Superoxide dismutase strongly inhibited the digitonin-mediated response, suggesting a key role of O2-* in the signalling cascade . Furthermore, cells treated with enzymatically produced O2-* generated by xanthine oxidase (0.015 U/ml) gave a similar extensin response and again, SOD exerted a strong inhibitory effect on the response . On the other hand, H2O2 (2 mM) or the enzymatic H2O2 generator, glucose oxidase (0.34 U/ml), elicited the accumulation of only three of the four transcripts (1.5, 2.6 and 4.0 kb), indicating that the corresponding genes could be regulated either by H2O2 or O2-* but that the gene encoding the 6.1 kb transcript was exclusively expressed in response to O2-* . Finally, it was shown that lipid peroxidation, which was only induced when cells were exposed to H2O2, did not participate in the AOS-mediated gene expression for extensin . It can be concluded from these results that tomato cells are able to discriminate H2O2 from O2-* and they probably sense the latter by the specific oxidation of an extracellular component.

Neurosci Lett, 1999 Mar 26, 263(2-3), 109 - 12
Ischemia-induced changes in 2'-5'-oligoadenylate synthethase mRNA levels in rat brain: comparison with changes produced by perturbations of endoplasmic reticulum calcium homeostasis in neuronal cell cultures; Paschen W et al.; 2'-5' Oligoadenylate synthetase (OAS) expression is induced by interferon or viral infection of cells . To better understand ischemia-induced changes in gene expression and to elucidate the possible underlying mechanisms, changes in OAS mRNA levels were evaluated after 30 min four-vessel occlusion and 2, 4, 8 or 24 h recovery and compared to the temporal profile of changes in mRNA levels induced by a transient depletion of endoplasmic reticulum (ER) calcium stores in primary neuronal cell cultures . OAS mRNA levels dropped during early recovery both in vivo and in vitro . After 6 h recovery from ER calcium pool depletion, OAS mRNA levels increased to about 350% of controls and returned to control levels after 24 h of recovery . After 24 h recovery from ischemia, OAS mRNA levels rose to about 390% of controls in the hippocampus and striatum and to 210% of the control value in the cortex . It is concluded that transient ischemia place cells into an antiviral state, most pronounced in the hippocampus and striatum, and that disturbances of ER calcium homeostasis may contribute to this process.

Mol Vis . 1999 Apr 19;5:4.
The golden age of retinal cell culture; Seigel GM; In the late 1950s, the study of retinal cells in vitro was in its infancy . Today, retinal cell and tissue culture is routinely used for studies of cell growth, differentiation, cytotoxicity, gene expression, and cell death . This review discusses the major classifications of retinal cell and tissue culture, including primary cell/explant models, retinoblastoma cell lines, and genetically engineered cell lines . These topics are addressed in an historical perspective, coupled with present-day applications for this continually-developing technology.

Virology, 1999 Apr 25, 257(1), 198 - 207
Induction of CD95 (Fas) and apoptosis in respiratory epithelial cell cultures following respiratory syncytial virus infection; O'donnell DR et al.; Respiratory syncytial virus (RSV) infection is associated with epithelial cell death and vigorous inflammation . In mouse models, and in immunosuppressed patients, CD8(+) T cells are necessary for RSV clearance . In vitro, RSV has been shown to induce expression of several proteins on the respiratory epithelial cell, including RSV proteins, ICAM-1, and MHC class I, that can potentially interact with CD8(+) T cells in initiating apoptosis of the target cell . One mechanism of T-cell-directed cell death is the interaction of FasL on the CD8(+) T lymphocytes and Fas expressed on the target cell . In order to determine the ability of RSV to induce Fas on the respiratory epithelium, we studied the RSV infection of a human respiratory epithelial cell line (A549) in vitro . Fas mRNA and protein levels are increased two-to-fourfold following RSV infection, and transcriptional upregulation of Fas was demonstrated using promoter/reporter gene constructs . RSV infection directly resulted in cellular apoptosis, and the frequency of apoptotic cells was further increased by cross-linking with antibodies to Fas . These data demonstrate that RSV infection induces cellular apoptosis and suggest that interactions of surface Fas with T cells may further augment this process in vivo .

Neuroreport, 1999 Mar 17, 10(4), 703 - 6
Enhancement of TNF-alpha production by ganglioside GM2 in human mononuclear cell culture; Mizutani K et al.; Some gangliosides have been regarded as autoantigens of immune-mediated neurological disorders such as Guillain-Barre syndrome (GBS), Miller Fisher syndrome and multifocal motor neuropathy . On the other hand, proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), may be important in the pathogenesis of some neuroimmunological disorders . To clarify the interactions between immune cells and gangliosides, we investigated the effects of gangliosides on the production of proinflammatory cytokines in peripheral blood mononuclear cell (PBMC) cultures . We found that ganglioside GM2 markedly enhances the production of TNF-alpha and that TNF-alpha induction by coated GM2 is still more marked . These findings suggest that immune cells, especially monocytes/macrophages, cause inflammation upon encountering GM2.

Parasitology, 1999 Mar, 118 ( Pt 3), 227 - 33
Development of Sarcocystis falcatula in cell cultures demonstrates that it is different from Sarcocystis neurona; Lindsay DS et al.; The development of Sarcocystis falcatula merozoites in bovine turbinate (BT) cell cultures is described and compared with development of Sarcocystis neurona merozoites . Merozoites of S . falcatula entered BT cell cultures and increased in size until 3 days post-inoculation when the nucleus of some merozoites developed lobes . Developing schizonts present at 4 days contained a lobed nucleus or appeared multinucleate . A single mature schizont was observed 4 days p.i . Schizonts were numerous 5 and 6 days p.i . Merozoites were produced from blastophores on the schizont . S . neurona merozoites developed to mature schizonts by 3 days p.i . in BT cells and a residual body was often present . Transmission electron microscopy revealed that S . falcatula merozoites possessed more micronemes than did S . neurona merozoites . Our study demonstrates that S . falcatula and S . neurona are not the same parasite.

Biochem Mol Biol Int, 1999 Mar, 47(3), 387 - 96
Degradation of an ubiquitin-conjugated protein is associated with myoblast differentiation in primary cell culture; Gardrat F et al.; At the early stages of myogenesis, myoblasts fuse to form multinucleated myotubes . This morphological differentiation is the result of dynamic changes in gene regulation and expression . The ubiquitin proteasome-dependent pathway has been reported to play an important role in many aspects of cellular functions such as regulation of growth and cell cycle progression . In this study, we showed that the amount of mRNA's corresponding to the iota subunit of the 20S proteasome, the level of the S4 subunit of the 19S complex and the 20S and 26S proteasomes peptidase activities increased during myoblast fusion . Cell permeable 20S proteasome inhibitor prevented fusion with concomitant accumulation of ubiquitin-conjugated protein . On the other hand, inhibition of ubiquitin ligase E3 enzymes prevented the formation of ubiquitin conjugate and decreased the fusion process . These results strongly support the involvement of the ubiquitin-proteasome proteolytic pathway in the events leading to myoblast fusion.

Nutrition, 1999 Mar, 15(3), 200 - 7
Mouse extensor digitorum longus muscle preparation as a tool in nutrition research: a quantitative comparison to in vivo and cell culture experiments; Svanberg E et al.; Incubated restrained and unrestrained extensor digitorum longus (EDL) muscles from adult non-growing mice were evaluated as a tool in non-steady state nutrition experiments . Energy state was determined by nucleotide determinations in muscles . Protein synthesis was estimated by the amount of L-{U-14C}phenylalanine incorporated into proteins, and protein balance was measured by tyrosine release from muscle proteins . Confluent cultured L6 rat muscle cells served as a reference system in steady state without hypoxia being sensitive to growth factors and regulatory peptides at physiologic concentrations . Irrespective of medium composition, incubated EDL muscles remained in negative protein balance, being unrelated to the resting tension of the incubated muscles . Energy-rich phosphates were not restored to normal levels during incubation, but protein synthesis was not attenuated by the decline in energy state . Fractional protein synthesis (0.05-0.15%/h) remained constant for up to 6 h of EDL incubation, and was comparable to protein synthesis in cultured confluent non-proliferating myocytes (0.20-0.30%/h) and to mixed leg muscles measured in vivo (0.10-0.20%/h) . Protein synthesis in incubated EDL muscles reflected alterations in muscle peptide formation in vivo following either oral provision of food or parenteral injection of insulin . EDL muscles were sensitive to in vitro exposure to both insulin (60-125 microU/mL) and insulin-like growth factor 1 (IGF-1) (1000 ng/mL) . The sensitivity to insulin seemed to be modified by the nutritional state (starved/fed) of the animals before sacrifice . Protein synthesis in EDL muscles was less responsive to serum-containing growth factors (IGF-1, epidermal growth factor {EGF}, platelet-derived growth factor {PDGF}) compared to confluent L6 muscle cells, which probably reflected different receptor expression . Our results demonstrate that protein metabolism in incubated unrestrained mouse EDL muscles reflects in vivo protein metabolism.

Neuroscience, 1999 Jan, 88(2), 571 - 83
Stress induces neurogenesis in non-neuronal cell cultures of adult olfactory epithelium; Feron F et al.; Among the basal cells of the olfactory epithelium is a stem cell which divides and whose progeny differentiate into new sensory neurons throughout adult life . Olfactory neurogenesis is highly regulated, for example it is stimulated by epithelial damage . Previous reports implicate several growth factors in progenitor cell proliferation and neuronal differentiation in vitro but these studies differ in growth conditions and age of donors making it difficult to determine precisely the roles of neurogenic stimuli and their sites of action . The aims of the present study were to develop purified basal cell cultures from adult olfactory epithelium and to stimulate neurogenesis in defined growth conditions in order to elucidate the cellular mechanisms by which neurogenesis is stimulated after epithelial damage . We show here that differentiated olfactory sensory neurons arise after biochemical or mechanical stress of rat and mouse olfactory epithelial cell cultures in the absence of growth factors, complex media (e.g., serum, conditioned media, pituitary and hypothalamic extracts), or other cells (e.g., explants, feeder layers of glia, or other non-epithelial cells) . Prior to the stress, these cultures contained basal cells and supporting cells but not neurons . After the stress, some cells differentiated into bipolar neurons expressing a number of neuronal proteins including olfactory marker protein . Bromodeoxyuridine experiments show that the differentiated neurons arose from recently divided cells which did not divide again before differentiating . We conclude that stress disrupts cell surface contacts to induce the immediate neuronal precursors to undergo final differentiation into olfactory sensory neurons . This may be a mechanism for enhanced neurogenesis after epithelial damage.

J Endod, 1999 Jan, 25(1), 24 - 9
An in vitro pulp chamber with three-dimensional cell cultures; Schmalz G et al.; To better simulate the in vivo situation, a three-dimensional fibroblast cell culture was introduced into an in vitro pulp chamber model . The system was evaluated by testing a series of dental filling materials . After a 24-h exposure with (0.3 or 5 ml/h) and without perfusion of the pulp chamber, the tissues were subjected to a routine MTT assay . Zinc phosphate cement, conventional glass ionomer cements, a silicone impression material, and zinc oxide-eugenol did not influence cell viability, compared with untreated controls; but, a light-curing glass ionomer cement significantly reduced cell survival . Perfusion of the chambers did not significantly influence the results, but perfusion conditions of 5 ml/h lead to a general decrease of cell vitality . The three-dimensional cell culture system in an in vitro pulp chamber seems to be a substantial improvement, because zinc oxide-eugenol does not evoke a cellular reaction (as is the case in vivo), and the test system is sensitive enough to detect other toxicants.

Cell Biol Toxicol, 1999 Feb, 15(1), 63 - 75
Use of skin cell cultures for in vitro assessment of corrosion and cutaneous irritancy; Roguet R; Skin cell culture is one of the most promising tools for in vitro evaluation of both cutaneous irritancy and corrosion . New culture methodologies, including three-dimensional reconstruction of skin, allow the evaluation of a wide range of compounds and complex formulations . A number of tests have already been developed for the evaluation of cytotoxicity and many end-points are now currently used, including cell viability, alteration of cell growth or cell function . In recent years parameters more closely related to in vivo irritancy effects such as synthesis of inflammatory mediators and/or their release by keratinocytes after exposure to potential skin irritants have been evaluated . This paper reviews technological aspects and results of validation using skin cell culture for in vitro assessment of corrosion and skin irritancy . Advantages and limits of skin cell cultures are also presented . Current questions about the validation process of cutaneous irritation and corrosion are also considered.

Biomed Tech (Berl), 1999 Jan-Feb, 44(1-2), 6 - 11
{Standardized testing of bone implant surfaces with an osteoblast cell culture system . II . Titanium surfaces of different degrees of roughness}; Noth U et al.; The effect of titanium surfaces with different degrees of roughness on osteoblast proliferation and differentiation was investigated using a standardised cell culture system . Human foetal osteoblasts (hFOB 1.19) were cultured on polished (Ti pol), sandblasted (Ti sb) and sandblasted/heat treated (Ti sb-ht) titanium surfaces for 17 days . Cell culture quality polystyrene (Ps) was used as a control . Cell number and viability were determined for assessment of proliferation . Alkaline phosphatase activity, collagen I and osteocalcin production were measured as parameters for osteoblast differentiation . In the early phase, higher proliferation values were measured on Ti pol . However, on Ti sb and Ti sb-ht higher proliferation was found in the late phase . The activity of the early differentiation marker alkaline phosphatase was higher on Ti pol . No differences were seen for the late differentiation parameters collagen I and osteocalcin . The test system permits the influence of the surface structure on the dynamics of the osteoblast development cycle to be determined . The larger surface area of rough materials leads to an initially delayed, but then prolonged cell proliferation . This model correlates with recent in vivo findings, and confirms the use of rough surfaces for implants in direct contact with bone, even at the cellular level.

J Biotechnol, 1999 Feb 19, 68(2-3), 89 - 99
Production of ginseng and its bioactive components in plant cell culture: current technological and applied aspects; Wu J et al.; Ginseng (the root of Panax ginseng CA Mayer) is a valuable oriental herb, which has been used in traditional Chinese medicine for thousands of years, both as a disease-healing drug and a general tonic . The medicinal value of ginseng is now also widely recognized in the west and the world ginseng market is expanding . The current supply of ginseng depends mainly on field cultivation, which is a slow and laborious process . Plant cell and tissue culture methods have been explored as potentially more efficient alternatives for the mass production of ginseng and its bioactive components . Research into ginseng cell and tissue cultures started in the early 1960s and commercial applications have been underway since the late 1980s . The ginseng cell culture has continued to attract considerable research and development effort in recent years as scientists seek to understand and optimize the culture conditions . In this paper, we review recent studies on ginseng cell culture processes, focusing on the physiological and bioengineering factors affecting the productivity of ginseng biomass and useful metabolites (e.g . ginseng saponin and polysaccharide) and the progress and concerns in large-scale applications.

J Appl Biomater, 1994, 5(3), 203 - 13
Corrosion and cell culture evaluations of nickel-chromium dental casting alloys; Bumgardner JD et al.; In this study, the corrosion and surface properties of four commercially available nickel-chromium dental casting alloys, were evaluated using electrochemical corrision testing and Auger electron microscopy . The corrosion tests were conducted under cell culture conditions of 5% CO 2 humidified atmosphere at 37 degrees C in minimum essential medium (MEM) balanced salt solution, 95% MEM-5% FBS (fetal bovine serum) cell culture media, and in 95% MEM-5% FBS media after cold solution sterilization of test samples . The results of the surface and corrision analyses were correlated to cytotoxicity and metal ion release from the alloys using agarose overlay and direct contact cell culture tests . The surface and electrochemical corrision analyses demonstrated that the non-beryllium containing alloys were more resistant to accelerated corrosion processes as compared to the beryllium-containing alloys . All alloys demonstrated decreased corrision rates in cell culture solutions after cold solution sterilization treatment . The corrision products released from the nickel-based alloys failed to alter the cellular morphology and viability of human gingival fibroblasts, however they did cause reductions in cellular proliferation . The potential for accelerated corrision and the exposure of local and systemic tissues to elevated levels of corrision products raises concerns over the biocompatibility of these alloys.

Biotechnol Bioeng, 1998 Dec 20, 60(6), 689 - 98
Capture of human monoclonal antibodies from cell culture supernatant by ion exchange media exhibiting high charge density; Necina R et al.; A shortcut purification sequence for therapeutic proteins should consist of three steps: capture, purification, and polishing . Special emphasis has been put on direct capture of human monoclonal antibodies from culture supernatants with ion-exchangers avoiding pretreatment steps such as desalting, dilution, and other means to reduce the ionic strength . CM-HyperD, a cation-exchanger composed of an inorganic macroporous support filled with a viscoelastic gel with a high charge density was used . Capture of monoclonal antibodies from clarified hybridoma cell culture grown in media supplemented with fetal calf serum was investigated . Screening of different pH conditions and buffers for the load step showed that monoclonal antibodies were efficiently bound by CM-HyperD at pH 4.0 and 5.0 at an ionic strength equivalent to culture supernatant . Combination of negative purification with Q-Sepharose FF and capturing with CM-HyperD gave sufficient yield and resolution . Implementation of wash steps with higher conductivity did not improve the purity, but decreased the yield . Interestingly, high flow rates improved the purity . When antibodies were captured from serumfree culture supernatant the antibody could be eluted in a single peak with substantial reduction of contaminants . Capturing of antibodies by ion-exchange sorbents from culture supernatant is possible despite the high salt content .

Biotechnol Bioeng, 1998 Jun 20, 58(6), 642 - 8
Improvement of interferon-gamma sialylation in Chinese hamster ovary cell culture by feeding of N-acetylmannosamine; Gu X et al.; Because the presence of sialic acid can extend circulatory lifetime, a high degree of sialylation is often a desirable feature of therapeutic glycoproteins . In this study, the incomplete intracellular sialylation of interferon-gamma (IFN-gamma), produced by Chinese hamster ovary cell culture, was minimized by supplementing the culture medium with N-acetylmannosamine (ManNAc), a direct intracellular precursor for sialic acid synthesis . By introducing 20 mM ManNAc into the culture medium, incompletely sialylated biantennary glycan structures were reduced from 35% to 20% at the Asn97 glycosylation site . This effect was achieved without affecting cell growth or product yield . The intracellular pool of CMP-sialic acid, the nucleotide sugar substrate for sialyltransferase, was also extracted and quantified by HPLC . Feeding of 20 mM ManNAc increased this intracellular pool of CMP-sialic acid by nearly thirtyfold compared with unsupplemented medium . When radiolabeled ManNAc was used to trace the incorporation of the precursor, it was found that supplemental ManNAc was exclusively incorporated into IFN-gamma as sialic acid and that, at 20 mM ManNAc feeding, nearly 100% of product sialylation originated from the supplemental precursor .

Hum Reprod, 1999 Feb, 14(2), 338 - 44
Relaxin secretion by human granulosa cell culture is predictive of in-vitro fertilization-embryo transfer success; Stewart DR et al.; We have developed a cell culture system for human luteinizing granulosa cells which supports the timely and dynamic secretion of oestrogen, progesterone and relaxin in patterns that mimic serum concentrations of these hormones during the luteal phase of the menstrual cycle . There was a wide variation in the amount of relaxin secreted by the cultured cells for the 69 patients studied . As relaxin production was generally maximal by day 10 of culture, comparisons were made at this time point . It was observed that most of the conceptions occurred in patients with higher relaxin secretion in vitro . All cycles with relaxin > 800 pg/ml on day 10 had a term pregnancy while only 13% of cycles with relaxin < 200 pg/ml had term pregnancies . A limited number of cycles from donor/recipient cycles did not show similar results . Steroid concentrations were not predictive of conception . These results demonstrated that in-vitro production of relaxin is predictive of implantation success in in-vitro fertilization (IVF)-embryo transfer cycles . This supports the hypothesis that relaxin may be involved in implantation and that lowered relaxin concentrations may be a partial cause of poor pregnancy rates after IVF.

Chin J Physiol, 1998 Dec 31, 41(4), 203 - 9
Free radicals are involved in methylmethacrylate-induced neurotoxicity in human primary neocortical cell cultures; Chen MS et al.; Methylmethacrylate monomer (MMA), a highly volatile material, has been extensively used for the construction of complete or partial dental prostheses . While previous studies have indicated a variety of complications and untoward side-effects associated with its use, the possible neurotoxicity induced by this monomer has not been addressed . In this study, we have investigated the MMA-produced neuronal injury in human neuron-enriched primary culture . Embryonic brain tissue (8-10 weeks postconception) was used for the primary neuron-enriched culture . Phase-contrast microscopy was used to evaluate morphological changes of cultured neurons . Extracellular concentrations of lactate dehydrogenase (LDH) and nitrite was measured from the culture medium to assess the magnitude of neuronal damage and nitric oxide formation, respectively . Neocortical neurons exposed to the monomer (1/200, Vmonomer/Vglycerol) for two days resulted in a significant increase in the LDH level but monomer (1/20000, 1/2000, or 1/200; Vmonomer/Vglycerol) failed to increase the nitrite level . Morphologically, the neurons subjected to monomer treatment exhibited irregular shrunken cell bodies with dystrophic and/or fragmented neurities, or even cell lysis . Moreover, superoxide dismutase plus catalase or vitamin C pretreatment protected against monomer-induced neurotoxicity . Our results suggest that this neurotoxicity can not likely be attributed to the cytotoxic effects of nitric oxide but may be mediated through the toxicity of superoxide and other free radicals . This is the first time, to our knowledge, that neurotoxicity induced by MMA has been demonstrated in human cortical neurons.

Chin J Physiol, 1998 Dec 31, 41(4), 195 - 202
Ethyl isopropylamiloride downregulates Na,K-ATPase gene expression which confers cytotoxicity in primary proximal tubule cell cultures; Chiu HM et al.; Our original attempt was to examine whether inhibition of Na/H exchange in proximal tubule would affect the expression of basolateral membrane protein Na,K-ATPase . Three amiloride analogues were tested within the range of 10(-6) M to 10(-4) M in primary cultures of proximal tubule cells . Only ethylisopropyl amiloride (EIPA) dose-dependently downregulated Na,K-ATPase activity in cultured proximal tubule cells . The time course study revealed that EIPA (10(-4) M) significantly decreased Na,K-ATPase alpha- and alpha-mRNA abundance within 4 hr and suppressed Na,K-ATPase alpha- and beta-mRNA levels by 76.3 +/- 4.5% and 85.5 +/- 5.8%, respectively, within 24 hr . The decrease in Na,K-ATPase mRNA was followed by a decrease in Na,K-ATPase activity by 22.5 +/- 10.8% and 48.8 +/- 5.9% within 12 and 24 hr, respectively, which could be reflected by a coordinate decrease in levels of both alpha- and mature beta-protein . The cell viability was not affected until 20 hr of EIPA treatment, when an increase in LDH release and cell detachment was observed . Because EIPA rapidly decreased intracellular pH (pHi) to 6.7 within 2 hr and raising pHi to 6.6 by metabolic acidosis could not elicit changes in Na,K-ATPase activity, EIPA-induced downregulation of Na,K-ATPase should not be mediated through H+ . In view of the time course of EIPA effects on Na,K-ATPase subunit mRNA, protein, activity and cell toxicity, the cytotoxic effect is likely resulted from a decrease in Na,K-ATPase activity . Take together, we conclude that EIPA induces downregulation of Na,K-ATPase expression via both pre- and post-translational mechanisms, which confers cytotoxic effects on proximal tubule cells.

J Nat Prod, 1999 Mar, 62(3), 481 - 3
Activities of plant-derived phenols in a fibroblast cell culture model
Koganov MM, Dueva OV, Tsorin BL.
Fourteen plant phenols of five structural groups (flavones, flavonols, flavan-3-ol, isoflavones, and phenylpropanoids) demonstrated concentration-dependent inhibitory or modulatory effects in a fibroblast cell culture model . The most potent inhibitory activity in this investigation was exhibited by apigenin (4), with flavone (1), chrysin (2), and genistein (9) being of somewhat lesser potency . These findings help to provide a better understanding of the action of these plant phenols in inflammatory/immune responses.

J Nat Prod, 1999 Mar, 62(3), 481 - 3
Activities of plant-derived phenols in a fibroblast cell culture model; Koganov MM et al.; Fourteen plant phenols of five structural groups (flavones, flavonols, flavan-3-ol, isoflavones, and phenylpropanoids) demonstrated concentration-dependent inhibitory or modulatory effects in a fibroblast cell culture model . The most potent inhibitory activity in this investigation was exhibited by apigenin (4), with flavone (1), chrysin (2), and genistein (9) being of somewhat lesser potency . These findings help to provide a better understanding of the action of these plant phenols in inflammatory/immune responses.

Brain Res Mol Brain Res, 1999 Mar 20, 66(1-2), 211 - 6
Inducible transcription factor expression in a cell culture model of apoptosis; Woodgate A et al.; We have developed a model of nerve cell death based on the toxicity of okadaic acid, a compound that triggers apoptosis in PC12 cells via a protein synthesis-dependent mechanism . The cell death process is accompanied by induction of JunB, c-Jun, JunD and Fos proteins . Phosphorylation-specific antibodies were used to demonstrate that c-Jun is phosphorylated at serine 63 and serine 73 . Electrophoretic gel mobility shift and pAP1-Luc luciferase assays showed that expression of ITFs is associated with increases in AP-1 binding and in AP-1 transcriptional activity . In addition, dose response and time course studies provided strong correlative evidence that Fos and Jun proteins are involved in the apoptotic death cascades . Thus, this model provides a useful system to investigate the role of inducible transcription factor proteins in apoptosis .

Biochem Mol Biol Int, 1999 Jan, 47(1), 107 - 15
Presence and comparison of angiotensin converting enzyme in commercial cell culture sera; Bramucci M et al.; This study was conducted to determine the presence of the angiotensin converting enzyme in commercial sera used in cell culture medium . The aim of the research was to bring the presence of proteinases (angiotensin converting enzyme) to cell culture users' knowledge and to give some data for solving problems about the development of peptides as useful drugs . The enzymes, purified from foetal bovine, adult bovine, foetal equine, adult equine, and human sera, showed molecular weights of about 170 kDa . Captopril and lisinopril inhibited enzyme activities at nanomolar concentrations . The enzymes were able to hydrolyze, with different efficiency, angiotensin I, bradykinin and epidermal mitosis inhibiting pentapeptide . The heat inactivation of commercial sera at 56 degrees C for 30 min showed a reduction of ACE activity of about 35-80% . Therefore, the presence of ACE activity in commercial sera can influence the activity of biological peptides tested on cell lines cultured "in vitro."

J Virol Methods, 1999 Feb, 77(2), 139 - 51
Characterization of primary cell cultures as potential target cells for analysis of bovine cytotoxic T lymphocytes; Earnest-DeYoung JV et al.; In domestic animal species, assessment of cell-mediated immune responses to virus infection is hampered by the requirement for class I MHC compatibility between target and effector cells . Additional complicating factors can include an inability to infect target cells in vitro, or virus-induced lysis of infected target cells . One way to circumvent these problems is to use virus-mediated gene transfer to deliver individual viral genes to autologous primary target cells . Several primary bovine cell cultures were assessed as potential target cells for cytotoxic T lymphocyte (CTL) assays by measuring their levels of class I MHC expression and susceptibilities to retroviral gene delivery . High levels in both class I MHC expression and susceptibility to gene delivery were seen in adherent cell cultures isolated from peripheral blood (PBAC) . PBAC, which arose as an outgrowth of adherent peripheral blood mononuclear cell cultures, had morphology, protein expression patterns, and response to functional assays characteristic of high endothelial cells . Expression of viral vector-delivered genes in PBAC cells was confirmed with a recombinant retrovirus carrying the green fluorescent protein (GFP) gene . The use of vector-mediated delivery of viral genes to bovine high endothelial cells is a promising method for assessment of cell-mediated immunity in cattle.

Toxicol Lett, 1999 Mar 8, 105(1), 47 - 57
A comparison of the effects of verocytotoxin-1 on primary human renal cell cultures; Williams JM et al.; Infection with verocytotoxin-producing Escherichia coli causes haemolytic uraemic syndrome (HUS) . Verocytotoxin-1 (VT1) is cytopathic to renal microvascular endothelial cells in culture, supporting the hypothesis that the vasculopathy of HUS is caused directly by the toxic action of VT1 on cells . We provide evidence that VT1 inhibits protein synthesis in primary cultures of glomerular epithelial cells (GE), cortical tubular epithelial cells (CTE) and mesangial cells (MC) . Using 100 pg/ml of VT1 we saw a decrease in protein synthesis to 14.3+/-1.9% in vero cells (a primate cell line), 1.7+/-0.3% in GE, 0.9+/-0.4% in CTE and 74.8+/-1.3% in MC at 24 h . The human renal epithelial cells are at least as sensitive as vero cells to the protein synthesis inhibitory effects of VT1 if not more so . Cell viability decreased in all cultures as measured by MTT reduction, neutral red incorporation and lactate dehydrogenase release and followed the same pattern of susceptibility as for protein synthesis inhibition . However, unlike vero cells, death occurred without DNA fragmentation . Cell sensitivity was greatest in cells which bound more VT1.

Neurotoxicology, 1999 Feb, 20(1), 41 - 8
The naturally occurring food mycotoxin fumonisin B1 impairs myelin formation in aggregating brain cell culture; Monnet-Tschudi F et al.; The effects of subchronical applications of the mycotoxin Fumonisin B1 (FB1) were analyzed in vitro, using aggregating cell cultures of fetal rat telencephalon as a model . As cells in the aggregates developed from an immature state to a highly differentiated state, with synapse and compact myelin formation, it was possible to study the effects of FB1 at different developmental stages . The results showed that FB1 did not cause cell loss and it had no effects on neurons . However it decreased strongly the total content of myelin basic protein, the main constituent of the myelin sheath, during the myelination period (DIV 18-28) . The loss of myelin was not accompanied by a loss of oligodendrocytes, the myelinating cells . However FB1 had effects on the maturation of oligodendrocytes, as revealed by a decrease in the expression of galactocerebroside, and on the compaction of myelin, as shown by a reduction of the expression of the mnyelin/oligodendrocyte glycoprotein MOG . The content of the cytoskeletal component glial fibrillary acidic protein (GFAP) was decreased in differentiated astrocytes, exclusively, while neurons were not affected by 40 microM of FB1 applied continuously for 10 days . In summary, FB1 selectively affected glial cells . In particular, FB1 delayed oligodendrocyte development and impaired myelin formation and deposition.

Eur J Biochem, 1999 Feb, 260(1), 156 - 65
Origin of the integrin-mediated signal transduction . Functional studies with cell cultures from the sponge Suberites domuncula; Wimmer W et al.; Sponges (phylum Porifera) represent the phylogenetically oldest metazoan animals . Recently, from the marine sponge Geodia cydonium a first cDNA encoding a putative integrin receptor molecule was isolated . In the present study basic functional experiments have been conducted to test the hypothesis that in sponges integrin polypeptides also function as adhesion molecules and as outside-in signaling molecules . The sponge Suberites domuncula has been used for the experiments because from this sponge only has a cell culture been established . Here we report that aggregation factor (AF)-mediated cell-cell adhesion is blocked by the RGDS peptide which is known to interact with beta integrin . Both RGDS and AF were found to stimulate DNA synthesis within 24 h . The beta subunit of the integrin receptor was cloned from S . domuncula; the estimated 91-kDa molecule comprises the characteristic signatures . Evolutionary conservation of the beta integrin was assessed by comparison with corresponding beta integrin subunits from evolutionary higher metazoan taxa . Addition of RGDS or of AF to isolated cells of S . domuncula causes a rapid (within 1-2 min) increase in the intracellular Ca2+ concentration which is further augmented in the presence of Ca2+ . Furthermore, incubation of the cells with RGDS or AF causes an activation of the GTP-binding protein Ras . In addition it is shown that after a prolonged incubation of the cells with RGDS and AF the expression of the genes coding for Ras and for calmodulin is upregulated . These results suggest that the integrin receptor functions in the sponge system not only as adhesion molecule but also as a molecule involved in outside-in signaling.

J Med Assoc Thai, 1999 Feb, 82(2), 167 - 72
The expression of cyclooxygenase-2 in human umbilical vein endothelial cell culture from preeclampsia; Akarasereenont P et al.; We have shown the HUVEC from normal pregnancy contained COX-1 protein but not COX-2 protein and released 6-keto-PGF1 alpha 277 +/- 5 ng/ml (for 24 h) . In contrast, HUVEC from preeclampsia contained both COX-1 and COX-2 protein and released significantly lesser amounts of 6-keto-PGF1 alpha (159 +/- 8 ng/ml for 24 h; p < 0.05) . Thus, COX-2 is expressed in HUVEC from preeclampsia but not in normal pregnancy and affects the release of prostacyclin suggesting the involvement of COX-2 in the pathogenesis of preeclampsia . The development of selective inhibitors of COX-2 may have a potential role in prevention and treatment of preeclampsia.

Brain Res, 1999 Feb 13, 818(2), 212 - 20
Biotransformation of nociceptin/orphanin FQ by enzyme activity from morphine-naive and morphine-treated cell cultures; Vlaskovska M et al.; The biotransformation of nociceptin/orphanin FQ (NOFQ) by enzyme activity isolated from U1690 human lung carcinoma and SH-SY5Y human neuroblastoma cell lines, and from rat brain cortex cells in primary culture was investigated . The identification and quantification of the cleavage products were performed using electrospray ionization mass spectrometry linked to size-exclusion chromatography . The effect of chronic morphine treatment of the cells (5 days) on NOFQ biotransformation was also studied . It was found that major products generated from NOFQ were the amino-terminal peptides N1-9 and N1-13 . The pattern of NOFQ biotransformation was quite similar for all three cell cultures . However, different proportions of the formed peptides were noted . The cleavage was inhibited by EDTA, PMSF, Hg2+, Cu2+ and Zn2+ . Dynorphin A2-13 inhibited NOFQ cleavage in a manner suggesting competition of the two peptides for the same enzyme . Chronic morphine treatment of the cell cultures resulted in a substantial increase in the enzyme activity, leading to higher levels of the major fragments and accumulation of N1-12 and the shorter peptides N1-5, N1-6 . Since the effect of morphine treatment of the cells was blocked by naloxone, it is likely that it was receptor specific . Taken together, the findings suggest that a metallosensitive endopeptidase, the activity of which is increased by chronic morphine treatment of the cells, is responsible for the biotransformation of NOFQ with fragments N1-9 and N1-13 being the major products .

Antibiot Khimioter, 1998, 43(11), 21 - 3
{Interferon-inducing action of polysaccharide-containing biopolymers from ginseng root and cell culture}; Smolina TP et al.; Induction of interferon (IF) and tumor necrosis factor (TNF) under the action of two polysaccharide preparations of ginseng i.e . panaxan-1 (from ginseng root) and panaxan-2 (from ginseng cell culture) was studied . Both the preparations induced production of TNF and IF in human leukocytes . By its properties and the typing results the induced IF proved to be gamma-IF . The preparation from the ginseng cell culture in the doses used had a higher IF inducing activity which could be explained by the difference in the polysaccharide composition of the preparations.

Biochim Biophys Acta, 1999 Jan 27, 1410(1), 77 - 84
Visualization of cyclosporin A and Ca2+-sensitive cyclical mitochondrial depolarizations in cell culture; Fall CP et al.; Mitochondria not only facilitate chemiosmotic energy transduction, but also are excitable organelles that are important participants in intracellular Ca2+ signaling and are obligate participants in the active cell death cascade known as apoptosis . Underlying these functions is the cyclosporin A (CSA)-sensitive mitochondrial permeability transition pore (MTP), which can open transiently in a low conductance mode (MTPL) to relieve excess Ca2+, and irreversibly during the initiation of apoptosis . Here we image for the first time CSA- and Ca2+-sensitive cyclical mitochondrial depolarizations in cultures of the SH-SY5Y human neuroblastoma cell . In addition, we show that mitochondrial transmembrane potential (DeltaPsi) increases in response to CSA, indicating a baseline channel activity . Moreover, networks of mitochondria are shown to behave as an excitable system that may use Ca2+ as a diffusible messenger to recruit neighboring mitochondria to depolarize . We propose that these depolarizations represent MTPL activity . Our data further reinforce the notion that mitochondria are excitable organelles and suggest coordinated activation of MTPL.

Toxicol Lett, 1999 Feb 22, 104(3), 239 - 48
Bgugaine, a pyrrolidine alkaloid from Arisarum vulgare, is a strong hepatotoxin in rat and human liver cell cultures; Rakba N et al.; Toxicity of bgugaine, a pyrrolidine alkaloid extracted from the tubers of Arisarum vulgare, was studied in three different liver cell culture models: (1) the rat hepatocyte primary culture; (2) a liver epithelial cell line; and (3) the human hepatoblastoma cell line HepG2 . Cytotoxicity was evaluated by LDH release, MTT reduction and MDA production . DNA fragmentation was analysed by flow cytometry or DNA gel-electrophoresis . In hepatocyte and epithelial cell cultures, drug toxicity appeared at 30 microM and was evaluated by an increase in LDH release, a decrease in MTT reduction and a higher level of MDA production . Bgugaine concentrations lower than 30 microM did not induce changes in these parameters . In HepG2 cells, bgugaine treatment also induced LDH release at concentrations of 40 and 50 microM . DNA fragmentation, analysed in the HepG2 cell line by flow cytometry, was observed in cultures exposed to 50 microM bgugaine . However, using DNA gel-electrophoresis, we demonstrated that lower bgugaine concentrations (10, 20 and 30 microM) also induced DNA damage . Our results show that: (1) bgugaine induces an important hepatotoxicity; (2) bgugaine toxicity is not mediated by a metabolic derivative; and (3) bgugaine induces a significant DNA damage . Therefore, our data suggest that the alkaloid bgugaine contained in Arisarum vulgarae may be involved in the toxicologic symptoms observed after consumption of this plant tubers by humans and animals.

Vet Microbiol, 1999 Mar 1, 65(2), 103 - 13
Strains of bovine herpesvirus 1 that do not express an epitope on glycoprotein E in cell culture still induce antibodies that can be detected in a gE-blocking ELISA; van Oirschot JT et al.; Two bovine herpesvirus 1 (BHV1) field strains that do not express an epitope on glycoprotein E (gE) in cell culture were inoculated into calves to examine whether their sera became positive in a gE-blocking ELISA that detects antibodies against gE . This gE-blocking ELISA uses one monoclonal antibody that is directed against the above mentioned epitope . All calves, except one, infected with these gE-epitope negative BHV1 strains, became positive in this gE-blocking ELISA, about two weeks later than in another gE-ELISA and a gB-ELISA . However, cattle infected with BHVI strains that do express this particular gE-epitope showed a similar type of antibody responses . These findings demonstrate that BHV1 strains that do not express a particular gE-epitope in cell culture, still can induce antibodies that are detected in a blocking ELISA that measures antibodies against that epitope.

J Acquir Immune Defic Syndr Hum Retrovirol, 1999 Mar 1, 20(3), 215 - 9
Oxandrolone, used for treatment of wasting disease in HIV-1-infected patients, does not diminish the antiviral activity of deoxynucleoside analogues in lymphocyte and macrophage cell cultures; Segal DM et al.; Antiviral agents are the primary therapy for patients infected with HIV-1 . However, supportive therapies are often necessary in addition to antiviral drugs because of the devastating wasting process associated with HIV-1 infection and AIDS . Oxandrolone, an anabolic steroid, is used in promoting weight gain and, most important lean body mass (LBM), in patients with HIV-1 disease . We investigated whether oxandrolone interferes with the antiviral activity of zidovudine (ZDV), dideoxyinosine (ddI), and dideoxycytidine (ddC) on HIV-1 replication in peripheral blood lymphocytes and macrophage-monocytes . The nucleoside analogues had nanomolar 50% inhibitory concentrations (IC50) in peripheral lymphocytes . Combinations of nucleoside analogues and oxandrolone did not result in increased IC50 values . Oxandrolone used alone exhibited micromolar IC50 values in peripheral blood lymphocytes . Lack of interference was consistent for nucleoside concentrations up to 5 microM and for oxandrolone concentrations up to 100 microM in several combinations of drugs, viral strains, and peripheral lymphocytes and macrophages . We conclude that oxandrolone can be used for the promotion of weight gain in patients with AIDS-related wasting without interference with the antiviral effects of ZDV, ddI, or ddC.

J Clin Microbiol, 1999 Apr, 37(4), 958 - 63
Multicenter comparison of the digene hybrid capture CMV DNA assay (version 2.0), the pp65 antigenemia assay, and cell culture for detection of cytomegalovirus viremia; Mazzulli T et al.; We compared the Digene Hybrid Capture CMV DNA Assay version 2.0, the pp65 antigenemia assay, traditional tube culture, and shell vial culture for the detection of cytomegalovirus (CMV) viremia in several patient populations at three centers . Of 561 blood specimens collected from 402 patients, complete clinical and laboratory data were available for 489 . Using consensus definitions for true positives and true negatives, the sensitivities of the Hybrid Capture assay, antigenemia, shell vial, and tube culture were 95, 94, 43, and 46%, respectively . The specificities of the Hybrid Capture assay and antigenemia were 95 and 94%, respectively . At all three study sites, the detected level of CMV viremia was significantly higher with the Hybrid Capture assay or antigenemia than with shell vial and tube culture . In a group of 131 healthy nonimmunosuppressed volunteers, the Hybrid Capture assay demonstrated a specificity of over 99% . The Hybrid Capture assay is a standardized assay that is simple to perform and can utilize whole blood specimens that have been stored for up to 48 h . The high sensitivity and specificity of the Hybrid Capture assay along with its simplicity and flexibility make it a clinically useful assay for the detection of CMV viremia in immunocompromised or immunosuppressed patients . Further evaluation to determine its role in predicting CMV disease and for monitoring the therapeutic response to anti-CMV therapy is needed.

Prenat Diagn, 1999 Jan, 19(1), 12 - 6
Comparison of cell cultures, chromosome quality and karyotypes obtained after chorionic villus sampling and early amniocentesis with filter technique; Sundberg K et al.; 548 cell cultures and karyotypes obtained by early amniocentesis with filtration technique (EAF) at a mean gestational age of 12 1/2 weeks were compared with 555 obtained by transabdominal chorionic villus sampling (CVS) at a mean gestational age of 11 weeks . The number of abnormal karyotypes, culture failure rate and harvest time were evaluated . The results were then compared with three similar studies from the literature evaluating early amniotic fluid cultures obtained with conventional techniques compared with chorionic villus cultures . Further, the quality of the chromosome preparations from chorionic villi and early amniotic fluid respectively was compared . More abnormal karyotypes were found among the CVS cultures, which was expected due to earlier sampling and presence of confined placental mosaicism . No ambiguous results were present after EAF . The lowest culture failure rate of 0.2 per cent was found after EAF compared with 0.9 per cent among CVS . EAF also showed a significantly lower culture failure rate when compared with the literature, where early amniocentesis (EA) had been carried out by standard techniques . The time from sampling to harvest was longer in the EAF group (mean 9.5 days) compared with CVS (mean 6.1 days), in accordance with the literature . Nevertheless, the culture time of EAF was significantly shorter than the mean of EA from the comparative studies, whereas CVS culture times showed no differences . Rates of pseudomosaicism, maternal cell contamination, single cell aberrations, number of chromosome bands, mitoses counted and number of primary cultures needed for each karyotype were also compared . We concluded that EAF carried out around 12 1/2 weeks of gestation is a successful method with a lower culture failure rate compared with CVS cultures from 11-week gestations and with a significantly lower culture failure rate when compared with EA from similar studies . EAF provides chromosome preparations of high quality, and the risk of ambiguous karyotypes is very low.

Zhongguo Yao Li Xue Bao, 1997 Jul, 18(4), 299 - 303
Actions of 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate in vascular smooth muscle cell cultures; Chen Z et al.; AIM: To study the effects of 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate (TMB-8) on vascular smooth muscle (VSM) cells A7r5 . METHODS: The effects of TMB-8 were investigated in A7r5 cell cultures with 45CaCl2 . RESULTS: TMB-8 reduced the intracellular free Ca2+ concentration, {Ca2+}i in a Ca(2+)-free medium and blocked Ca2+ entry from the extracellular site in a regular Ca2+ medium . The equilibrated total cellular binding of Ca2+ was increased by TMB-8 whereas 45Ca2+ entry activated by both NE and KCl was inhibited . However, the NE-activated Ca2+ entry was not blocked by TMB-8 if TMB-8 was added together with 45Ca2+ at a later time instead of by pretreatment . Similar to actions of NE and KCl, depletion of Ca2+ from sarcoplasmic reticulum (SR) would also activate Ca2+ entry, which was blocked by TMB-8 . When TMB-8 was rinsed out alone or together with NE after pretreatment with NE plus TMB-8 in VSM cells, the inhibitory effect of TMB-8 was not affected . CONCLUSION: TMB-8 not only blocks Ca2+ entry from the extracellular site, but also enhances Ca2+ uptake into SR which, indirectly inhibits Ca2+ entry from the extracellular site.

Methods, 1998 Nov, 16(3), 320 - 44
Microglia in cell culture and in transplantation therapy for central nervous system disease; Dobrenis K; The central nervous system (CNS) is host to a significant population of macrophage-like cells known as microglia . In addition to these cells which reside within the parenchyma, a diverse array of macrophages are present in meningeal, perivascular, and other peripheral locations . The role that microglia and other CNS macrophages play in disease and injury is under intensive investigation, and functions in development and in the normal adult are just beginning to be explored . At present the biology of these cells represents one of the most fertile areas of CNS research . This article describes methodology for the isolation and maintenance of microglia in cell cultures prepared from murine and feline animals . Various approaches to identify microglia are provided, using antibody, lectin, or scavenger receptor ligand . Assays to confirm macrophage-like functional activity, including phagocytosis, lysosomal enzyme activity, and motility, are described . Findings regarding the origin and development of microglia and results of transplantation studies are reviewed . Based on these data, a strategy is presented that proposes to use the microglial cell lineage to effectively deliver therapeutic compounds to the CNS from the peripheral circulation.

J Invertebr Pathol, 1999 Mar, 73(2), 199 - 205
Yield, biological activity, and field performance of a wild-type Helicoverpa nucleopolyhedrovirus produced in H . zea cell cultures; Chakraborty S et al.; This paper reports studies of the in vitro production of a virus from Helicoverpa armigera (HaSNPV) and its possible use as a specific Helicoverpa/Heliothis larvicide . Growth kinetics of Helicoverpa zea (H . zea) cells and virus occlusion body yields were compared in three SF900II-based media, namely, SF900II (serum-free), SF900II + 1% serum, or SF900II + 10% serum . Viable cell densities were usually higher in the media supplemented with serum than in the serum-free medium; however, in the serum-free medium, cell diameters were 1.7 times greater (i.e., individual cell volumes were five times larger) . Both volumetric production of virus occlusion bodies and production per cell were higher in the serum-free medium than in the media supplemented with serum . However, the infectivity of the occlusion bodies from the serum-free medium was less than that with those from the medium supplemented with 10% serum, when compared in bioassays employing newly hatched larvae . The infectivity of the in vitro produced occlusion bodies was also less than that of in vivo produced occlusion bodies in a commercially available virus product, GemStar . High levels of infection of H . armigera larvae obtained in a preliminary field assessment on preflowering tomatoes using the in vitro produced occlusion bodies indicated the suitability of the in vitro process for biopesticide production .

J Membr Biol, 1999 Mar 1, 168(1), 77 - 89
Select de novo gene and protein expression during renal epithelial cell culture in rotating wall vessels is shear stress dependent; Kaysen JH et al.; The rotating wall vessel has gained popularity as a clinical cell culture tool to produce hormonal implants . It is desirable to understand the mechanisms by which the rotating wall vessel induces genetic changes, if we are to prolong the useful life of implants . During rotating wall vessel culture gravity is balanced by equal and opposite hydrodynamic forces including shear stress . The current study provides the first evidence that shear stress response elements, which modulate gene expression in endothelial cells, are also active in epithelial cells . Rotating wall culture of renal cells changes expression of select gene products including the giant glycoprotein scavenger receptors cubulin and megalin, the structural microvillar protein villin, and classic shear stress response genes ICAM, VCAM and MnSOD . Using a putative endothelial cell shear stress response element binding site as a decoy, we demonstrate the role of this sequence in the regulation of selected genes in epithelial cells . However, many of the changes observed in the rotating wall vessel are independent of this response element . It remains to define other genetic response elements modulated during rotating wall vessel culture, including the role of hemodynamics characterized by 3-dimensionality, low shear and turbulence, and cospatial relation of dissimilar cell types.

Proc Natl Acad Sci U S A, 1999 Mar 2, 96(5), 2153 - 8
Dynamics and elasticity of the fibronectin matrix in living cell culture visualized by fibronectin-green fluorescent protein; Ohashi T et al.; Fibronectin (FN) forms the primitive fibrillar matrix in both embryos and healing wounds . To study the matrix in living cell cultures, we have constructed a cell line that secretes FN molecules chimeric with green fluorescent protein . These FN-green fluorescent protein molecules were assembled into a typical matrix that was easily visualized by fluorescence over periods of several hours . FN fibrils remained mostly straight, and they were seen to extend and contract to accommodate movements of the cells, indicating that they are elastic . When fibrils were broken or detached from cells, they contracted to less than one-fourth of their extended length, demonstrating that they are highly stretched in the living culture . Previous work from other laboratories has suggested that cryptic sites for FN assembly may be exposed by tension on FN . Our results show directly that FN matrix fibrils are not only under tension but are also highly stretched . This stretched state of FN is an obvious candidate for exposing the cryptic assembly sites.

Biochem Biophys Res Commun, 1999 Feb 16, 255(2), 360 - 6
Physico-chemical modeling of the role of free radicals in photodynamic therapy . IV . Quantitative aspects of photodynamic effects on free radicals generated in cell cultures; Nemeth A et al.; Production and the mechanism of the interactions of free radicals generated by stimulated macrophages in the presence of luminol and a free radical inhibitor was investigated to determine the possibility of using luminol-dependent chemiluminescence for studying photodynamic effects in biology . Earlier measurements have been revisited and additional experiments performed indicating that oxidation products of luminol neither inhibit the in vitro formation of radicals nor quench CL . Simulation based on the mechanism suggested revealed that the likely value for the rate constant of the primary step between luminol and superoxide anion radicals producing luminol radicals is 5x10(2)-1x10(3) M-1s-1 . It has been established that the ratio of the concentration of radicals generated by the biological system to that formed by oxidation of luminol exceeds 10(3); that is, the contribution of the latter is negligible and the system is appropriate to measure quantitatively the effect of excited photosensitizers on free radicals .

Cytokine, 1998 Dec, 10(12), 993 - 6
Impaired production of interleukin 6 and tumour necrosis factor alpha in whole blood cell cultures of patients with multiple myeloma; Filella X et al.; Cytokine production in whole blood cell cultures can be an indicator of immune cellular status in neoplastic patients . Production of interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-alpha) after stimulation with lipopolysaccharide was measured in 1/10 diluted whole blood culture of patients with monoclonal gammopathies {monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM)} . With this system, lower production of IL-6 and TNF-alpha at 4, 24 and 48 h in total blood cultures in patients with symptomatic MM was observed . Thus, the capacity of cytokine production was greater in control subjects and in patients with MGUS and MM in response than in symptomatic MM (P=0.005 at 4 h and P=0 . 006 at 24 and 48 h for IL-6 and P<0.0001 at 24 h and P<0.001 at 48 h for TNF-alpha) . These differences remained significant after adjustment on the basis of the lymphocyte count (P<0.001 for IL-6 and TNF-alpha at 24 and 48 h) . The impaired IL-6 and TNF-alpha production in patients with symptomatic MM is probably due to a tumour-related immunosuppressive status .

J Neuroendocrinol, 1999 Feb, 11(2), 145 - 52
Effects of osmotic pressure and brain-derived neurotrophic factor on the survival of postnatal hypothalamic oxytocinergic and vasopressinergic neurons in dissociated cell culture; Kusano K et al.; Neurons from hypothalamic paraventricular nuclei (PVN) and supraoptic nuclei (SON) from postnatal day 6-8 rats were enzymatically dissociated and separately maintained in monolayer cultures for 14 days . The osmotic pressure of the culture medium, based on Neurobasal medium (Life Technologies), was varied (255, 300 and 330 mOsm/l) by adjustment using mannitol . The survival of oxytocin (OT), vasopressin (VP) and oxytocin-vasopressin (OT/VP) coexpressing neurons were studied under these varied conditions, and the identification of the cell phenotypes in the cultures was carried out by using double-label immunofluorescence . Under control osmolar conditions (300 mOsm/l) equivalent numbers of OT and VP neurons were found in the SON (P = 0.8398) and PVN (P = 0.4721) cultures . The OT neurons' survival did not change in 255 or 330 mOsm media in the SON cultures, but the VP neurons in the SON cultures were significantly increased in 255 mOsm/l medium as compared to control (300 mOsm/l) medium (P = 0.0088) . No significant changes were found in VP neuron survival in SON cultures between the 300-330 mOsm/l media (P = 0.2372) . Similar data were obtained for the VP neurons in PVN-derived cultures, but the OT neurons in these cultures survived significantly better at 300 mOs/l than at 255 mOsm/l (P<0.0001), but were not significantly different at 330 mOsm/l (P = 0.1208) . In general, the VP neurons were more vulnerable than OT neurons to increases of culture medium osmolarity with respect to their survival . The number of OT/VP coexpressing neurons was greater in SON-derived cell cultures as compared to PVN-derived cell cultures, and their numbers were higher in the lower osmolarity media . The effects of adding brain-derived neurotrophic factor (BDNF) to the culture medium on survival were determined . BDNF significantly increased the numbers of all three types of neurons in both PVN and SON cell cultures (P = 0.0001-0.0060) . The phenotypically identified cells, cultured in the 300 mOsm/l medium, responded by depolarization or hyperpolarization when transferred to hypertonic or hypotonic perfusion salines, respectively.

Biomed Tech (Berl), 1998 Dec, 43(12), 354 - 9
{Standardized tests of bone implant surfaces with an osteoblast cell culture system . I . Orthopedic standard materials}; Merklein F et al.; The effect of standard orthopaedic materials on proliferation and differentiation of osteoblasts was examined using a standardised cell culture system . Osteoblasts hFOB 1.19 were cultured on stainless steel (SS), a chromium-cobalt-molybdenum alloy (CrCoMb) and commercially pure titanium (cpTi) for 12 days . Cell culture polystyrene (PS) was used as a reference . Cell numbers and cell viability were used as parameters of proliferation . Cell differentiation was assessed using alkaline phosphatase activity, collagen I and osteocalcin production . The parameters of proliferation showed earlier maximum values on PS and cpTi, while proliferation was delayed on SS and CrCoMb . The highest values of differentiation were found on cpTi . The development of alkaline phosphatase activity showed two peaks reflecting apoptosis and redifferentiation . The cell culture system hFOB 1.19 is thus suitable for revealing differences in proliferation and differentiation of osteoblasts on standard orthopaedic materials . The results correlate with previous in vivo findings . Using this system, the dynamic effect of the material surface on the differentiation process of osteoblasts can be demonstrated.

Anal Biochem, 1999 Mar 1, 268(1), 110 - 6
Enzyme-based flow injection analysis system for glutamine and glutamate in mammalian cell culture media; Mayer C et al.; We present the setup of a flow injection analysis system designed for on-line monitoring of glutamate and glutamine . These amino acids represent a major energy source in mammalian cell culture . A cycling assay consisting of glutamate dehydrogenase and aspartate aminotransferase produces NADH proportional to the glutamate concentration in the sample . NADH is then measured spectrophotometrically . Glutamine is determined by conversion to glutamate which is fed into the cycling assay . The conversion of glutamine to glutamate is catalyzed by asparaginase . Asparaginase was used in place of glutaminase due to its relatively high reactivity with glutamine and a pH optimum similar to that of glutamate dehydrogenase . The enzymes were immobilized covalently to activated controlled pore glass beads and integrated into the flow injection analysis system . The application of the immobilized enzymes and the technical setup are presented in this paper .

Synapse, 1999 Mar 1, 31(3), 186 - 95
Agonist- and antagonist-induced plasticity of rat 5-HT1A receptor in hippocampal cell culture; Nishi M et al.; We examined the response and regulation of 5-HT1A receptor on hippocampal cultured fetal neurons grown in the absence of serotonin and steroids using three experimental designs: 1) functional response using an antibody against phosphorylated cyclic adenosine monophosphate response element binding protein (pCREB); 2) transcriptional regulation using in situ hybridization; and 3) translational expression using antipeptide 5-HT1A receptor antibody . Pretreatment of cultured hippocampal cells with the agonist 8-hydroxy-2-(di-N-propylamino)-tetralin (8-OH-DPAT) (10(-8) M) or ipsapirone (IPS) (10(-9) M) for 10 min blocked the forskolin-stimulated increase in pCREB immunoreactivity . In situ hybridization radioautography revealed that IPS (10(-9) M) decreased the 5-HT1A receptor mRNA expression (-33%) after a 24-h treatment . The decrease in 5-HT1A receptor mRNAwas accompanied by a change in protein immunoreactivity using a 5-HT1A receptor antipeptide antibody . Computer-assisted morphometric analyses showed a reduction in the 5-HT1A receptor immunoreactive (IR) intensity as compared to control 24 h after treatment with 8-OH-DPAT (10(-7)-10(-12) M) and IPS (10(-9) M) . Thus, fetal hippocampal neurons have a functional 5-HT1A receptor that is downregulated at both the transcription and translation levels . In addition, we found increased 5-HT1A receptor-IR intensity (+17% approximately +39%) 24 h after treatment with the antagonist N-{2-{4-(2-methoxyphenyl)-1-piperazinyl}ethyl}-N-(2-pyridinyl) cyclohexane carboxamide (WAY 100635) (10(-7)-10(-12) M) . Our results indicate that the 5-HT1A receptor is sensitive to both agonists (downregulation) and antagonists (upregulation) in hippocampal fetal neurons grown in the absence of serotonin and steroids.

J Virol Methods, 1999 Jan, 77(1), 75 - 9
Sheep poxvirus identification by PCR in cell cultures; Mangana-Vougiouka O et al.; A simple, rapid and specific diagnostic polymerase chain reaction (PCR) method was developed for sheep poxvirus identification . The primers used were from the sequenced genomes of the capripox viruses KS-1 and InS-1 . Six different sheep pox isolates were tested against two orf (parapox) and three animal herpesviruses as controls . Material from uninfected cell cultures was also used as control . The sensitivity of the PCR was approximately equivalent with each of the two primers and for the six sheep pox isolates . All the negative control virus DNAs were negative and differed clearly from those of the sheep pox strains.

Exp Hematol, 1999 Feb, 27(2), 370 - 9
A comparative study of the cell cycle status and primitive cell adhesion molecule profile of human CD34+ cells cultured in stroma-free versus porcine microvascular endothelial cell cultures; Chute JP et al.; Porcine microvascular endothelial cells (PMVECs) plus cytokines support a rapid proliferation and expansion of human CD34+CD38- cells that are capable of multilineage engraftment within the bone marrow of a secondary host . CD34+CD38- cells contain the self-renewing, long-term culture-initiating cells (LTC-IC) that are ideal targets for retroviral gene transfer experiments . Previous experiments attempting retroviral infection of CD34+CD38- cells have failed partly because these cells do not enter cell cycle in response to cytokine combinations . In this study, we determined the cell cycle status and the cell adhesion molecule profile on purified CD34+ cells and the CD34+CD38- subset before and after ex vivo expansion on PMVECs . Purified human CD34+ cells were cocultured with PMVECs for 7 days in the presence of optimal concentrations of granulocyte/macrophage-colony-stimulating factor (GM-CSF) + interleukin (IL)-3 + IL-6 + stem cell factor (SCF) + Flt-3 ligand . The total CD34+ population and the CD34+CD38- subset increased 8.4- and 67-fold, respectively, with absolute increases in the number of colony-forming unit-granulocyte macrophage (CFU-GM) (28.2-fold), CFU-Mix (8.7 fold), and burst-forming unit-erythroid (BFU-E) (4.0-fold) progenitor cells . After 7 days of coculture with PMVECs, 44% of the CD34+CD38+ subset were found to be in G1, and 51% were in G2/S/M phase of the cell cycle . More remarkably, 53% of the CD34+CD38- subset were in G1, and 17% were in G2/S/M phase after 7 days of PMVEC coculture . In contrast, only 22% of the CD34+CD38- subset remaining after 7 days of stroma-free culture were in G1, and 6% were in G2/S/M phase . Despite the high level of cellular activation and proliferation induced by PMVEC coculture, the surface expression of adhesion molecules CD11a (LFA-1), CD11b, CD15s (sialyl-Lewis x), CD43, and CD44 (HCAM) on the total CD34+ population was maintained, and the surface expression of CD49d (VLA-4), CD54 (ICAM), CD58, and CD62L (L selectin) increased after ex vivo expansion . In contrast, CD34+ cells expanded on stroma-free cultures showed lower and more variable expression of CD62L and CD15s . These findings demonstrate that the primitive CD34+CD38- subset of marrow progenitor cells can be induced to enter cell cycle and can be significantly expanded ex vivo on a hematopoietic supportive microenvironment (PMVECs) while preserving the expression of cell adhesion molecules that may be important in stem cell homing and engraftment.

J Chromatogr A, 1999 Jan 22, 831(1), 63 - 72
Purification of monoclonal antibodies from cell culture supernatants using a modified zirconia based cation-exchange support; Clausen AM et al.; A method suitable for the isolation of monoclonal antibodies (Mabs) is described . The protocol utilizes a zirconia based column modified with ethylenediamine-N,N'-tetra(methylenephosphonic) acid to create a novel cation-exchange chromatographic support . Initial experiments using a linear salt gradient demonstrate the ability of this support to efficiently separate Mab from transferrin and bovine serum albumin in a model matrix . Results of the purification of Mab from an actual cell culture supernatant over a range in protein concentrations are also shown . Analyses by enzyme-linked immunosorbent assay and gel electrophoresis demonstrate that Mabs can be recovered from a cell culture supernatant at high yield (92-98%) and high purity (> 95%) in a single chromatographic step.

J Neurobiol, 1999 Jan, 38(1), 65 - 81
Retinoic acid and neurotrophins collaborate to regulate neurogenesis in adult-derived neural stem cell cultures; Takahashi J et al.; ABSTRACT: The adult rat hippocampus contains fibroblast growth factor 2-responsive stem cells that are self-renewing and have the ability to generate both neurons and glia in vitro, but little is known about the molecular events that regulate stem cell differentiation . Hippocampus-derived stem cell clones were used to examine the effects of retinoic acid (RA) on neuronal differentiation . Exposure to RA caused an immediate up-regulation of NeuroD, increased p21 expression, and concurrent exit from cell cycle . These changes were accompanied by a threefold increase in the number of cells differentiating into immature neurons . An accompanying effect of RA was to sustain or up-regulate trkA, trkB, trkC, and p75NGFR expression . Without RA treatment, cells were minimally responsive to neurotrophins (NTs), whereas the sequential application of RA followed by brain-derived neurotrophic factor or NT-3 led to a significant increase in neurons displaying mature y-a-minobutyric acid, acetylcholinesterase, tyrosine hydroxylase, or calbindin phenotypes . Although NTs promoted maturation, they had little effect on the total number of neurons generated, suggesting that RA and neurotrophins acted at distinct stages in neurogenesis . RA first promoted the acquisition of a neuronal fate, and NTs subsequently enhanced maturation by way of RA-dependent expression of the Trk receptors . In combination, these sequential effects were sufficient to stimulate stem cell-derived progenitors to differentiate into neurons displaying a variety of transmitter phenotypes.

J Neurosci, 1999 Mar 1, 19(5), 1657 - 62
Ischemic tolerance in murine cortical cell culture: critical role for NMDA receptors; Grabb MC et al.; Murine cortical cultures containing both neurons and glia (days in vitro 13-15) were exposed to periods of oxygen-glucose deprivation (5-30 min) too brief to induce neuronal death . Cultures "preconditioned" by sublethal oxygen-glucose deprivation exhibited 30-50% less neuronal death than controls when exposed to a 45-55 min period of oxygen-glucose deprivation 24 hr later . This preconditioning-induced neuroprotection was specific in that neuronal death induced by exposure to excitotoxins or to staurosporine was not attenuated . Neuroprotection was lost if the time between the preconditioning and severe insult were decreased to 7 hr or increased to 72 hr and was blocked if the NMDA antagonist 100 microM 3-((D)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid was applied during the preconditioning insult . This was true even if the duration of preconditioning was increased as far as possible (while still remaining sublethal) . A similar preconditioning effect was also produced by sublethal exposure to high K+, glutamate, or NMDA but not to kainate or trans-1-aminocyclopentane-1, 3-dicarboxylic acid.

Dent Mater, 1998 Mar, 14(2), 158 - 63
Release of elements from dental casting alloys into cell-culture medium over 10 months; Wataha JC et al.; OBJECTIVE: The release of elements from eight types of commonly used dental casting alloys into cell-culture medium was measured over a 10-month period . The release of elements was determined to provide information about the long-term biological risk these alloys may pose to the oral tissues . The current work extends previous studies of shorter time periods, and is more relevant to the in vivo situation, where dental alloys are present intraorally for years . METHOD: The alloys were Au-, Ag-, Pd-, and Ni-based with nobilities ranging from 0 to 95 at.% . Alloy samples (n = 12) were exposed to cell-culture medium . The medium was changed every 30 days for 10 months . Elemental release into the medium was measured by means of atomic absorption spectrophotometry . Differences in mass release were determined using ANOVA and Tukey multiple comparison intervals (alpha = 0.05) . RESULTS: The release of elements continued through 10 months, and it appeared that the release was constant throughout most of the experiment . Higher initial rates were suspected but not verified . Most alloys reached a constant rate after < 100 days of exposure to the medium . Long-term element release was not generally intuitive based on the amount of an element in an alloy or the overall nobility of the alloy . Total mass lost over the 10-month period ranged from < 2 micrograms/cm2 for the Au-Pd alloy to 55 micrograms/cm2 for the Au-Ag-Cu alloy (Tukey interval at alpha = 0.05 was 0.8 microgram/cm2) . By comparison, pure Cu lost 4500 micrograms/cm2 during this period . SIGNIFICANCE: Tests which assess biological risk from elemental release must consider longer-term release because elemental release continues for extended periods . Longer-term mass loss from a given alloy is often not intuitive based on its overall composition or noble metal content.

Clin Neuropathol, 1999 Jan-Feb, 18(1), 1 - 8
MGMT- and P450 3A-inhibitors do not sensitize glioblastoma cell cultures against nitrosoureas; Kirches E et al.; AIM, MATERIAL AND METHODS: Using RT-PCR and immunohistochemistry the expression of cytochrome P450 was evaluated in a series of 22 glioblastomas and 4 anaplastic astrocytomas (WHO grade III) . Since rat liver P450 can catalyze the denitrosation of the nitrosourea compound BCNU in vitro, cell culture experiments were performed to test a possible sensitizing effect of P450 3A inhibitors (tiamulin and ketoconazole) in BCNU treatment of glial tumor cells . O6-benzylguanine (BG), an inhibitor of the DNA repair enzyme O6-methylguanine-DNA-methyltransferase (MGMT), was used in parallel experiments, since MGMT is discussed as a main mechanism in nitrosourea resistance . RESULTS: RT-PCR reactions with primers designed according to the sequences for CYP1A1 and CYP2E1 were always positive, while those for CYP1A2 and CYP2D6 were negative . The strongest PCR products were detected with CYP3A primers, but CYP3A expression was heterogeneous within the tumor samples . Antibodies to human liver CYP3A4 stained a subfraction of tumor cells (18% of the cells in glioblastomas and 14% in grade III astrocytomas) and to some extend neurons in normal brain areas, while astrocytes were negative . For cell culture experiments with P450 3A and MGMT inhibitors, early passages of 3 glioblastomas, a late passage of an immortalized cell line derived from a reoccurring glioblastoma, and the human glioblastoma line LN405 were used . The sensitivity of the tumor cells for both nitrosourea compounds was very low, when low concentrations were applied (comparable to the achievable blood concentrations in glioma patients) . Strong effects did only occur when the concentrations were raised 9-fold or 27-fold . CONCLUSION: In no case could a significant sensitizing effect of P450 3A- and MGMT inhibitors be demonstrated.

Eur J Neurosci, 1999 Jan, 11(1), 327 - 34
Zn2+ entry produces oxidative neuronal necrosis in cortical cell cultures; Kim EY et al.; Evidence has accumulated that Zn2+ plays a central role in neurodegenerative processes following brain injuries including ischaemia or epilepsy . In the present study, we examined patterns and possible mechanisms of Zn2+ neurotoxicity . Inclusion of 30-300 microM Zn2+ for 30 min caused neuronal necrosis apparent by cell body and mitochondrial swelling in cortical cell cultures . This Zn2+ neurotoxicity was not attenuated by antiapoptosis agents, inhibitors of protein synthesis or caspase . Blockade of glutamate receptors or nitric oxide synthase showed no beneficial effect against Zn2+ neurotoxicity . Interestingly, antioxidants, trolox or SKF38393, attenuated Zn(2+)-induced neuronal necrosis . Pretreatment with insulin or brain-derived neurotrophic factor increased the Zn(2+)-induced free radical injury . Kainate or AMPA facilitated Zn2+ entry and potentiated Zn2+ neurotoxicity in a way sensitive to trolox . Reactive oxygen species and lipid peroxidation were generated in the early phase of Zn2+ neurotoxicity . These findings indicate that entry and accumulation of Zn2+ result in generation of toxic free radicals and then cause necrotic neuronal degeneration under certain pathological conditions in the brain.

J Clin Microbiol, 1999 Mar, 37(3), 785 - 7
Isolation of Legionella pneumophila by centrifugation of shell vial cell cultures from multiple liver and lung abscesses; La Scola B et al.; A 7-year-old girl was admitted to the hospital with acute lymphoblastic leukemia and was treated with allogenic cord blood transplantation . At day 30 after graft, she developed a fever and multiple nodular lesions disseminated in the liver and lungs . All bacterial cultures attempted on liver and lung biopsy specimens and blood remained sterile on standard axenic media . However, inoculation of liver and lung biopsy specimens on eukaryotic cell monolayers by the centrifugation-shell vial technique (M . Marrero and D . Raoult, Am . J . Trop . Med . Hyg . 40:197-199, 1989) led to the recovery of a strain of Legionella pneumophila serogroup 1, identified by 16S rRNA gene amplification and sequencing and serotyping . Our findings demonstrate that the centrifugation-cell culture method, which has previously been useful for the isolation of other strictly or facultatively intracellular bacteria, can also serve as a method for the recovery of L . pneumophila from clinical material.

J Med Chem, 1999 Feb 11, 42(3), 393 - 9
The presence of substituents on the aryl moiety of the aryl phosphoramidate derivative of d4T enhances anti-HIV efficacy in cell culture: A structure-activity relationship; Siddiqui AQ et al.; New substituted-aryl phosphoramidate derivatives of the anti-HIV drug d4T were synthesized as membrane-soluble intracellular prodrugs for the free bioactive phosphate to establish relationship(s) between compound structure and in vitro antiviral activity . The majority of compounds demonstrated an elevation of in vitro potency relative to that of the parent nucleoside, and unlike d4T, all retained full activity in thymidine kinase-deficient cells . The compound bearing a p-chloro aryl group (8e) expressed nanomolar activity in vitro, a 14-fold increase in activity relative to that of the unsubstituted phosphoramidate (100-fold compared to d4T) . An assay using pig liver esterase was used to establish the stability of the compounds to enzymatic degradation . While there was no apparent correlation between in vitro activity and half-life of enzymatic degradation, there was a close correlation between compound lipophilicity, determined by octanol/water partition coefficient, and in vitro potency . We suggest that substitutions made to the aryl moiety of the aryl phosphoramidate of d4T that result in enhancing lipophilicity may serve to increase the cellular uptake of the prodrug by passive diffusion, leading to the expression of antiviral potency at reduced prodrug concentrations.

Trop Anim Health Prod, 1998 Dec, 30(6), 341 - 9
Susceptibility to tropical theileriosis of calves born to dams immunized with Theileria annulata (Hisar) cell culture vaccine; Beniwal RK et al.; The susceptibility/immune status to tropical theileriosis of calves born of immunized dams was evaluated . Six cows were vaccinated with the Theileria annulata cell culture vaccine in the eighth month of pregnancy . Sera from the immunized dams exhibited very high post-vaccination antibody titres as determined by the indirect fluorescent antibody (IFA) test . The calves born to these dams did not show antibodies against T . annulata at the time of birth (IFA titres of < 1:20) . The new-born calves were fed colostrum from their mothers and were challenged with T . annulata-infected ground tick supernate at 5-7 days of age . All the calves developed fever (from day 5-6 onwards) and parasitological reactions (from day 8-9 onwards) after challenge . There was a significant decrease in the haemoglobin and packed cell volume of the calves after challenge . All the calves showed signs of acute theileriosis by day 9-10 after challenge and had to be treated with buparvaquone in order to save their lives . The study indicated that detectable levels of anti-theilerial antibodies were not transferred from immune dams to their offspring . All the calves born to immunized dams were fully susceptible to theileriosis and thus themselves needed vaccination.

Clin Orthop, 1999 Jan, (358), 235 - 43
Marrow cell culture on poly-L-lactic acid fabrics; Hasegawa Y et al.; Bone marrow cells from rat femurs were cultured in Eagle Minimum Essential Medium containing 15% fetal calf serum until confluence . After the cells were trypsinized, they were subcultured on fabrics made of biodegradable poly-L-lactic acid for 2 weeks in the medium containing fetal calf serum, ascorbic acid phosphate, beta-glycerophosphate, and with and without dexamethasone . In the presence of dexamethasone, the fabrics showed many mineralized nodules together with cuboidal shaped cells that had osteoblastic activity, as evidenced by high alkaline phosphatase activity and the appearance of osteocalcin messenger ribonucleic acid . However, in the absence of dexamethasone, nodules did not form and many fibroblastic cells appeared with no evidence of osteoblastic activity . These results indicate the possibility of making a hybrid ligament substitute having an in vitro prefabricated bone anchor.

Plant Physiol, 1999 Feb, 119(2), 705 - 12
Purification and cDNA cloning of isochorismate synthase from elicited cell cultures of Catharanthus roseus; van Tegelen LJ et al.; Isochorismate is an important metabolite formed at the end of the shikimate pathway, which is involved in the synthesis of both primary and secondary metabolites . It is synthesized from chorismate in a reaction catalyzed by the enzyme isochorismate synthase (ICS; EC 5.4.99.6) . We have purified ICS to homogeneity from elicited Catharanthus roseus cell cultures . Two isoforms with an apparent molecular mass of 64 kD were purified and characterized . The Km values for chorismate were 558 and 319 microM for isoforms I and II, respectively . The isoforms were not inhibited by aromatic amino acids and required Mg2+ for enzyme activity . Polymerase chain reaction on a cDNA library from elicited C . roseus cells with a degenerated primer based on the sequence of an internal peptide from isoform II resulted in an amplification product that was used to screen the cDNA library . This led to the first isolation, to our knowledge, of a plant ICS cDNA . The cDNA encodes a protein of 64 kD with an N-terminal chloroplast-targeting signal . The deduced amino acid sequence shares homology with bacterial ICS and also with anthranilate synthases from plants . Southern analysis indicates the existence of only one ICS gene in C . roseus.

J Pharm Sci, 1999 Feb, 88(2), 180 - 4
Permeability characteristics of novel mydriatic agents using an in vitro cell culture model that utilizes SIRC rabbit corneal cells; Goskonda VR et al.; The purpose of this study was to evaluate the permeability characteristics of a previously reported in vitro corneal model that utilizes SIRC rabbbit corneal cells and to investigate the permeability of three novel esters of phenylephrone chemical delivery systems (CDS) under different pH conditions using this in vitro model . The SIRC rabbit corneal cell line was grown on transwell polycarbonate membranes, and the barrier properties were assessed by measuring transepithelial electrical resistance (TEER) using a voltohmmeter . The permeabilities of esters of phenylephrone CDS across the SIRC cell layers were measured over a pH range 4.0-7 . 4 . The esters tested include phenylacetyl (1), isovaleryl (2), and pivalyl (3) . The SIRC rabbit corneal cell line, when grown on permeable filters, formed tight monolayers of high electrical resistance with TEER values increasing from 71.6 +/- 20.8 Omega.cm2 at day 3 in culture to 2233.42 +/- 15.2 Omega.cm2 at day 8 in culture and remained constant through day 14 in culture . The transepithelial permeability coefficients (Papp) at pH 7.4 ranged from 0.58 x 10(-6) cm/s for the hydrophilic marker, mannitol, to 43 . 5 x 10(-6) cm/s for the most lipophilic molecule, testosterone . The Papp at pH 7.4 for phenylephrine was 4.21 x 10(-6) cm/s . The Papp values and the lag times of the three esters of phenylephrone were pH dependent . The Papp for 1, 2, and 3 at pH 7.4 were 14.76 x 10(-6), 13.19 x 10(-6), and 12.86 x 10(-6) cm/s, respectively and the permeabilities decreased at conditions below pH 7.4 . The lag times at pH 7.4 were 0.10, 0.17, and 0.12 h for 1, 2, and 3, respectively, and the values increased at lower pH conditions . The TEER values of SIRC cell line observed at day 8 to day 14 in the present investigation are similar to the resistance value reported for rabbit cornea (2 kOmega.cm2) . All the esters showed significantly (p < 0.05) higher permeabilities than phenylephrine at pH 7.4 . The rate and extent of transport of the drugs across the cell layers were influenced by the fraction of ionized and un-ionized species and the intrinsic partition coefficient of the drug . The results indicate that the permeability of ophthalmic drugs through ocular membranes may be predicted by measuring the permeability through the new in vitro cell culture model.

Mol Biol Cell, 1999 Feb, 10(2), 313 - 27
Interaction of fibroblast growth factor-2 (FGF-2) with free gangliosides: biochemical characterization and biological consequences in endothelial cell cultures; Rusnati M et al.; Exogenous gangliosides affect the angiogenic activity of fibroblast growth factor-2 (FGF-2), but their mechanism of action has not been elucidated . Here, a possible direct interaction of sialo-glycolipids with FGF-2 has been investigated . Size exclusion chromatography demonstrates that native, but not heat-denatured, 125I-FGF-2 binds to micelles formed by gangliosides GT1b, GD1b, or GM1 . Also, gangliosides protect native FGF-2 from trypsin digestion at micromolar concentrations, the order of relative potency being GT1b > GD1b > GM1 = GM2 = sulfatide > GM3 = galactosyl-ceramide, whereas asialo-GM1, neuraminic acid, and N-acetylneuramin-lactose were ineffective . Scatchard plot analysis of the binding data of fluorochrome-labeled GM1 to immobilized FGF-2 indicates that FGF-2/GM1 interaction occurs with a Kd equal to 6 microM . This interaction is inhibited by the sialic acid-binding peptide mastoparan and by the synthetic fragments FGF-2(112-129) and, to a lesser extent, FGF-2(130-155), whereas peptides FGF-2(10-33), FGF-2(39-59), FGF-2(86-96), and the basic peptide HIV-1 Tat(41-60) were ineffective . These data identify the COOH terminus of FGF-2 as a putative ganglioside-binding region . Exogenous gangliosides inhibit the binding of 125I-FGF-2 to high-affinity tyrosine-kinase FGF-receptors (FGFRs) of endothelial GM 7373 cells at micromolar concentrations . The order of relative potency was GT1b > GD1b > GM1 > sulfatide a = sialo-GM1 . Accordingly, GT1b,GD1b, GM1, and GM2, but not GM3 and asialo-GM1, prevent the binding of 125I-FGF-2 to a soluble, recombinant form of extracellular FGFR-1 . Conversely, the soluble receptor and free heparin inhibit the interaction of fluorochrome-labeled GM1 to immobilized FGF-2 . In agreement with their FGFR antagonist activity, free gangliosides inhibit the mitogenic activity exerted by FGF-2 on endothelial cells in the same range of concentrations . Also in this case, GT1b was the most effective among the gangliosides tested while asialo-GM1, neuraminic acid, N-acetylneuramin-lactose, galactosyl-ceramide, and sulfatide were ineffective . In conclusion, the data demonstrate the capacity of exogenous gangliosides to interact with FGF-2 . This interaction involves the COOH terminus of the FGF-2 molecule and depends on the structure of the oligosaccharide chain and on the presence of sialic acid residue(s) in the ganglioside molecule . Exogenous gangliosides act as FGF-2 antagonists when added to endothelial cell cultures . Since gangliosides are extensively shed by tumor cells and reach elevated levels in the serum of tumor-bearing patients, our data suggest that exogenous gangliosides may affect endothelial cell function by a direct interaction with FGF-2, thus modulating tumor neovascularization.

Int J Cancer, 1999 Jan 5, 80(1), 98 - 103
Protein-kinase-Cmu expression correlates with enhanced keratinocyte proliferation in normal and neoplastic mouse epidermis and in cell culture; Rennecke J et al.; In order to gain insight into the biological function of a PKC iso-enzyme, the protein kinase Cmu, we analyzed the expression pattern of this protein in mouse epidermis and keratinocytes in culture . Daily analysis of neonatal mouse epidermis immediately after birth showed a time-dependent reduction in the PKCmu content . Expression of the proliferating-cell nuclear antigen (PCNA), indicative of the proliferative state of cells, was reduced synchronously with PKCmu as the hyperplastic state of the neonatal tissue declined . In epidermal mouse keratinocytes, fractionated according to their maturation state, PKCmu expression was restricted to PCNA-positive basal-cell fractions . In primary cultures of those cells, growth arrest and induction of terminal differentiation by Ca2+ resulted in strongly reduced PKCmu expression, concomitantly with the loss of PCNA expression . Treatment of PMK-R1 keratinocytes with 100 nM of the mitogen 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in activation of PKCmu, reflected by translocation from the cytosolic to the particulate fraction and by shifts in electrophoretic mobility . DNA synthesis was significantly inhibited by the PKCmu inhibitor Goedecke 6976, while Goedecke 6983 did not inhibit PKCmu . Carcinomas generated according to the 2-stage carcinogenesis protocol in mouse skin consistently exhibited high levels of PKCmu . These data correlate PKCmu expression with the proliferative state of murine keratinocytes and point to a role of PKCmu in growth stimulation . A correlation between PKCmu expression and enhanced cell proliferation was also observed for NIH3T3 fibroblasts transfected with and overexpressing human PKCmu.

Int J Cancer, 1999 Jan 29, 80(3), 448 - 54
Neurotensin and a non-peptide neurotensin receptor antagonist control human colon cancer cell growth in cell culture and in cells xenografted into nude mice; Maoret JJ et al.; The intestine is a large endocrine organ, but the dependence of colon cancer on hormones remains unknown . We show here that neurotensin, a paracrine/endocrine peptide in the gut, and the neurotensin receptor antagonist SR 48692 control colon cancer cell growth in vitro and in vivo by interacting with receptors that are ectopically expressed in colon cancers . In cell culture, neurotensin stimulates the growth of human colon cancer cell lines (SW480, SW620, HT29, HCT116 and Cl.19A) expressing the neurotensin receptor NTR1 but does not change the growth of Caco2 cells, which do not express NTR1 . In SW480 cells, neurotensin is active in the 10(-10) to 10(-6) M concentration range (ED50 = 0.47 nM) while the neurotensin fragment (I-II) is inactive . Neurotensin also enhances the cellular cloning efficiency of SW480 cells in soft agar by inducing a 50% increase of colony formation . This effect is blocked by SR 48692, which alone does not alter colony formation . Subcutaneous delivery of neurotensin (0.54 micromol/kg every 24 hr) by osmotic pumps to nude mice that have been xenografted with SW480 cells results in a significant increase of tumor volume, i.e., up to 255% of control at day 20 of treatment . SR 48692 administered alone (1.7 micromol/kg every 24 hr) by daily i.p . injections reduces the development of tumors formed by xenografting SW480 cells in nude mice . A significant mean reduction of tumor volume of 38% is observed during the 22-day period of treatment . SR 48692 alone is also active at reducing tumor volume after xenografting HCT116 cells in nude mice . Our results support the notion that colon cancer growth may be dependent on blood-borne neurotensin and suggest that non-peptide neurotensin antagonists, such as SR 48692, may be useful for the development of novel therapeutic strategies of colon cancer.

Eur Urol, 1999 Jan, 35(1), 70 - 80
Pro-inflammatory and T cell inhibitory cytokines are secreted at high levels in tumor cell cultures of human renal cell carcinoma; Lahn M et al.; OBJECTIVES: The objectives of this study were to assess cytokine secretion in human renal cell carcinoma (RCC) and to identify cytokines contributing to the immunomodulatory effect of tumor cells . METHODS: Cytokine secretion in the supernatant of primary tumor cell cultures (PTCC) and corresponding cell lines (CL) was assayed using ELISA . Tumor cells were characterized by morphology, immunocytochemistry, and flow-cytometric analysis . Tumor-cell-induced T cell activation was determined by coculture of gamma delta and alpha beta T cell clones with tumor CL . RESULTS: We assessed the cytokine secretion of tumor cells from 27 PTCC and their corresponding CL (3/27) of RCC . We found that RCC predominantly produced both pro-inflammatory and T-cell-inhibitory cytokines, such as IL-8, IL-6, GM-CSF, TNF-alpha, IL-10 and TGF-beta 1 . CL were adapted to serum-free medium which may prove as a useful tool in future studies of cytokine secretion in RCC . In addition, we used gamma delta and alpha beta T cell clones to assess the immunomodulatory effect of tumor cells from RCC and found that predominantly gamma delta T cells were activated by RCC . CONCLUSIONS: Our data suggest that RCC produce large amounts of both pro-inflammatory and T-cell-inhibitory cytokines that potentially could influence the immune response of the host, especially tumor-specific cytotoxic T cells.

Circ Res, 1999 Feb 5, 84(2), 166 - 78
Calcification of vascular smooth muscle cell cultures: inhibition by osteopontin; Wada T et al.; Calcification of vascular tissue is a common complication in aging, atherosclerosis, diabetes, renal failure, aortic stenosis, and prosthetic valve replacement . Osteopontin is a noncollagenous adhesive protein routinely found at sites of dystrophic calcification and synthesized at high levels by macrophages in calcified aortic valves and atherosclerotic plaques . In the present study, we have characterized the calcification of bovine aortic smooth muscle cell (BASMC) cultures in vitro and have studied the effects of exogenous osteopontin on mineral deposition . Induction of calcification in BASMC cultures was alkaline phosphatase-dependent and was characterized by a multilayer cell morphology . Mineral deposition occurred in the basal matrix of multilayered areas as indicated by von Kossa staining, and transmission electron microscopy and electron diffraction identified the mineral as apatite . Ultrastructural analysis of the cultures showed the presence of extracellular matrix vesicles, calcifying collagen fibrils, and nodular-type calcifications similar to those found in calcified heart valves and atherosclerotic plaques . Purified osteopontin (0.05 to 5 microgram/mL) dose dependently inhibited calcification of BASMC cultures, whereas vitronectin and fibronectin had no effect . In contrast to the inhibitory mechanism of levamisole on mineral deposition, osteopontin did not inhibit alkaline phosphatase activity or reduce phosphorus levels in the culture medium . Addition of calcium to the cultures overcame the inhibitory effect of osteopontin on BASMC culture calcification and resulted in decreased levels of calcium in the culture medium and increased levels in the cell layer . Moreover, using high-resolution, colloidal-gold immunocytochemistry, osteopontin was found intimately associated with growing apatite crystals . These data indicate that the effect of osteopontin, although calcium-dependent, was not mediated by simple calcium chelation but most likely by direct interaction of osteopontin with crystal surfaces . These studies suggest that BASMCs can be used to model vascular calcification in vitro and that soluble osteopontin released near sites of vascular calcification may represent an adaptive mechanism aimed at preventing vascular calcification.

Histochem Cell Biol, 1999 Jan, 111(1), 61 - 9
Immunohistochemical investigations on the differentiation marker protein E11 in rat calvaria, calvaria cell culture and the osteoblastic cell line ROS 17/2.8; Schulze E et al.; Until now, many extracellular matrix proteins, e.g . osteopontin and osteonectin, have been used to determine a cell's osteogenic maturation . The disadvantage in evaluation of these proteins is their relative wide-ranging appearance throughout the osteogenic differentiation process . Thus, the aim of this study was to establish an immunohistochemical setup using E11, a marker that binds selectively to cells of the late osteogenic cell lineage . In addition, the histochemical expression of the bone matrix proteins osteonectin, osteopontin and fibronectin was compared to that of E11 using monoclonal antibodies . For light microscopical detection of osteogenic markers in cultured cells we developed a simple paraffin technique using a fibrin glue as embedding medium . This allows the handling of cultured cells such as a tissue sample and includes the use of stored biological specimens for further immunohistochemical experiments . We used newborn rat calvariae for whole tissue preparations and for isolation and cultivation of bone cells . In addition, we included the rat osteosarcoma cell line ROS 17/2.8 in this study . For the first time, we have localised E11 in osteocytes of rat calvaria preparations at the electron microscopical level . E11 was detected at plasma membranes of osteocytes and their processes, but not at those of osteoblasts . Accompanying experiments with cultured newborn rat calvaria cells and ROS 17/2.8 cells revealed E11 reactivity on a subset of cells . The results obtained confirm the suitability of the differentiation marker E11 as a sensitive instrument for the characterisation of bone cell culture systems.

Arch Virol, 1998, 143(12), 2471 - 85
Transmissible gastroenteritis virus induced apoptosis in swine testes cell cultures; Sirinarumitr T et al.; Transmissible gastroenteritis virus (TGEV) is a coronavirus which causes severe gastroenteritis and atrophy of intestinal villous epithelial cells in piglets . However, the mechanism of cell death caused by TGEV is not known . In this study, we report that TGEV induces cell death by apoptosis . TGEV-induced apoptosis was demonstrated by agarose gel electrophoresis, electron microscopy, and terminal deoxytransferase digoxigenin-dUTP nick end labeling (TUNEL) . Double labeling experiment confirmed the result from electron microscopy and showed that most of the apoptotic cells were bystander cells as they were negative for TGEV nucleic acids . Results of this study indicate that TGEV induces apoptosis in vitro and that most of the cells undergoing apoptosis are bystander cells, thus amplifying the cytopathic effect of TGEV.

Plant Cell, 1999 Feb, 11(2), 273 - 87
Rapid Avr9- and Cf-9 -dependent activation of MAP kinases in tobacco cell cultures and leaves: convergence of resistance gene, elicitor, wound, and salicylate responses; Romeis T et al.; The Cf-9 resistance (R) gene from tomato confers resistance to the fungal pathogen Cladosporium fulvum expressing the corresponding, pathogen-derived avirulence gene product Avr9 . To understand how an initial R/Avr recognition event is transmitted and triggers the induction of plant defenses, we investigated early Avr9/Cf-9-dependent activation of protein kinases in transgenic tobacco expressing the Cf-9 gene . We identified two protein kinases of 46 and 48 kD, using myelin basic protein as substrate, that became rapidly activated in a strictly gene-for-gene manner within 2 to 5 min after Avr9 elicitation in both Cf9 tobacco plants and derived cell cultures . Studies with pharmacological inhibitors and effectors revealed that Ca2+ influx and a phosphorylation event(s) are required for kinase activation, but neither enzyme is involved in the Avr9-dependent synthesis of active oxygen species . The activation of both kinases is achieved via post-translational mechanisms, and the activation but not inactivation step includes tyrosine phosphorylation . Using specific antibodies, we found that the 46- and 48-kD kinases were similiar to WIPK (for wound-induced protein kinase) and SIPK (for salicylic acid-induced protein kinase), two previously characterized mitogen-activated protein (MAP) kinases from tobacco . In addition, Cf9 tobacco plants and cell cultures showed an Avr9-dependent accumulation of the WIPK transcript . Cf9 tobacco suspension cultures are thus a unique system in which to analyze the earliest events in R gene function . These data indicate that (1) the R/Avr-mediated induction of plant defense is accomplished via several parallel signaling mechanisms, and (2) R/Avr-dependent signal transduction pathways are interlinked at MAP kinases with responses of plants not only to non-race-specific elicitors but also to abiotic stimuli, such as wounding and mechanical stress.

Int J Parasitol, 1998 Dec, 28(12), 1875 - 80
A Cryptosporidium parvum oocyst low molecular mass fraction evokes a CD4+ T-cell-dependent IFN-gamma response in bovine peripheral blood mononuclear cell cultures; de Graaf DC et al.; T-Cell antigens that induce the in-vitro interferon-gamma response during Cryptosporidium parvum infection of neonatal calves were identified . A total oocyst extract was separated into a high and a low Mr fraction by a microfiltration technique . Both the high and low Mr fractions evoked an in-vitro interferon-gamma response in naturally infected animals, although strong individual differences between the hosts were observed . Using a complement-mediated technique CD4+ T-cells or WC1+gammadelta T-cells were depleted, whereupon the remaining lymphocyte cultures were stimulated with the different antigen preparations . It was shown that the in-vitro interferon-gamma response of Cryptosporidium-infected calves is CD4+ T-cell-dependent.

Cancer Chemother Pharmacol, 1999, 43(3), 213 - 20
Measurement of delivery and metabolism of tirapazamine to tumour tissue using the multilayered cell culture model; Kyle AH et al.; PURPOSE: Efficient extravascular penetration is essential for the optimal activity of most anticancer drugs and is particularly relevant to bioreductive cytotoxins which target hypoxic cells that can be located distal to functional blood vessels within tumours . Tirapazamine (3-amino-1,2,4-benzotriazine-1,4-di-N-oxide; Triazone; SR 259075; formerly SR 4233) is a lead bioreductive cytotoxin currently undergoing clinical evaluation . It exhibits preferential cytotoxicity towards cells at reduced oxygen tension, and could complement existing anticancer therapies where hypoxic cells are believed to constitute a refractory population . We assessed the ability of tirapazamine to penetrate tumour tissue using an in vitro multilayered cell culture (MCC) model . METHODS: Diffusion of tirapazamine through oxic and hypoxic multilayered cell cultures composed of SiHa . human cervical carcinoma cells, was measured using a dual reservoir diffusion apparatus from which samples were quantified via HPLC . Drug concentration kinetics from both reservoirs were analysed using a mathematical model for diffusion and metabolism within the MCC . Results were then applied to a second mathematical model which described extravascular drug penetration within a tumour cord, the sheath of cells surrounding a blood vessel . RESULTS: The diffusion coefficient of tirapazamine within SiHa MCCs was determined as 7.0+/-0.5 x 10(-7) cm2/s and the maximal metabolic rate for hypoxic MCCs, Vmax, as 1.5+/-0.4 microM/s . The thickness of individual tissue cultures was determined by diffusion of tritiated water (HTO) . A linear relationship was shown to exist between tissue thickness and the inverse of permeability to HTO . Experimental results were used to simulate drug distribution within a tumour cord . These simulations indicate that, when tirapazamine is administered via intravenous infusion, a stable tirapazamine distribution throughout the cord occurs within 15 min with cells most peripheral to the blood vessel exposed to only 10% of the blood drug concentration . Under these conditions, the simulations predict cell kill to be limited to the first 75 microm of tissue surrounding a blood vessel . CONCLUSION: This study indicates that extravascular penetration of tirapazamine to peripheral cells existing at low oxygen tension may be limited by the metabolism of tirapazamine by more proximal cells existing at moderate oxygen tension . Simulations found that tirapazamine reached only 10% of the blood concentration at cells most peripheral to blood vessels . These results indicate that tirapazamine would be significantly cytotoxic only to cells located within approximately 75 microm of blood vessels . Further MCC-based modelling of extravascular drug penetration would serve as a means of identifying new antitumour agents with location-specific activity.

Biochem Biophys Res Commun, 1999 Jan 8, 254(1), 138 - 42
Isolation of a low-molecular-weight growth inhibitory factor from hybridoma cell cultures; Roe O et al.; A low-molecular-weight growth inhibitory factor was produced by hybridoma cells . The number of viable cells in hybridoma cell cultures reached a maximum of about 5 x 10(5) cells/ml when the inhibitory factor had accumulated to a critical level, after which the number of viable cells declined with a concomitant increase in the number of dead cells . The growth inhibitory factor was purified to apparent homogeneity by ultrafiltration, reverse-phase chromatography, passage through cation exchangers, and gel filtration . Analysis by reverse-phase chromatography and micellar electrokinetic chromatography using a capillary electrophoresis system indicated that the final inhibitory fraction was pure . The factor had a molecular weight of 500 or less, as judged by ultrafiltration, and its behavior upon ion-exchange chromatography indicated that it was uncharged . Its absorbance maximum at 263 nm indicated that it was not a peptide, but that it may have a conjugated system of carbon-carbon double bonds .

Brain Res, 1999 Feb 6, 818(1), 84 - 95
Selective neurodegeneration induced in rotation-mediated aggregate cell cultures by a transient switch to stationary culture conditions: a potential model to study ischemia-related pathogenic mechanisms; Pardo B et al.; Aggregating brain cell cultures at an advanced maturational stage (20-21 days in vitro) were subjected for 1-3 h to anaerobic (hypoxic) and/or stationary (ischemic) conditions . After restoration of the normal culture conditions, cell loss was estimated by measuring the release of lactate dehydrogenase as well as the irreversible decrease of cell type-specific enzyme activities, total protein and DNA content . Ischemia for 2 h induced significant neuronal cell death . Hypoxia combined with ischemia affected both neuronal and glial cells to different degrees (GABAergic neurons>cholinergic neurons>astrocytes) . Hypoxic and ischemic conditions greatly stimulated the uptake of 2-deoxy-D-glucose, indicating increased glucose consumption . Furthermore, glucose restriction (5.5 mM instead of 25 mM) dramatically increased the susceptibility of neuronal and glial cells to hypoxic and ischemic conditions . Glucose media concentrations below 2 mM caused selective neuronal cell death in otherwise normal culture conditions . GABAergic neurons showed a particularly high sensitivity to glucose restriction, hypoxia, and ischemia . The pattern of ischemia-induced changes in vitro showed many similarities to in vivo findings, suggesting that aggregating brain cell cultures provide a useful in vitro model to study pathogenic mechanisms related to brain ischemia .

Growth Dev Aging, 1998 Autumn, 62(3), 107 - 18
Influence of thyroxine and hydrocortisone in vivo on porcine preadipocyte recruitment, development and expression of C/EBP binding proteins in fetal stromal vascular (S-V) cell cultures; Hausman GJ et al.; At 90 days of gestation hypophysectomized (hypox) porcine fetuses were treated with hydrocortisone (HC) or thyroxine (T4) and S-V cultures prepared from intact, treated and untreated hypox fetuses at 105 days of gestation . After 24 h, cultures were stained for the AD-3 antigen and the transcription factors, C/EBP alpha and delta . The proportion of preadipocytes (AD-3+) was doubled in cultures derived from HC treated hypox fetuses compared to cultures from intact, untreated and T4 treated hypox fetuses . Compared to cultures from intact fetuses, proportions of C/EBP delta reactive cells (approximately 40%) were increased similarly in cultures from untreated and HC treated hypox fetuses . Compared to day 0-1, preadipocyte proportions were markedly reduced in cultures from untreated and HC treated fetuses and increased in cultures from intact and T4 treated fetuses after three days of fetal bovine serum (FBS; day 0-3) . After six days of insulin (FBS day 0-3-->insulin, transferrin and selenium; ITS), fat cell numbers were 5 fold higher in cultures from T4 treated fetuses compared to cultures from untreated and HC treated hypox fetuses . Other cultures were seeded and plated in FBS + 80 nM dexamethasone (DEX; day 0-3) followed by insulin (day 3-9) . Preadipocyte development was independent of the source of S-V cells in cultures treated with FBS + DEX-->insulin . These data indicate that, compared to preadipocyte recruitment, hormonal regulation of adipocyte differentiation may be more complex and involve more interactions.

Biochimie, 1998 Oct, 80(10), 871 - 81
Effect of nitric oxide synthase induction on the expression of mitochondrial respiratory chain enzyme subunits in mixed cortical and astroglial cell cultures; Nicoletti VG et al.; In the present study we evaluated the effects of NO synthase (NOS) induction on the regulation of cytochrome c oxidase (CO) and F0F1-ATPase subunit expression in astroglial and mixed cortical cell cultures . In mixed cortical cell cultures, 18 h of treatment with lipopolysaccharide (LPS, 0.1 microgram/mL) plus interferon-gamma (INF-gamma, 10 U/mL) caused an increase of mRNAs for CO-I, F0F1-ATPase 6 and also for iNOS at 20 DIV . The induction of both CO-I and F0F1-ATPase 6 was abolished by the NOS inhibitor N-monomethyl-L-arginine (NMMA) or by the enzymatic scavenger superoxide dismutase/catalase (SOD/CAT) . In primary astroglial cell cultures, treatment for 18 h with increasing concentrations of LPS and INF gamma, produced an increase in the amount of mitochondrial encoded CO-I and -II subunits, with no significant modifications of nuclear encoded subunit IV . An increase was also observed at level of transcription for CO-I and -II, and F0F1-ATPase 6 mRNAs . These effects were abolished by addition of NMMA or SOD/CAT . mRNA induction of CO-I was higher in mixed cortical than in astroglial cell cultures while that of F0F1-ATPase 6 was similar in both cell types . These results suggest that the expression of mitochondrial encoded subunits (CO-I, CO-II and F0F1-ATPase 6) is up-regulated in response to oxygen and NO reactive species . The activity of cytochrome c oxidase decreased after LPS/INF gamma treatment in both astroglial and mixed cortical cultures . The activity of ATP synthase was unmodified, while ATP content drastically decreased after LPS/INF gamma treatment, in both astroglial and mixed cortical cultures . The enzymatic activities of catalase and Mn-SOD (mitochondrial) showed a significant increase after LPS/INF gamma treatment, which was abolished by NMMA.

Anticancer Res, 1998 Nov-Dec, 18(6A), 4147 - 58
Cell culture as spheroids: an approach to multicellular resistance; Desoize B et al.; Cells cultured as spheroids present an heterogeneity similar to that of tumours in vivo . In the spheroid peripheral layers, cells are proliferating, deeper cells are non-cycling, when in the aggregate centre, cells form often a necrotic core . A multicellular resistance is related on the cell contact to other cells or to the extracellular matrix . The mechanism of this resistance remains unknown . It seems to be linked to the spheroid centre hypoxia, quiescence of a large fraction of the cell population and to the apoptose inhibition . The "classical" or "unicellular" mechanisms of resistance, as mdr1, MRP, can coexist but are not responsible of this type of resistance . This culture model is a good opportunity to study a resistance which looks close to the patient tumour resistance . A new class of therapeutic molecules appears that can reverse multicellular resistance, inhibit tumours growth and preclude metastases . The mechanism of action of this new pharmacological class is the disruption of the cell adhesion forces.

Anticancer Res, 1998 Nov-Dec, 18(6A), 4067 - 70
Differential growth response to exogenous calcium in normal and carcinogen-exposed primary human keratinocyte cell cultures; Steele VE et al.; The purpose of these studies was to examine an early carcinogen-induced change in primary human epithelial cell cultures and to attempt to reverse this change with retinoic acid . Primary cultures of human foreskin keratinocytes were prepared and exposed to the carcinogen, propane sultone . After each passage, a portion of cells were plated into medium containing increasing amounts of calcium . In a series of experiments it became evident that carcinogen exposed cells continued to grow in the presence of added calcium . Solvent control cell growth was decreased under such conditions . This new phenotype became apparent after the third subculture, but was pronounced after the fourth subculture . The addition of retinoic acid to the culture medium at each medium change reduced this effect and the keratinocytes grew more slowly, similar to control cells, in the presence of added calcium . The results suggest that carcinogen-exposed human keratinocytes acquire a resistance to calcium-induced differentiation or growth cessation and that retinoic acid can ameliorate this process . Although the mechanism of retinoic acid's inhibition remains unclear, these studies do provide a human cell model system which can be used to screen potential chemopreventive agents and for further mechanistic research.

Biotechnol Annu Rev, 1998, 4, 113 - 76
Use of plant cell cultures in biotechnology; Muhlbach HP; Plant cell cultures are being widely used in scientific studies on the physiology, biochemistry and molecular biology of primary and secondary metabolism, developmental regulation and cellular responses to pathogens and stress . In this chapter the significance of plant cell cultures in biotechnology is discussed with special emphasis on commercial production of secondary metabolites and pharmaceuticals, the potential of genetically transformed cell cultures, photosynthetically active cell cultures, production of somatic embryos, and novel assay systems based on the use of plant cells . Future aspects of biotechnical applications with respect to the potentials and limitations of these approaches are assessed, particularly in comparison with the productivity of lower eucaryotes.

Endocr Res, 1998 Aug-Nov, 24(3-4), 753 - 7
Basal catecholamine and cortisol secretion in primary chromaffin cell cultures before and after purification and retroviral transfection; Uhlmann K et al.; We have investigated the effects of supraphysiological concentrations of catecholamines on glucocorticoid secretion in vitro . These effects were analyzed in adrenocortical cells shown to be present in chromaffin cell cultures as well as in cortical cells cocultured with transfected chromaffin cells that overproduce catecholamines . Cortisol release from residual cortical cells in chromaffin cell cultures was found to be 2.5 times higher than from isolated adrenocortical cells . Removal of the adrenocortical cells from the chromaffin cells resulted in an almost complete cessation of cortisol secretion . Catecholamine overproduction was achieved by transfecting chromaffin cells with the blank retroviral vector pSAM-EN . Coculture of adrenocortical cells with these transfected chromaffin cells further enhanced the stimulating effect of chromaffin cells on cortisol 2.3-fold compared to normal cocultures . In conclusion, cortical cells in chromaffin cell cultures secrete significant amounts of cortisol, which should be considered when evaluating the endocrine function of these cell cultures and which can be abolished by purification . The hormonal activity of adrenocortical cells is highly increased in an environment of catecholamine overproduction, which is of both basic and clinical importance.

Brain Res, 1999 Jan 23, 816(2), 544 - 53
Electrophysiological characterization of 5-HT receptors on rat petrosal neurons in dissociated cell culture; Zhong H et al.; The petrosal ganglion supplies chemoafferent pathways via the glossopharyngeal (IXth) nerve to peripheral targets which release various neurotransmitters including serotonin (5-HT) . Here, we combined rapid 5-HT application with patch clamp, whole-cell recording to investigate whether 5-HT receptors are expressed on isolated petrosal neurons (PN), cultured from 7-12 day-old rat pups . In responsive cells, the dominant effect of 5-HT was a rapid depolarization associated with a conductance increase in approximately 43% of the neurons (53/123); however, in a minority population ( approximately 6%; 8/123), 5-HT caused membrane depolarization associated with a conductance decrease . In the former group, 5-HT produced a transient inward current (I5-HT) in neurons voltage-clamped near the resting potential ( approximately -60 mV); the effect was mimicked by the 5-HT3 receptor-specific agonist, 2-methyl-5-HT, suggesting it was mediated by 5-HT3 receptors . Further, I5-HT was selectively inhibited by the 5-HT3 receptor-specific antagonist MDL72222 (1-10 microM), but was unaffected by either 5-HT1/5-HT2 receptor antagonist, spiperone, or by 5-HT2 receptor-specific antagonist, ketanserin (50-100 microM) . I5-HT displayed moderate inward rectification and had a mean reversal potential (+/-S.E.M.) of -4.3+/-6.6 mV (n=6) . Application of 5-HT (dose range: 0.1-100 microM) produced a dose-response curve that was fitted by the Hill equation with EC50= approximately 3.4 microM and Hill coefficient= approximately 1.6 (n=8) . The activation phase of I5-HT (10 microM 5-HT at -60 mV) was well fitted by a single exponential with mean (+/-S.E.M.) time constant of 45+/-30 ms (n=6) . The desensitization phase of I5-HT was best fitted by a single exponential with mean (+/-S.E.M.) time constant of 660+/-167 ms (n=6) . Fluctuation analysis yielded an apparent mean single-channel conductance (+/-S.E.M) of 2.7+/-1.5 pS (n=4) at -60 mV . In the minority ( approximately 6%) population of neurons which responded to 5-HT with a conductance decrease, the depolarization was blocked by the 5-HT2 receptor antagonist, ketanserin (50 microM) . Taken together, these results suggest that 5-HT3 receptors are the major subtype expressed by rat petrosal neurons, and therefore are candidates for facilitating chemoafferent excitation in response to 5-HT released from peripheral targets .

Biochem Biophys Res Commun, 1998 Dec 18, 253(2), 506 - 13
Effects of leukemia inhibitory factor and oncostatin M on bone mineral formed in in vitro rat bone-marrow stromal cell culture: physicochemical aspects; Bohic S et al.; Leukemia inhibitory factor (LIF) and oncostatin M (OSM), two pleiotropic cytokines involved in bone remodeling, have both anabolic and catabolic activities . This study analyzed the effects of LIF and OSM on the physicochemical characteristics of mineral phases formed in a rat bone-marrow stromal cell culture model . Stromal cells were cultured for three weeks in the presence of 10(-8) M dexamethasone, 50 microgram/mL ascorbic acid and 10 mM Na beta-glycerophosphate with or without 10 ng/ml LIF or OSM . Subsequently, the physicochemical characteristics of the mineralization nodules formed were analyzed by energy dispersive X ray microanalysis (EDX) and Fourier transform-infrared (FT-IR) and FT-Raman spectroscopy . EDX and FT-IR spectroscopy revealed the influence of LIF and OSM on the physicochemical characteristics of mineral phases . FT-Raman spectroscopy showed modifications of the main vibrational modes of the organic matrix . These alterations induced by growth factors could help define new strategies for the prevention and treatment of skeletal disorders .

Cell Biol Int, 1998, 22(2), 115 - 25
Induction of cellular processes containing collagenase and retinoid by integrin-binding to interstitial collagen in hepatic stellate cell culture; Sato M et al.; Cultured hepatic stellate cells were induced to elongate long, multipolar cellular processes by interstitial collagen gel used as a substratum, as compared to flattened or round cell shapes on polystyrene surface or on Matrigel containing the basement membrane components, respectively . The process induction was inhibited by several reagents as follows: (1) anti-integrin alpha2 antibody; (2) an oligopeptide, DGEA, an integrin-binding sequence in type I collagen molecule; (3) wortmannin, a phosphatidylinositol 3-kinase inhibitor . Protein tyrosine phosphorylation was enhanced throughout cells including cellular processes by culturing on type I collagen gel . Dual fluorescence staining showed that the core of the processes contained microtubules, whereas the periphery of the processes comprised fibrillar actin . Thus, the process extension was found to depend on integrin-binding to type I collagen fibres, followed by signal transduction and cytoskeleton assembly . The cellular processes included interstitial collagenase and vitamin A-containing lipid droplets . The lipid droplets and vitamin A-autofluorescence were increased by retinyl acetate addition to the culture medium, suggesting an important role of processes in hepatic stellate cell function .

Am J Respir Cell Mol Biol, 1999 Jan, 20(1), 43 - 52
Restoration of the mucous phenotype by retinoic acid in retinoid-deficient human bronchial cell cultures: changes in mucin gene expression; Koo JS et al.; Retinoid-deficient cultures of airway epithelial cells undergo squamous differentiation . Treatment of such cultures with retinoic acid (RA) leads to restoration of the mucous phenotype . The purpose of our study was to characterize the cellular and molecular changes following RA treatment of retinoid-deficient human tracheobronchial epithelial cell cultures . Of particular interest was to determine when during the conversion of the squamous to the mucous phenotype the mucin genes MUC2, MUC5AC, and MUC5B were expressed . We used cornifin alpha and secreted mucin as markers to monitor the squamous and mucous phenotypes, respectively . Our studies showed that the RA responsiveness of the cultures progressively decreased with protracted retinoid deficiency, requiring higher RA concentrations to restore the mucous phenotype . Within 12 h after the start of RA treatment, cornifin alpha expression decreased, signaling the beginning of a change in cellular phenotype . At 24 h after addition of RA to the cultures, a significant number of mucous cells appeared, and at 72 h mucin was secreted in measurable amounts . Induction of mucin gene expression occurred sequentially: MUC2, MUC5AC, and MUC5B mRNAs were upregulated at 24, 48, and 72 h, respectively . When cultures maintained in 10(-8) M RA were treated with 10(-6) M RA, MUC2 but not MUC5AC and MUC5B mRNA levels were upregulated within 6 h . Our study indicates that MUC2 mRNA is an early marker of mucous differentiation, whereas MUC5AC and MUC5B mRNAs are expressed during more advanced stages of mucous differentiation . Our studies further suggest that each of the mucin genes is regulated by distinct mechanisms.

Plant Mol Biol, 1998 Dec, 38(6), 1101 - 11
Differential induction by methyl jasmonate of genes encoding ornithine decarboxylase and other enzymes involved in nicotine biosynthesis in tobacco cell cultures; Imanishi S et al.; A cDNA of tobacco BY-2 cells corresponding to an mRNA species which was rapidly induced by methyl jasmonate (MeJA) in the presence of cycloheximide (CHX) was found to encode ornithine decarboxylase (ODC) . Another cDNA from a MeJA-inducible mRNA encoded S-adenosylmethionine synthase (SAMS) . Although these enzymes could be involved in the biosynthesis of polyamines, the level of putrescine, a reaction product of ODC, increased slowly and while the levels of spermidine and spermine did not change following treatment of cells with MeJA . However, N-methylputrescine, which is a precursor of pyrrolidine ring of nicotine, started to increase shortly after MeJA-treatment of cells and the production of nicotine occured thereafter . The levels of mRNA for arginine decarboxylase (ADC), an alternative enzyme for putrescine synthesis, and that for S-adenosylmethionine decarboxylase (SAMDC), required for polyamine synthesis, were not affected by MeJA . In addition to mRNAs for ODC and SAMS, mRNA for putrescine N-methyltransferase (PMT) was also induced by MeJA . Unlike the MeJA-induction of ODC mRNA, MeJA-induction of SAMS and PMT mRNAs were blocked by CHX . The level of ODC mRNA declined after 1 to 4 h following MeJA treatment, while the levels of mRNAs for SAMS and PMT continued to increase . Auxin significantly reduced the MeJA-inducible accumulation of mRNAs for ODC, SAMS and PMT . These results indicate that MeJA sequentially induces expression of a series of genes involved in nicotine biosynthesis by multiple regulatory mechanisms.

Cell Biochem Biophys, 1998, 29(3), 307 - 31
A novel fluorescence chamber for the determination of volume changes in human CaSki cell cultures attached on filters; Gorodeski GI et al.; The objective of the study was to test the hypothesis that, in the cultured human cervical epithelium, CaSki, the effect of calcium mobilizing agents on transepithelial electrical conductance (GTE), is the result of cell volume decrease . CaSki cells attached on filters were loaded with fura-2, and measurements of fluorescence at the isosbestic wavelength 360 nm (excitation/emission {F360/510}) were made in a newly designed fluorescence chamber; this design allowed us also to determine changes in cytosolic calcium ({Ca2+}i) . The experimental conditions were similar to those used to measure changes in paracellular permeability in the Ussing chamber, and they enabled us to compare the time-course of changes in {Ca2+}i, in F360/510, and in GTE . Hypertonicity increased, and hypotonicity decreased F360/510 and GTE, without having an effect on {Ca2+}i, and the changes in F360/510 and in GTE correlated linearly . Metabolism, bleaching, and extrusion of intracellular fura-2 were minimal, indicating that the changes in F360/510 reflect changes in dye concentration . Hypertonicity decreased, and hypotonicity increased the size of dispersed CaSki cells, suggesting that osmolarity-induced changes in F360/510 reflect changes in size of the attached cells . Ionomycin increased {Ca2+}i, F360/510, and GTE, but the increases in {Ca2+}i preceded those in F360/510 and GTE . The calcium chelator BAPTA blocked the ionomycin-induced increase in {Ca2+}i, F360/510, and in GTE . Preincubation with 4-acetamido-4'isothiocyanatostilbene-2,2'disulfonic acid (SITS) augmented the ionomycin-induced increase in {Ca2+}i, but blocked the increases in F360/510 and in GTE . Pretreatment of cells with hypertonic solution abrogated the increases in F360/510 and in GTE in response to ionomycin, but had little effect on the ionomycin-induced increase in {Ca2+}i . On the basis of these results we suggest that the ionomycin-induced increase in GTE is mediated by {Ca2+}i-dependent chloride secretion and osmotic water loss.

Infect Immun, 1999 Jan, 67(1), 102 - 7
Comparison of surface proteins of Anaplasma marginale grown in tick cell culture, tick salivary glands, and cattle; Barbet AF et al.; Anaplasma marginale, a tick-borne rickettsial pathogen of cattle, infects bovine erythrocytes, resulting in mild to severe hemolytic disease that causes economic losses in domestic livestock worldwide . Recently, the Virginia isolate of A . marginale was propagated in a continuous tick cell line, IDE8, derived from embryonic Ixodes scapularis . Development of A . marginale in cell culture was morphologically similar to that described previously in ticks . In order to evaluate the potential of the cell culture-derived organisms for use in future research or as an antigen for serologic tests and vaccines, the extent of structural conservation of the major surface proteins (MSPs) between the cell culture-derived A . marginale and the bovine erythrocytic stage, currently the source of A . marginale antigen, was determined . Structural conservation on the tick salivary-gland stage was also examined . Monoclonal and monospecific antisera against MSPs 1 through 5, initially characterized against erythrocyte stages, also reacted with A . marginale from cell culture and tick salivary glands . MSP1a among geographic A . marginale isolates is variable in size because of different numbers of a tandemly repeated 28- or 29-amino-acid peptide . The cell culture-derived A . marginale maintained the same-size MSP1a as that found on the Virginia isolate of A . marginale in bovine erythrocytes and tick salivary glands . Although differences were observed in the polymorphic MSP2 antigen between culture and salivary-gland stages, MSP2 did not appear to vary, by two-dimensional gel electrophoresis, during continuous passage in culture . These data show that MSPs of erythrocyte-stage A . marginale are present on culture stages and may be structurally conserved during continuous culture . The presence of all current candidate diagnostic and vaccine antigens suggests that in vitro cultures are a valuable source of rickettsiae for basic research and for the development of improved diagnostic reagents and vaccines against anaplasmosis.

Biotechniques, 1998 Dec, 25(6), 990 - 4, 996
Colloidal silica-coated tissue culture dishes for primary cell cultures: growth of rabbit renal proximal tubule cells; Taub M et al.; The use of colloidal silica as a substratum for primary cultures of differentiated cells has significant advantages over classic tissue culture polystyrene . In this report, the growth and the level of expression of differentiated function of primary rabbit renal proximal tubule (RPT) cell cultures on colloidal silica is examined, using hormonally defined serum-free medium . Primary RPT cells grew to confluence more rapidly on colloidal silica than on tissue culture polystyrene (TC+) . Moreover, following three passages, the RPT cells increased in number threefold more than parallel cultures on TC+ . The morphology of primary RPT cells on colloidal silica were found by means of transmission electron microscopy to possess a polarized morphology with a brush border, and differentiated markers were retained even after passaging, including the Na+/glucose cotransport system and Glut 7.

J Biomater Sci Polym Ed, 1998, 9(11), 1187 - 205
Retinal pigment epithelium cell culture on thin biodegradable poly(DL-lactic-co-glycolic acid) films; Lu L et al.; Thin films of 50:50 and 75:25 poly(DL-lactic-co-glycolic acid) (PLGA) were manufactured with a controlled thickness of less than 10 microm . The effect of PLGA copolymer ratio on in vitro cell attachment, proliferation, morphology, and tight junction formation was evaluated using a human D407 retinal pigment epithelium (RPE) cell line . Almost complete cell attachment was achieved on both PLGA films after 8 h of cell seeding, which was comparable to that on tissue culture polystyrene (TCPS) controls . The initial cell seeding density affected attachment, and the optimal value for 50:50 PLGA was 25000 cells cm(-2) . After 7 days of in vitro culture, cell density on 50:50 and 75:25 PLGA films increased 45 and 40 folds, respectively, and a 34-fold increase was observed on TCPS . The RPE cells cultured on PLGA films at confluence had a characteristic cobblestone morphology . Confluent RPE cells also developed normal tight junctions in vitro which were concentrated mainly at the apical surfaces of cell-cell junctions . These results demonstrated that thin biodegradable PLGA films can provide suitable substrates for human RPE cell culture, and may serve as temporary carriers for subretinal implantation of organized sheets of RPE.

Microsc Res Tech, 1998 Dec 1, 43(5), 379 - 84
Adhesion-guided in vitro morphogenesis in pure and mixed cell cultures; Powers MJ et al.; The ability to understand and control the morphogenesis of mammalian cells is a fundamental objective of cell and developmental biology and tissue engineering research . Numerous processes, both biochemical and biophysical in nature, have been studied in an attempt to elucidate the mechanisms underlying this behavior . We focus here on the contributions of biophysical phenomena to the morphogenetic behavior of pure and mixed cell populations on solid surfaces in vitro . These principles are illustrated using characteristic liver tissue cells as a model system . The studies discussed demonstrate that cell-substratum and cell-cell adhesive forces are critical determinants of the ultimate morphology, cytoarchitecture, and organization achieved by these cells in vitro.

Prenat Diagn, 1998 Nov, 18(11), 1191 - 4
Further observations of true mosaic trisomy 17 ascertained in amniotic fluid cell cultures; Djalali M et al.; Three new cases of true mosaic trisomy 17 (MT17) were diagnosed in amniotic fluid cells . Postnatal chromosome analysis from lymphocytes did not confirm the trisomic cell line, and follow-up studies showed normal psycho-motor development of the children, in one case up to the age of 4 1/2 years . We suggest that there are similarities between MT17 and MT20, in which the majority of pregnancies result in deliveries of healthy babies.

Bone, 1998 Dec, 23(6), 511 - 20
Matrix mineralization in MC3T3-E1 cell cultures initiated by beta-glycerophosphate pulse; Fratzl-Zelman N et al.; MC3T3-E1 cells, grown in the presence of serum and ascorbate, express alkaline phosphatase and produce an extensive collagenous extracellular matrix that can be mineralized by the addition of beta-glycerophosphate (beta-GP) . In the present work, we study the influence of concentration and duration of beta-GP treatment on the mineralization pattern in 4-week-old cell cultures . Amount and structure of mineral deposition were monitored by von Kossa staining, light, and electron microscopy, as well as small-angle X-ray scattering (SAXS) of unstained specimens . SAXS measures the total surface of the mineral phase and is therefore preferentially sensitive to very small crystals (typically <50 nm) . It was used to determine the ratio (M) of small crystals to collagen matrix . A variety of mineralization patterns was observed to occur simultaneously, some associated with collagen within nodules or in deeper layers of the cultures and some independent of it . At a beta-GP concentration of 10 mmol, mineralization was initiated after about 24 h and continued to increase, irrespective of whether the high level of beta-GP was maintained or reduced to 2 mmol . With shorter pulses (<24 h), no significant mineralization was observed in the week following beta-GP pulse . With continuous treatment at 5 mmol beta-GP, the first signs of mineralization were detected 14 days after the beginning of treatment in the 4-week-old cultures, but no mineralization at all occurred at lower beta-GP concentrations . When cells were grown without ascorbic acid for 4 weeks, only two cell layers without collagen matrix were found . In these cultures, no mineralization detectable by SAXS could be induced with beta-GP . These data indicate that, in viable cells, high doses of beta-GP are essential for the nucleation of mineral crystals, but not for the progression of mineralization once crystals had been nucleated . In contrast, when 4-week-old cell cultures were devitalized, M was found to increase immediately, even at 2 mmol beta-GP . These results suggest that, in MC3T3-E1 cell cultures, cell viability is essential for prevention of spontaneous mineralization of the extracellular matrix.

Rapid Commun Mass Spectrom, 1998, 12(22), 1765 - 8
Identification of oxytocin and vasopressin from neurohypophyseal cell culture; Janaky T et al.; Our observation that dispersed cultures of neurohypophysis obtained from adult rats are capable of synthesizing and releasing oxytocin and vasopressin is unexpected, because in whole animals these hormones are known only to be stored, not to be produced in the posterior lobe of the pituitary . The hormone content of cell culture medium was elevated from 0 to 129 +/- 14 pg/mg protein for oxytocin and from 0 to 42 +/- 4 pg/mg protein for vasopressin during two weeks as determined by specific radioimmunoassay . By molecular mass and structure determination (tandem mass spectrometry) we have proved that the supernatant of the cell cultures contains not only immunologically but mass spectrometrically identified neurohypophyseal hormones.

Int J Dev Biol, 1998, 42(7), 917 - 25
Using EC and ES cell culture to study early development: recent observations on Indian hedgehog and Bmps; Grabel L et al.; Despite great technological advances in the study of mammalian development in the past two decades, certain problems in early development, such as how the extraembryonic lineages are established, have remained intractable . We suggest that teratocarcinoma (EC) and embryonic stem cells (ES) remain useful in vitro tools for studying some of these problems . We present a continuation of our studies on the role of IHH-based signaling in early development and demonstrate that the IHH N-peptide is expressed in the outer visceral endoderm cells of both the EC and ES-derived embryoid body . We also show that Bmp2 is upregulated and Bmp4 downregulated during the differentiation of F9 EC cells into embryoid bodies, whereas both genes are upregulated when J7 ES cells differentiate into embryoid bodies . We also examine the spatial localization of Ihh, Bmp2, and Bmp4 in day 6.5-7.0 and 7.5-8.0 embryos by in situ hybridization analysis . These data support the EC temporal expression data in that all 3 genes are expressed in visceral endoderm . Bmp4 expression appears to be limited to extraembryonic regions, where mesoderm as well as visceral endoderm are stained . Ihh and Bmp2 are expressed in extraembryonic tissues and the embryo proper . Functional roles for the observed expression patterns are discussed.

Chem Biol Interact, 1998 Oct 2, 115(3), 167 - 74
Growth inhibitory factor and zinc affect neural cell cultures in a tissue specific manner; Bruinink A et al.; Deficiency of neuronal growth inhibitory factor (GIF) and abnormalities in zinc homeostasis have been suggested to play a role in the neuropathogenesis of Alzheimer's disease . We report here that embryonic chick cerebral cell cultures zinc and copper containing GIF in the presence of marmoset hippocampal extract reduces significantly and concentration dependently mitochondrial succinate dehydrogenase activity (MTT) and cell mass . In contrast, no indications could be found that GIF affected neural retina cell cultures . Our results suggest that the observed effects of GIF are not elicited by zinc.

J Biol Rhythms, 1998 Dec, 13(6), 479 - 93
Day/night differences in the stimulation of adenylate cyclase activity by calcium/calmodulin in chick pineal cell cultures: evidence for circadian regulation of cyclic AMP; Nikaido SS et al.; In chick pineal cell culture, stimulation of adenylate cyclase with the diterpene forskolin was greater during the subjective night than during the subjective day . This rhythm of cyclic AMP (cAMP) stimulation mimicked the rhythm of unstimulated cAMP measured previously during LD cycles from flow-through culture . Direct measurement of adenylate cyclase activity in permeabilized cells revealed an adenylate cyclase activity activated by Ca2+/calmodulin during the night but not during the day . However, this difference in adenylate cyclase activity at two times of the circadian cycle is apparent only when permeabilized cells were prewashed with buffer containing GTE When cAMP was measured from flow-through cultures maintained in continuous darkness to determine whether a circadian clock may regulate cAMP, a low-amplitude rhythm was measured . The circadian rhythm of cAMP was similar to the cAMP rhythm previously measured on LD cycles except that the rhythm in darkness had a lower amplitude . Similar to the suppression of melatonin, cAMP was suppressed by light presented during the middle of the night . LD differences in nocturnal cAMP levels were abolished with dipyridamole, an inhibitor of cyclic GMP (cGMP) phosphodiesterase . These results suggest that the rhythm of cAMP in chick pineal cells involves the stimulation of adenylate cyclase by Ca2+/calmodulin during the night and a GTP-dependent suppression of adenylate cyclase activity during the day . The photic suppression of cAMP at night involves the activation of a dipyridamole-sensitive, cGMP phosphodiesterase.

Eur J Cancer, 1998 Jun, 34(7), 1086 - 90
Inhibition of growth of primary human tumour cell cultures by a 4-anilinoquinazoline inhibitor of the epidermal growth factor receptor family of tyrosine kinases; Baguley BC et al.; The epidermal growth factor receptor (EGFR) is thought to mediate the action of the mitogens EGF and tumour growth factor-alpha (TGF-alpha) in a variety of cancers, including those of the lung, breast and ovary . A number of new selective inhibitors of EGFR tyrosine kinase have now been developed as potential new antitumour agents . We used a potent inhibitor of this tyrosine kinase, 6-amino-4-{(3-bromophenyl)amino}-7-(methylamino)quinazoline (SN 25531; PD 156273), to determine the responses of primary cultures derived from patients with cancer of the lung, ovary, breast, cervix and endometrium . Cells were cultured in 96-well plates and proliferation assessed by incorporation of 3H-thymidine . Measured growth inhibitory concentrations IC50 values) varied from 1 nM to 14 microM with a 1000-fold differential between sensitive and resistant cultures . Results were compared with rates of proliferation, estimated using a paclitaxel-based method . We also measured the IC50 values for the tyrosine kinase inhibitor using a number of established human cell lines, and compared them with EGFR content using fluorescent antibody staining and flow cytometry . The presence of EGFR was found to be necessary, but not sufficient, for in vitro response . Only a small number of cell lines (3 of 7 for lung, 1 of 7 for ovarian, 2 of 3 squamous cell and 0 of 12 for melanoma) were sensitive to the tyrosine kinase inhibitor . In contrast, 40 of the 50 primary cultures (including 14 of 15 lung cancer samples and 14 of 19 ovarian cancer samples) were sensitive.

Anat Rec, 1998 Dec, 252(4), 554 - 67
Morphological and immunocytochemical characterization of primary osteogenic cell cultures derived from fetal rat cranial tissue; Irie K et al.; Enzymatic digestion of bone tissue potentially releases a mixture of precursor, differentiating, and mature cells . Conceptually, early fetal osteogenic tissue should provide a more uniform population of cells than late embryonic or newborn bone in which cells have already differentiated . In this context, we have applied sequential enzymatic digestion to obtain and culture cells from 15-16-day fetal rat cranial tissue, a developmental age where deposition of bone matrix has not yet started at this site . These cultures were compared with those of osteogenic cells isolated from newborn rat calvariae and grown under similar conditions . Matrix production and composition were examined by colloidal gold immunocytochemistry using antibodies to bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN) . The plated cells formed mineralized nodules by day 14 . The presence of mineral was determined by von Kossa staining and backscattered electron imaging (BEI), and the accumulation of calcium and phosphorus within the nodules was demonstrated by X-ray microanalysis and elemental mapping . At early time intervals, cells were generally cuboidal in shape and showed a well-developed Golgi apparatus, which occasionally was immunoreactive for OPN . Labeling for BSP and OPN was found over mineralization foci and electron-dense material within, and at the periphery, of larger mineralized masses and over accumulations of afibrillar matrix at the dish surface . Osteocalcin immunoreactivity was also associated with electron-dense portions of the bone-like matrix . These data demonstrate the potential of presumptive fetal rat calvarial cells to form a bone-like matrix in vitro and suggest that the assembly and mineralization pattern show similarities to the process of intramembranous ossification . Such a culture system is of interest not only for studying cellular and matrix events of bone formation, but also factors which influence mesenchymal cells in committing themselves to the osteogenic pathway.

J Pharmacol Toxicol Methods, 1998 Jun, 39(4), 211 - 20
In vitro taurine uptake into cell culture influenced by using media with or without CO2; Lelong IH et al.; Buffers used to incubate cells for pharmacological or toxicological studies are usually of very simple composition, far from the composition of biological fluids or cell culture media . Comparative studies on taurine uptake levels by cultured cells show that a new CO2-Independent Medium (CIM) is suitable for incubating cells in place of the Krebs-Ringer buffer (KR) usually used . Basal uptake level of taurine was lower for cells incubated in CIM or in other culture media when compared to those incubated whether in KR or in other "physiological buffers." Isoproterenol depressed similarly the taurine uptake in cells incubated in CIM or KR . The same uptake modulation by beta-alanine, GES, GABA, or HEPES was observed for cells incubated in CIM or KR . C6 cells growth in CIM was dependent on the starting cell density when classically vented T-flasks were used, growth being notably reduced at low density . In tightly closed flasks cells grew in CIM similarly to control cultures maintained in M199 medium or DMEM.

Mediators Inflamm, 1998, 7(1), 31 - 3
Effects of lipoxygenase metabolites of arachidonic acid on the growth of human mononuclear marrow cells and marrow stromal cell cultures; Desplat V et al.; The effects of various lipoxygenase metabolites of arachidonic acid (AA) were investigated on the growth of freshly isolated human bone marrow mononuclear cells and marrow stromal cell cultures . LTB4, LXA4, LXB4, 12-HETE and 15-HETE (1 microM) decreased {3H}-thymidine incorporation on marrow stromal cell cultures without affecting cell number . Only 12-HETE showed a dose-response effect on {3H}-thymidine incorporation . While LTB4 (1 microM) decreased thymidine incorporation on marrow mononuclear cells, LTC4, LXA4, LXB4, 12-HETE and 15-HETE had no effect . The lipoxygenase inhibitor NDGA had no effect on both cell types suggesting no role of endogenous lipoxygenase metabolites on cell growth . These results suggest no important role of lipoxygenase metabolites of AA on the proliferation of human marrow mononuclear cells and marrow stromal cell cultures.

Biochem Biophys Res Commun, 1998 Nov 27, 252(3), 552 - 5
Expression of a NOS-III-like protein in human astroglial cell culture; Colasanti M et al.; Evidence for the presence of a type-III nitric oxide synthase-like protein (NOS-III-like protein) in astroglial cells is reported . The mRNA of a NOS-III-like protein is constitutively expressed in human astrocytoma T67 cells, taken as an astroglial model . The nucleotide sequence of the PCR product (422 bp) shares more than 99% identity with the cDNA (from 1588 to 2009) of the human endothelial nitric oxide synthase (NOS-III) . The molecular mass of the astroglial NOS-III-like protein is about 140 kDa, as observed for human NOS-III . Moreover, the astroglial NOS-III-like protein is constitutively tyrosine-phosphorylated and associated with caveolin-1 . The astroglial NOS-III-like protein is apparently inactive, as reported for phosphorylated human NOS-III associated with caveolin-1 .

Pharmacol Toxicol, 1998 Nov, 83(5), 194 - 9
Cytoprotection by deprenyl and tolcapone in a cell culture model of cerebral ischaemia; Ekblom J et al.; Foetal rat brain aggregation cultures were exposed to a single episode of anoxia and hypoglycaemia for 30 min . Lactate dehydrogenase specific activity was estimated in the culture medium after ischaemia as a marker of lost cell integrity . Release of lactate dehydrogenase was most prominent during the first 24 hr period after the ischaemic damage, then it gradually declined . Immediately after ischaemic exposure, the cultures were treated with different concentrations of L-deprenyl or tolcapone . Significantly lower amounts of lactate dehydrogenase leaked into the culture medium during the first 24 hr after the ischaemic episode in cultures treated with deprenyl or tolcapone (1-100 nM) . These results suggest that deprenyl and tolcapone may reduce cell damage after ischaemia, at doses causing enzyme inhibition.

J Cell Biochem, 1998 Dec 1, 71(3), 382 - 91
Dexamethasone stimulates osteogenic differentiation in vertebral and femoral bone marrow cell cultures: comparison of IGF-I gene expression; Milne M et al.; Osteoblast-like cell cultures have been established from the marrow of adult rat vertebrae . We have simultaneously examined the response to dexamethasone (dex) treatment in cultures of young adult female vertebral and femoral marrow cells . Alkaline phosphatase (AP) activity was analyzed as well as the expression of mRNAs for osteocalcin (OC) and insulin-like growth factor I (IGF-I) . The vertebral and femoral marrow cells were maintained for 7 days in primary culture with or without 10(-8) M dex and then 6 days in secondary culture without dex or with 10(-8) M or 10(-7) M dex . All cells were examined on day 6 of secondary culture . Vertebral and femoral cultures each expressed the highest AP enzyme levels when grown with dex in primary culture (10(-8) M) and secondary culture (10(-7) M) . Under all experimental conditions, vertebral cultures had lower AP enzyme activity than femoral cultures . When dex was omitted from secondary culture, OC gene expression was not detected in either vertebral or femoral passaged cells even if dex was present in primary culture . For dex conditions where OC was expressed, vertebral cultures had higher OC mRNA steady-state levels than femoral cultures . IGF-I gene expression was detected by Northern analysis in both vertebral and femoral secondary cultures . However, vertebral marrow cultures had much higher IGF-I mRNA levels compared to femoral cultures whether or not dex was present in primary culture . These findings demonstrate that dex supports the differentiation of both vertebral and femoral adult marrow osteogenic cells into osteoblasts . Our results support the hypothesis that osteoblastic marrow cultures differ depending upon which location in the skeleton they are from and that there are skeletal site-dependent differences in the insulin-like growth factor system components.

J Cell Biochem, 1998 Dec 1, 71(3), 351 - 62
Insulin-like growth factor binding proteins localize to discrete cell culture compartments in periosteal and osteoblast cultures from fetal rat bone; Chen Y et al.; Insulin-like growth factor (IGF)-I and IGF-II are expressed at biologically effective levels by bone cells . Their stability and activity are modulated by coexpression of IGF binding proteins (IGFBPs) . Secreted IGFBPs may partition to soluble, cell-associated, and matrix-bound compartments . Extracellular localization may sequester, store, or present IGFs to appropriate receptors . Of the six IGFBPs known, rat osteoblasts synthesize all but IGFBP-1 . Of these, IGFBP-3, -4, and -5 mRNAs are induced by an increase in cAMP . Little is known about extracellular IGFBP localization in bone and nothing about IGFBP expression by nonosteoblastic periosteal bone cells . We compared basal IGFBP expression in periosteal and osteoblast bone cell cultures and assessed the effects of changes in cAMP-dependent protein kinase A or protein kinase C . Basal IGFBP gene expression differed principally in that more IGFBP-2 and -5 occurred in osteoblast cultures, and more IGFBP-3 and -6 occurred in periosteal cultures . An increase in cAMP enhanced IGFBP-3, -4, and -5 mRNAand accordingly increased soluble IGFBP-3, -4, and -5 and matrix-bound IGFBP-3 and -5 in both bone cell populations . In contrast, protein kinase C activators suppressed IGFBP-5 mRNA, and its basal protein levels remained very low . We also detected low Mr bands reactive with antisera to IGFBP-2, -3, and -5, suggesting proteolytic processing or degradation . Our studies reveal that various bone cell populations secrete and bind IGFBPs in selective ways . Importantly, inhibitory IGFBP-4 does not significantly accumulate in cell-associated compartments, even though its secretion is enhanced by cAMP . Because IGFBPs bind IGFs less tightly in cell-bound compartments, they may prolong anabolic effects by agents that increase bone cell cAMP.

Hum Gene Ther, 1998 Nov 1, 9(16), 2363 - 73
The presence of human coxsackievirus and adenovirus receptor is associated with efficient adenovirus-mediated transgene expression in human melanoma cell cultures; Hemmi S et al.; Adenovirus (AdV)-mediated gene expression of immune stimulators represents a valuable in vivo approach for gene therapy of human cancer . The expression level of the therapeutic gene is of crucial importance for the efficacy of this type of treatment . Entry of AdV is dependent on the primary adenovirus receptor CAR and the secondary AdV receptor identified earlier to be a member of the integrin family of surface molecules . We have analyzed 14 different human melanoma cell cultures from different stages together with one melanoma cell line for their AdV-mediated transduction and expression efficiency . Recombinant viruses at various concentrations were used for expression of the B7-1 costimulatory molecule under the control of different promoters and the expression levels of B7-1 were analyzed by flow cytometry . AdV-mediated IL-12 expression was measured using a commercial ELISA . Levels of transgene expression were compared with the expression levels of HCAR, the alpha(v)beta3 and alpha(v)beta5 integrins, and HLA class I . In 4 of 14 cell cultures tested, the presence of the primary virus receptor CAR was associated with the high transduction efficiency phenotype when using the B7-1- and IL-12-expressing viruses at a relatively low multiplicity of infection (MOI) of 50 . Immunohistochemistry on cryosections from the original biopsies yielded a strong signal specific for CAR . In contrast, cell cultures expressing low or undetectable levels of CAR needed a 20- to 40-fold higher viral input to show comparable expression level of B7-1 or IL-12 . Expression levels of the transgenes hardly varied when using different promoters and no association was observed with the presence or absence of HLA class I molecules or with the expression levels of integrins.

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