Environ Health Perspect, 2002 Jul, 110(7), 641 - 5 Mitogen-activated protein kinase activation by oxidative and bacterial stress in an amphibian cell culture model; Carter LA et al.; The decline of many amphibian species could be caused by their susceptibility to environmental pollutants that cause cellular stress and cell death . A variety of intracellular signal transduction pathways are activated by environmental stress factors, which result in cell death . Mitogen-activated protein kinases are intracellular signaling molecules that include the extracellular signal-regulated kinases (ERK-1 and ERK-2) . We used cultured (italic)Xenopus(/italic) tadpole cells (XTC-2 cells) to investigate the activation of ERK by oxidative or bacterial stress, two environmental factors that could contribute to pollution in aquatic systems . We exposed XTC-2 cell monolayers to hydrogen peroxide or bacterial lipopolysaccharide and measured ERK activation by Western blotting using antibodies raised against phosphorylated ERK-1 and ERK-2 . Only ERK-2 was detected in XTC-2 cells . Both hydrogen peroxide and lipopolysaccharide caused ERK-2 phosphorylation in a time- and concentration-dependent manner . Hydrogen peroxide caused a 20- to 30-fold increase in ERK-2 activation that peaked 30 min after treatment, and lipopolysaccharide induced a 5- to 10-fold increase in ERK-2 activation that peaked 60 min after treatment . PD98059, an inhibitor of the ERK pathway, reduced the cytotoxic response of XTC-2 cells to hydrogen peroxide or lipopolysaccharide . These data suggest that ERK-2 is an intracellular target of oxidative and bacterial stress in amphibians that mediates, at least in part, the cytotoxic response to hydrogen peroxide or lipopolysaccharide . Moreover, the (italic)Xenopus(/italic) (XTC-2) cell culture system could serve as a useful model to identify agents that might threaten amphibian populations and human health. Neurol Res, 2002 Jul, 24(5), 479 - 82 The tissue level of dexamethasone in human brain tumors is about 1000 times lower than the cytotoxic concentration in cell culture; Nestler U et al.; In glioblastoma patients dexamethasone is routinely administered as an antiedematous drug . In contrast to its empirically proven effect, the biochemical way of action remains poorly understood . In order to assess whether a direct cytotoxic effect is present in vivo we compared dexamethasone levels in brain tumor specimens with its cytotoxic concentrations in cell culture . Biopsy specimens were taken during microsurgical tumor removal, homogenized and dexamethasone levels were measured by high pressure liquid chromatography . In cell culture we tested different concentrations of dexamethasone on A172, U87, U373 cells and on eleven primary glioblastoma cell lines . Furthermore a pilocytic astrocytoma I, an astrocytoma II and an oligodendroglioma III and a meningioma were examined . Cell viability was assessed using the Alamar Blue assay and the concentrations resulting in loss of 50% of the cell population were calculated (LD50) . The average brain tumor tissue concentration of dexamethasone was 225 nanogram g(-1) . The mean LD50 in cell culture ranged at 222 microgram ml(-1) . We conclude that a direct cytotoxic effect of dexamethasone on brain tumor cells is not present in vivo because the tissue levels of the drug are about 1000 times lower than the LD50 in cell culture. Biotechnol Bioeng, 2002 Aug 20, 79(4), 408 - 15 The effect of ajmalicine spiking and resin addition timing on the production of indole alkaloids from Catharanthus roseus cell cultures; Lee-Parsons CW et al.; The potential for the feedback inhibition of indole alkaloid synthesis was investigated by spiking suspension cultures of Catharanthus roseus with 0, 9, or 18 mg/L ajmalicine on day 0 . The production of ajmalicine, catharanthine, and serpentine were inhibited in a dose-dependent manner . The inhibition was transient as the exogenous ajmalicine was ultimately either metabolized in the medium or within the cell . The addition of neutral resin has previously been shown to enhance ajmalicine production . To minimize product inhibition and product metabolism, Amberlite XAD-7 resin was added to immobilized cultures of C . roseus starting on either day 0, 5, or 15, and fresh resin was exchanged for spent resin every 5 days . The addition of resin did not decrease the viability of the culture . Growth was reduced only in cultures with resin added on day 0 . Alkaloid production was enhanced to different extents by the timing of resin addition, suggesting that feedback inhibition or product metabolism was present throughout the culture period . Ajmalicine recovery was nearly 100% when the resin was added initially either on day 0 or day 5 . Ajmalicine recovery was reduced to 55% when the resin was added later in the culture period starting on day 15, presumably because of resin saturation or the inaccessibility of alkaloids trapped in the vacuole . Delaying the addition of XAD-7 resin until 5 days after the start of the culture resulted in the highest improvement in ajmalicine production, i.e approximately 70% and also resulted in the complete recovery of ajmalicine from the cell . Clin Cancer Res, 2002 Jul, 8(7), 2292 - 7 Differential expression of FEZ1/LZTS1 gene in lung cancers and their cell cultures; Toyooka S et al.; PURPOSE: The FEZ1/LZTS1 (FEZ1) gene, located on chromosome 8p22 (8p22), was identified recently as a candidate tumor suppressor gene . Because loss of heterozygosity at 8p21-22 is a frequent event in lung cancers, we studied FEZ1 alteration in short-term cultures of resected lung cancer tumors and cell lines . EXPERIMENTAL DESIGN: We examined FEZ1 expression in 17 non-small cell lung cancer (NSCLC), 19 small cell lung cancer (SCLC) cell lines, and 6 pairs of short-term cultures of resected NSCLCs and accompanying nonmalignant bronchial cells (NBECs) by reverse transcription-PCR and Western blotting . To investigate the mechanism for silencing, cells were cultured with 5-aza-2'-deoxycytidine or trichostatin A . We screened for genomic mutations by PCR-single-strand conformational polymorphism . RESULTS: Thirteen of 17 NSCLC (76%) and 3 of 19 SCLC (16%) of cell lines showed absent expression (P = 0.001) . Of the paired NSCLC-NBEC cultures, 3 of 6 showed loss of expression in tumor cell cultures . In the cell lines retaining expression, the amplicon products in SCLCs were more intense than those of NSCLCs and NBECs . Expression of FEZ1 was not restored by 5-aza-2'-deoxycytidine and trichostatin A . Although FEZ1 expression was moderately correlated with loss of heterozygosity of specific microsatellite makers at 8p21-22 in NSCLC cell lines, it was strongly correlated to D8S261 and LPL loci in SCLC cell lines . No mutation was found within cording region of FEZ1 by PCR-single-strand conformational polymorphism . CONCLUSIONS: We found differential FEZ1 expression in NSCLC and SCLC cell lines, and the absent expression in 3 of 6 short-term cultures of NSCLC tumors . FEZ1 may be related to tumorigenesis of lung cancer. J Neural Transm, 2002 May, 109(5-6), 651 - 61 Synthetic neuromelanin is toxic to dopaminergic cell cultures; Nguyen A et al.; In the present study, primary cultures of mesencephalic dopaminergic cells were exposed to synthetic dopamine neuromelanin (NM) for 48 hrs at concentrations of 0, 1, 10, 20, 50 and 100 microg NM/ml medium . Differently prepared synthetic NM with or without incorporated iron and NM oxidatively damaged by hydrogen peroxide were used . All NMs affected cellular structures e.g . as swelling of neural processes, rounding of cells, and occasional inclusion of neuromelanin particles . Cell numbers were uniformly and dose dependently reduced . Exposure to MPP(+) and ferric iron led to cytotoxic changes which could be further aggravated by oxidatively damaged NM, suggesting cytotoxicity of soluble compounds of NM in predamaged neurons. Arch Toxicol, 2002 Jul, 76(7), 414 - 22 Epub 2002 May 29. Effects of the dithiocarbamate fungicide propineb in primary neuronal cell cultures and skeletal muscle cells of the rat; Schmuck G et al.; After repeated-dose toxicity studies with the fungicide propineb, reversible effects on muscle functions were found . Therefore, mechanistic investigations should contribute to clarification of its mode of action in relation to disulfiram and diethyldithiocarbamate neurotoxicity or direct effects on muscle cells . In principle, besides the dithiocarbamate effects, two different mechanisms have been discussed for this fungicide . One mechanism is the degradation to carbon disulfide (CS(2)) and propylenthiourea (PTU) and the other are direct effects of zinc . Primary neuronal cell cultures of the rat are a well established model to identify neurotoxic compounds like n-hexane or acrylamide . In this cell culture model, endpoints such as viability, energy supply, glucose consumption and cytoskeleton elements were determined . Additionally, skeletal muscle cells were used for comparison . Propineb and its metabolite PTU were investigated in comparison to CS(2), disulfiram and diethyldithiocarbamate . The toxicity of zinc was tested using zinc chloride (ZnCl(2)) . It was clearly shown that propineb exerted strong effects on the cytoskeleton of neuronal and non-neuronal cell cultures (astrocytes, muscle cells) . This was similar to ZnCl(2,) but not to CS(2) . With CS(2) and disulfiram effects on the energy supply were more prominent . In conclusion, the toxicity of propineb is not comparable to disulfiram, diethyldithiocarbamate or CS(2) neurotoxicity . In regard to these findings, a direct reversible effect of propineb on skeletal muscle cells seems to be more likely. Am J Physiol Renal Physiol, 2002 Aug, 283(2), F302 - 8 Proliferation and osmotic tolerance of renal inner medullary epithelial cells in vivo and in cell culture; Zhang Z et al.; Renal inner medullary (IM) cells survive interstitial osmolality that ranges from 600 to 1,700 mosmol/kgH2O or more . In contrast, much smaller acute changes killed the cells previously studied in tissue culture, such as mouse IM collecting duct 3 (mIMCD3) cells, that are immortalized with SV40 and proliferate rapidly . Proliferation and DNA replication sensitize mIMCD3 cells to hypertonicity . In the present studies, we observed that proliferating cells were scarce in rat IM . Then, we prepared passage 2 mouse IM epithelial (p2mIME) cells . They have a much lower incidence of DNA replication than do mIMCD3 cells . p2mIME cells survive much greater acute increases in NaCl than do mIMCD3 cells and also tolerate significantly greater acute increases of urea and of NaCl plus urea, but still not to levels as high as occur in vivo . We conclude that immortalization and continued DNA replication account for part of the previously observed difference in osmotic tolerance between IM cells in vivo and in cell culture but that other factors must also be involved. Ther Apher, 2002 Jun, 6(3), 213 - 20 Affinity hemodialysis for antiviral therapy . I . Removal of HIV-1 from cell culture supernatants, plasma, and blood; Tullis RH et al.; We tested an affinity hemodialysis technique designed to efficiently remove HIV and toxic viral proteins from blood . Miniature polyethersulfone hollow-fiber dialysis cartridges (200-500 nm pore) were packed with anti-HIV antibodies covalently coupled to agarose beads and sealed inside the cartridge . Cell culture fluids, plasma, or infected blood (7-15 ml) containing HIV-1 were circulated over the cartridge at 0.7-10 ml/min and the rate of removal of HIV measured by PCR and p24 ELISA . The technique removed up to 98% of HIV-1 particles from cell culture supernatants . Affinity hemodialysis also efficiently captured cultured HIV from human blood plasma (90%) and native HIV from infected blood (83% to 100%) . Viral capture followed first-order kinetics (t(1/2) = 2.8 h) . Variations in antibody type, matrix linkage (protein G versus direct coupling), bead pore size, and temperature of operation (25-37 degrees C) had only small effects . Although some binding was nonspecific, direct binding to the immobilized antibodies appeared to be the predominant mechanism. Biochem Pharmacol, 2002 Jul 1, 64(1), 49 - 60 Analysis of rainbow trout Ah receptor protein isoforms in cell culture reveals conservation of function in Ah receptor-mediated signal transduction; Pollenz RS et al.; Two distinct aryl hydrocarbon receptor (AHR) cDNAs have been isolated from rainbow trout . The encoded receptor protein products termed rtAHR2alpha and rtAHR2ss are 97% identical at the amino acid level but are reported to have distinct functions with regard to AHR-mediated gene regulation . To test this hypothesis, the two proteins were evaluated functionally both in vitro and in a Chinese hamster lung cell line, E36 . To facilitate analysis, both rtAHR2 isoforms were tagged with the FLAG peptide and could be expressed and quantified in a rabbit reticulocyte lysate . However, both proteins failed to form functional complexes with mammalian or rainbow trout AHR nuclear translocator protein (ARNT) that could associate with xenobiotic response elements (XREs) in a ligand-dependent manner in vitro . In contrast, both proteins exhibited positive function on AHR-mediated signaling when expressed in the E36 cell line . Both rtAHR2 isoforms showed a cytoplasmic distribution in the unliganded state and could drive the expression of a reporter gene under control of the trout CYP1A3 promoter . Although both proteins induced reporter gene activity to the same magnitude, the EC(50) values of the two isoforms for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) differed by an order of magnitude, with the rtAHR2ss isoform less responsive to TCDD . When the functions of the rtAHR2 isoforms were tested in the context of the dominant negative rtARNT(a) protein, TCDD-mediated induction of reporter gene activity was reduced as the level of rtARNT(a) protein increased . In summary, both rtAHR2 isoforms appear to exhibit positive function in AHR-mediated signaling, suggesting conservation of function. Eur J Neurosci, 1990, 2(9), 762 - 768 Amoeboid Microglial Cells and not Astrocytes Synthesize TNF-alpha in Swiss Mouse Brain Cell Cultures; Hetier E et al.; The role of tumour necrosis factor (TNF-alpha) in brain physiology and pathology has been the focus of several studies . However, the source of this lymphokine in the central nervous system and the regulation of its synthesis is still poorly understood . We have therefore used purified astrocytes and brain macrophages in culture to compare the abilities of these two cell types to synthesize TNF-alpha and its mRNA . We find that, in the Swiss mouse, no significant TNF activity or TNF-alpha mRNA are produced by astrocytes, even following activation with lipopolysaccharides (LPS) . On the other hand, purified microglial cells express a cytotoxic activity able to kill TNF-sensitive LM cells . Part of this activity is released into the culture medium and part remains bound to the membrane after mild paraformaldehyde treatment, demonstrating the existence in the culture of the soluble and membrane-bound forms of TNF activity . The fact that amoeboid microglial cells, and not astrocytes, are the actual source of TNF in brain cultures was further demonstrated by Northern blot analysis and in situ hybridization using a TNF-alpha specific oligonucleotide probe . The definition of the cell type which, in the CNS, is responsible for TNF synthesis will allow the regulation of this lymphokine to be analysed and opens the way for a better understanding of the interactions between amoeboid microglial cells and the other cell types which make up the nervous system. Biomaterials, 2002 Aug, 23(15), 3235 - 45 In vitro investigation of titanium and hydroxyapatite dental implant surfaces using a rat bone marrow stromal cell culture system; Knabe C et al.; In this study, rat bone marrow cells (RBM) were used to evaluate different titanium and hydroxyapatite dental implant surfaces . The implant surfaces investigated were: a titanium surface having a porous titanium plasma-sprayed coating (sample code Ti-TPS), a titanium surface with a deep profile structure (sample code Ti-DPS), an uncoated titanium substrate with a machined surface (sample code Ti-ma) and a machined titanium substrate with a porous hydroxyapatite plasma-sprayed coating (sample code Ti-HA) . RBM cells were cultured on the disc-shaped test substrates for 14 days . The culture medium was changed daily and examined for calcium and phosphate concentrations . After 14 days specimens were examined by light microscopy, scanning electron microscopy, energy dispersive X-ray analysis and morphometry of the cell-covered substrate surface . All test substrates facilitated RBM growth of extracellular matrix formation . Ti-DPS and Ti-TPS to the highest degree, followed by Ti-ma and Ti-HA . Ti-DPS and Ti-TPS displayed the highest cell density and thus seem to be well suited for the endosseous portion of dental implants . RBM cells cultured on Ti-HA showed a delayed growth pattern . This may be related to its high phosphate ion release. J Neurosci, 2002 Jul 1, 22(13), 5403 - 11 Activation of group III metabotropic glutamate receptors inhibits the production of RANTES in glial cell cultures; Besong G et al.; The chemokine RANTES is critically involved in neuroinflammation and has been implicated in the pathophysiology of multiple sclerosis . We examined the possibility that activation of G-protein-coupled metabotropic glutamate (mGlu) receptors regulates the formation of RANTES in glial cells . A 15 hr exposure of cultured astrocytes to tumor necrosis factor-alpha and interferon-gamma induced a substantial increase in both RANTES mRNA and extracellular RANTES levels . These increases were markedly reduced when astrocytes were coincubated with l-2-amino-4-phosphonobutanoate (l-AP-4), 4-phosphonophenylglycine, or l-serine-O-phosphate, which selectively activate group III mGlu receptor subtypes (i.e., mGlu4, -6, -7, and -8 receptors) . Agonists of mGlu1/5 or mGlu2/3 receptors were virtually inactive . Inhibition of RANTES release produced by l-AP-4 was attenuated by the selective group III mGlu receptor antagonist (R,S)-alpha-methylserine-O-phosphate or by pretreatment of the cultures with pertussis toxin . Cultured astrocytes expressed mGlu4 receptors, and the ability of l-AP-4 to inhibit RANTES release was markedly reduced in cultures prepared from mGlu4 knock-out mice . This suggests that activation of mGlu4 receptors negatively modulates the production of RANTES in glial cells . We also examined the effect of l-AP-4 on the development of experimental allergic encephalomyelitis (EAE) in Lewis rats . l-AP-4 was subcutaneously infused for 28 d by an osmotic minipump that released 250 nl/hr of a solution of 250 mm of the drug . Detectable levels of l-AP-4 ( approximately 100 nm) were found in the brain dialysate of EAE rats . Infusion of l-AP-4 did not affect the time at onset and the severity of neurological symptoms but significantly increased the rate of recovery from EAE . In addition, lower levels of RANTES mRNA were found in the cerebellum and spinal cord of EAE rats infused with l-AP-4 . These results suggest that pharmacological activation of group III mGlu receptors may be useful in the experimental treatment of neuroinflammatory CNS disorders. Tsitologiia, 2002, 44(3), 235 - 41 {Mode of epithelium-fibroblast interaction in mixed heterotypic cell cultures}; Fetisova EK et al.; The interaction between epithelium (dog kidney epithelium MDCJ/clone 20) and fibroblasts (diploid human fibroblasts M19 and AG-1523) was studied in mixed heterotypic cell cultures . The mode of cell interaction depends on the manner of their collision . At collision of the epithelium lamella and the lateral side of fibroblast, the lamella was seen to creep under the lateral side to force back the fibroblast . At the frontal collision of epithelium and fibroblast lamellae, the mode of interaction depends on the local situation . With the presence of a free substratum around, the fibroblast formed a new lamella and moved aside from the place of collision . In the case, when the neighboring cells prevented fibroblast from moving, it migrated under the epithelium . In this work, we have first demonstrated the formation of specialized intercellular adhesions between epithelium and fibroblasts . The cultures were studied by phase contrast, interference reflection or video tape recording, using an image processing system (Hamamatsu) . For studying adhesion, immuno-fluorescent methods were performed. J Clin Virol, 2002 Jul, 25 Suppl 1, S13 - 8 Comparison of mixed cell culture containing genetically engineered BGMK and CaCo-2 cells (Super E-Mix) with RT-PCR and conventional cell culture for the diagnosis of enterovirus meningitis; Buck GE et al.; BACKGROUND: Enteroviral meningitis has traditionally been diagnosed by cell culture . More recently, molecular techniques and shell vials with a mixture of human colon carcinoma and genetically engineered buffalo green monkey kidney cells (BGMK cells) (Super E-Mix) have been described . OBJECTIVE: We compared the results of this new cell culture technique with two reverse transcriptase polymerase chain reaction (RT-PCR) techniques and conventional cell culture to assess the accuracy of these various methods . STUDY DESIGN: 2 ml of cerebrospinal fluid (CSF) was obtained from 72 patients with suspected viral meningitis . 600 microl was used for conventional cell culture and 200 microl was inoculated to each of two Super E-Mix vials . One vial was incubated for 24 h, then stained with pan-enterovirus antibody, and the other was examined for CPE daily for 5 days and stained when positive or at the end of incubation . The final aliquot (500 microl) was centrifuged at 25,000g, and the pellet in 140 microl of supernatant was used for ribonucleic acid (RNA) extraction and tested by commercial single-step (Chemicon, Temecula, CA) and two-step (Argene, Varilhes, France) RT-PCR methods . RESULTS: Conventional culture was 51% sensitive and 100% specific . The Super E-Mix was slightly higher (sensitivity 76%, specificity 100%) . The Chemicon and Argene PCR methods had a sensitivity of 93% and 88% and specificity of 90% and 97%, respectively . CONCLUSION: The Super E-Mix procedure had greater sensitivity than conventional cell culture, but RT-PCR was the most sensitive technique with this type of specimen . The RT-PCR methods performed equally. Reproduction, 2002 Jul, 124(1), 107 - 17 Induction of sperm maturation in vitro in epididymal cell cultures of the tammar wallaby (Macropus eugenii): disruption of motility initiation and sperm morphogenesis by inhibition of actin polymerization; Lin M et al.; A sperm-epididymal cell co-culture was shown to be capable of inducing the in vitro maturation of spermatozoa from a marsupial species, the tammar wallaby (Macropus eugenii) . This system was able to maintain wallaby epididymal epithelial cells in vitro for more than 2 months . The system also enabled immature wallaby spermatozoa to differentiate from a T-shaped to a streamlined form, accompanied by the development of progressive motility after co-culture with epididymal cell monolayers that had been cultured for 7 days . The addition of inhibitors of actin polymerization (latrunculin A or B) to the co-culture system showed that wallaby sperm maturation was impaired by the interruption of actin organization within the immature spermatozoa . These results indicate that actin filaments play a significant role in sperm transformation during post-testicular maturation in marsupials . These observations also indicate that the marsupial co-culture system has the potential to greatly increase understanding of sperm-epididymal cell interactions and the mechanism of sperm maturation in these species. Wien Klin Wochenschr, 2002 Apr 15, 114(7), 279 - 83 Medullary thyroid carcinomas in cell culture--models for future therapies; Pfragner R et al.; We report the successful establishment of seven human medullary thyroid carcinomas (MTC) as continuous cell lines . Characteristic features--such as the presence of neuroendocrine granules--and the positive immunoreactivity to antibodies to CT, CGRP, GRP, SRIF, 5-HT, NSE, PHE, LK2H10, ER and Pgr were followed throughout the cultivation . An overexpression of the antiapoptotic gene bcl-2 was detected in the cell lines . Deregulation of apoptosis plays an important role in multistep tumorigenesis . MTCs are known for the phenomenon of bcl-2-based chemo- and radioresistance . Our studies focus on influencing the growth rates and modulating the apoptotic rates by treatment with proliferation-modifying substances and anticancer drugs . Our MTC cell lines are useful models for these in vitro studies. Peptides, 2002 May, 23(5), 903 - 10 Family of hemorphins: co-relations between amino acid sequences and effects in cell cultures; Blishchenko EY et al.; Hemorphins, i.e . endogenous fragments of beta-globin chain segment (32-41) LVVYPWTQRY(F) suppress the growth of transformed murine fibroblasts L929 cell culture, the effect is due to cytotoxicity and inhibition of cell proliferation . The contribution of cytotoxicity depends on the presence of Leu(32): VV-hemorphins, except VV-hemorphin-4, exhibit cytotoxicity significantly higher than respective LVV-hemorphins . Decrease of cell number induced by hemorphins depend on the extent of N- and C-terminal degradation of hemorphins: VV-hemorphins in most cases are more active than LVV-, V-hemorphins, and hemorphins . In the group of VV-hemorphins the activity of VV-hemorphin-5 (valorphin) is significantly higher than of VV-hemorphin-7, VV-hemorphin-6, and VV-hemorphin-4, meaning that the presence of C-terminal Gln is important for suppressing of cell number . The amino acid sequence VVYPWTQ corresponding to valorphin was identified as important for manifestation of the both cytotoxic and antiproliferative effects. Virology, 2002 Jun 5, 297(2), 298 - 306 Mutations in NS5B polymerase of hepatitis C virus: impacts on in vitro enzymatic activity and viral RNA replication in the subgenomic replicon cell culture; Cheney IW et al.; Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) essential for virus replication . Several consensus sequence motifs have been identified in NS5B, some of which have been shown to be critical for its enzymatic activity . A unique beta-hairpin structure located between amino acids 443 and 454 in the thumb subdomain has also been shown to play an important role in ensuring terminal initiation of RNA synthesis in vitro . However, the importance of these sequence and structural elements in viral RNA replication in infected cells has not been established, mainly due to the lack of a reliable cell culture system for HCV . In this study, we investigated the effect of several single amino acid substitutions and beta-hairpin truncations in NS5B on viral RNA replication by using the subgenomic replicon cell culture system . A strong correlation between in vitro polymerase activity and viral RNA replication was observed with most of the substitutions . Interestingly, truncations of the beta-hairpin (by four and eight amino acid residues, respectively), which did not reduce the in vitro enzymatic activity, completely abolished the ability of the replicon RNA to replicate in Huh-7 cells, demonstrating its essential role in viral RNA replication . Furthermore, a conservative substitution in motif D, from an arginine residue (AMTR(345)), which is conserved among all HCV isolates, to a lysine residue, resulted in significant improvements in both transient RNA replication and colony formation efficiencies . This result also correlates with a previous observation that the enzymatic activity of NS5B increased by about 50% when the same NS5B substitution was introduced (V . Lohmann, F . Korner, U . Herian, and R . Bartenschlager, J . Virol . 1997, 71, 8416-8428). J Periodontol, 2002 Jun, 73(6), 585 - 90 The modulation of androgen metabolism by estradiol, minocycline, and indomethacin in a cell culture model; Tilakaratne A et al.; BACKGROUND: This investigation attempts to clarify the proanabolic effects of minocycline and indomethacin by studying their effects on androgen metabolism and mediation by estradiol . A cell culture model was used with androgen substrates because of the proanabolic effects of androgen metabolites . METHODS: Monolayer cultures of human gingival fibroblasts (HGF) derived from 6 patients were incubated in duplicate with 14C- testosterone or 14C-4-androstenedione as substrates and optimal concentrations of estradiol (E1,3 microgram/ml) and minocycline (M25 microgram/ml) or indomethacin (I, 1 microgram/ml) alone and in combination (E1,3+11 or E1,3+M25 microgram/ml); similar experiments were carried out with human oral periosteal fibroblasts (HPF), M, I, E, and the combinations . At the end of a 24-hour incubation period in Eagle's MEM, the medium was solvent extracted with ethyl acetate and the metabolites were separated by TLC in a benzene:acetone solvent system (4:1 v/v) . The separated metabolites were quantified using a radioisotope scanner . RESULTS: Both androgens were metabolized to 5alpha-dihydrotestosterone (DHT) and 4-androstenedione (4-A) or testosterone (T) at baseline and in response to the agents tested, by HGF and HPF . With HGF, there were significant increases in the yields of DHT and 4-A or T in response to M, E, and M+E, resulting in 50% to 2.4-fold increases in these metabolites over control incubations (n = 6; P<0.01) . The responses to I and combinations of I+E were similar . HPF also demonstrated significant increases of 29% to 4-fold in the yields of androgen metabolites in response to M, E, and M+E (n = 6; P<0.01) . I and E similarly increased the yields of androgen metabolites, alone and in combination . CONCLUSIONS: Adjunctive periodontal treatment with minocycline or indomethacin can contribute to hormone-modulated anabolic responses in males and females in gingival and periosteal fibroblasts derived from a chronically inflamed source. Cell Biol Toxicol, 2002, 18(3), 175 - 80 Suppression of sulfur mustard-increased IL-8 in human keratinocyte cell cultures by serine protease inhibitors: implications for toxicity and medical countermeasures; Cowan FM et al.; The toxicity of the chemical warfare blistering agent sulfur mustard (2,2'-dichlorodiethyl sulfide; SM) has been investigated for nearly a century; however, the toxicological mechanisms of SM remain obscure and no antidote exists . The similarity of dermal-epidermal separation caused by SM exposure, proteolysis, and certain bullous diseases has fostered the hypothesis that SM vesication involves proteolysis and/or inflammation . Compound screening conducted by the US Army Medical Research Institute of Chemical Defense established that topical application of three tested serine protease inhibitors could reduce SM toxicity in the mouse ear vesicant model . Although most of the drugs with efficacy for SM toxicity in rodent models are anti-inflammatory compounds, no in vitro assay is in current use for screening of potential anti-inflammatory SM antidotes . IL-8 is a potent neutrophil chemotactic cytokine that is increased in human epidermal keratinocyte (HEK) cell cultures following exposure to SM and has been proposed as a marker for SM-induced inflammation . This study was conducted to establish in vitro screening of IL-8 in SM-exposed HEK as a possible model for evaluating candidate compounds prior to in vivo testing . We chose two protease inhibitors, one from those shown as successful in the MEVM (ethyl p-guanidinobenzoate hydrochloride, ICD 1579) and a prototypic inhibitor of trypsin, N-tosyl-L-lysine chloromethyl ketone (TLCK) . TLCK (62.5 to 1000 micromol/L) or ICD 1579 (31.25 to 1000 micromol/L) was added to HEK cell cultures 1 h after SM exposure (200 micromol/L) and dose-dependently suppressed SM-increased IL-8 . The suppression of SM-increased IL-8 by a class of drug candidate compounds such as protease inhibitors may provide a mechanistic marker that helps predict future medical countermeasures for SM toxicity and reduces the need for testing in animal models. Menopause, 2002 Jul-Aug, 9(4), 273 - 81 Medroxyprogesterone acetate versus norethisterone: effect on estradiol-induced changes of markers for endothelial function and atherosclerotic plaque characteristics in human female coronary endothelial cell cultures; Mueck AO et al.; OBJECTIVE: Progestin addition to estradiol (E(2)) replacement therapy may lead to a deterioration of beneficial effects on the vasculature . The effect of the two clinically most common progestins, medroxyprogesterone acetate (MPA) and norethisterone (NET), during continuous combination with E(2) on the synthesis of markers for coronary endothelial function, atherosclerotic plaque initiation, and plaque formation was investigated in human female vascular cell cultures and compared with that of E(2) alone . DESIGN: Endothelial cell cultures from human female coronary arteries were used to evaluate the effect of progestin addition to E(2) on the production of the following endothelial markers: prostacyclin, endothelin, plasminogen activator inhibitor-1, E-selectin, intercellular adhesion molecule-1, monocyte chemoattractant protein-1 (MCP-1), and the precursor of matrix metalloproteinase-1 (pro-MMP-1) . E(2) was tested at 0.1 microM, 1 microM, and 10 microM alone and in equimolar combinations with MPA or NET . The markers were determined by enzyme immunoassays in the cell supernatant . RESULTS: E(2) induced a significant increase of endothelial prostacyclin production and was able to significantly decrease the synthesis of endothelin, plasminogen activator inhibitor-1, E-selectin, and intercellular adhesion molecule-1 . Neither MPA nor NET addition negatively interfered with these E(2)-induced benefits . However, MPA antagonized the E(2)-induced significant reduction of MCP-1 synthesis, with the difference between both progestins being significant (p < 0.01) . Interestingly, an enhancement of the positive E(2)-effect on pro-MMP-1 production was observed by the addition of both MPA and NET (p < 0.01) . CONCLUSION: E(2) can positively influence various markers of endothelial function . Addition of MPA or NET can elicit different effects, which has been demonstrated for the first time in human coronary cell cultures . No impact was found on markers representing primarily vasotonus and thrombogenicity . In terms of MMP-1, which is crucial for atherosclerotic plaque stability, an enhancement of the beneficial E(2) effect was observed . However, regarding MCP-1, contrary effects of progestins cannot be excluded . This indicates that progestins may differ in their effects, particularly in the early stages of atherosclerosis, which has also been supported by other studies. Cell Physiol Biochem, 2002, 12(2-3), 153 - 62 LLC-PK(1) cells maintained in a new perfusion cell culture system exhibit an improved oxidative metabolism; Felder E et al.; Cultured renal proximal tubule cells dedifferentiate from an oxidative metabolism to high rates of glycolysis over time . There are many reasons why cells in culture dedifferentiate, not least being a lack of homogenous nutrient supply and poor oxygenation . To this end we have developed a new cell culture device (EpiFlow), which combines continuous perfusion of medium with continuous oxygenation of cells grown on microporous supports . LLC-PK(1) cells cultured under EpiFlow conditions were compared with the same cells grown under conventional static conditions . EpiFlow maintained cells exhibited an improved oxidative metabolism as evidenced by 1) a decreased activity of glycolytic enzymes, 2) an increase in the activity of mitochondrial phosphate-dependent-glutaminase, 3) an increase in cellular ATP content, and 4) an improved morphology (increased cell height, mitochondrial density and an increased number and height of microvilli) . In addition, LLC-PK(1) cells maintained under perfusion conditions exhibited an increased sensitivity to the respiratory chain blocker antimycin A as assayed by mitochondrial membrane potential (JC-1) . We conclude that LLC-PK(1) cells maintained under EpiFlow conditions develop an improved oxidative metabolism that is more comparable to the in vivo situation . Proc Natl Acad Sci U S A, 2002 Jun 25, 99(13), 8660 - 5 Epub 2002 Jun 19. Dome formation in cell cultures as expression of an early stage of lactogenic differentiation of the mammary gland; Zucchi I et al.; The study of the development of the mammary gland at the molecular level in the animal is difficult because of the complex tissue organization of the gland . We have previously developed an in vitro system for genetic analysis of mammary cell differentiation, based on the cell line LA7 clonally derived from a rat mammary adenocarcinoma . This cell line, after induction with DMSO, differentiates forming structures called domes . This process is under strict gene regulation, and we have previously identified several of the genes involved . In the present paper, we have defined the meaning of dome formation in relation to mammary development, by showing that treatment of LA7 cells with the lactogenic hormones hydrocortisone and prolactin induces dome formation; in the animal, these hormones precede and accompany milk production . Moreover, dome formation is accompanied by expression within the cells of the milk protein genes WDMN1 and beta-casein, which are differentiation markers for the gland during pregnancy and lactation . We also show that two proteins, highly expressed in the mammary gland during lactation, HSP90-beta and annexin I, are strongly expressed in DMSO-induced LA7 cells . Both proteins are essential in the formation of domes because when their synthesis is blocked by antisense RNA oligonucleotides, dome formation is abolished . Thus our in vitro system is a model for lobulo-alveolar development, and the genes identified in the pathway of dome formation are likely to be involved in the early differentiation steps occurring in the rat mammary gland during pregnancy and lactation. Brain, 2002 Jul, 125(Pt 7), 1522 - 33 Mitochondrial dysfunction in a cell culture model of familial amyotrophic lateral sclerosis; Menzies FM et al.; The molecular mechanisms by which mutations in the gene for Cu/Zn superoxide dismutase (SOD1) lead to the selective death of motor neurones in familial amyotrophic lateral sclerosis (FALS) remain incompletely understood . Previous evidence has indicated that mitochondrial abnormalities may develop during motor neurone injury, but several important questions remain unanswered . We have developed a cell culture model of FALS in which a motor neurone cell line (NSC34) has been stably transfected to express normal or mutant human SOD1 at levels approximating to those seen in the human disease . The aims of the study were to: (i) investigate whether morphological mitochondrial abnormalities occur at expression levels of mutant SOD1 close to physiological levels; and (ii) determine whether the presence of mutant SOD1 causes abnormalities of mitochondrial respiratory chain function and changes in cellular bioenergetic parameters in motor neuronal cells . Using this cellular model, we demonstrate that the presence of mutant SOD1 results in the development of abnormally swollen and pale staining mitochondria . These morphological changes are accompanied by biochemical abnormalities with specific decreases in the activities of complexes II and IV of the mitochondrial electron transfer chain . These same complexes are inhibited when control NSC34 cells are subjected to oxidative stress induced by serum withdrawal . The decrease in respiratory chain complex activity in the presence of mutant SOD1 was not accompanied by decreased expression of representative proteins present in these complexes . Motor neuronal cells expressing mutant SOD1 showed increased cell death when exposed to oxidative stress by serum withdrawal, whereas the presence of normal human SOD1 exerted a protective effect . Under basal, unstressed culture conditions, no change in the ATP : ADP ratio was observed in the presence of mutant SOD1 . However, the mitochondrial changes associated with the presence of mutant SOD1 clearly had adverse cellular bioenergetic consequences as shown by increased cell death in the presence of pharmacological inhibition of the glycolytic pathway . We conclude that one important mechanism by which mutant SOD1 causes motor neurone injury involves inhibition of specific components of the mitochondrial electron transfer chain . Therapeutic measures aimed at protecting mitochondrial respiratory chain function may be useful in SOD1 related familial and possibly other forms of amyotrophic lateral sclerosis. Int J Parasitol, 2002 Jul, 32(8), 1053 - 64 Establishment of Besnoitia darlingi from opossums (Didelphis virginiana) in experimental intermediate and definitive hosts, propagation in cell culture, and description of ultrastructural and genetic characteristics; Dubey JP et al.; Besnoitia darlingi from naturally infected opossums (Didelphis virginiana) from Mississippi, USA, was propagated experimentally in mice, cats, and cell culture and was characterised according to ultrastructural, genetic, and life-history characteristics . Cats fed tissue cysts from opossums shed oocysts with a prepatent period of nine or 11 days . Oocysts, bradyzoites, or tachyzoites were infective to outbred and interferon-gamma gene knockout mice . Tachyzoites were successfully cultivated and maintained in vitro in bovine monocytes and African green monkey cells and revived after an 18-month storage in liquid nitrogen . Schizonts were seen in the small intestinal lamina propria of cats fed experimentally-infected mouse tissues . These schizonts measured up to 45 x 25 microm and contained many merozoites . A few schizonts were present in mesenteric lymph nodes and livers of cats fed tissue cysts . Ultrastructurally, tachyzoites and bradyzoites of B . darlingi were similar to other species of Besnoitia . A close relationship to B . besnoiti and an even closer relationship to B . jellisoni was indicated for B . darlingi on the basis of the small subunit and ITS-1 portions of nuclear ribosomal DNA. Differentiation, 2002 May, 70(2-3), 77 - 83 Mucociliary differentiation according to time in human nasal epithelial cell culture; Yoon JH et al.; Knowledge of the state of differentiation, cell phenotype, and expression of genes for mucus production at the time of study is important because these may vary at different times during the culture period . The primary purpose of this study was to determine whether the number of ciliated cells increases as a function of differentiation in NHNE cells . If we observed an increase in the number of ciliated cells, the composition ratio of ciliated and secretory cells according to the culture duration was determined . The levels of mucin and lysozyme secretion and their gene expression at this time were also examined . The presence of ciliated cells was not evident up to 2 days after confluence . However, 3.1 +/- 0.2 %, 7.4 +/- 0.5 %, and 14.5 +/- 0.6 % of the cells were ciliated on the 7th, the 14th, and the 28th day after confluence, respectively . Meanwhile, the percentage of secretory cells were 35.6 +/- 2.8 %, 32.8 +/- 2.5 %, 32.8 +/- 2.5 %, and 49.4 +/- 1.4 % on the 2nd, the 7th, 14th, and 28th day after confluence . The amount of secreted mucin showed an abruptly increasing pattern by the 14th day after confluence but showed no significant changes thereafter . The amount of secreted lysozyme increased as a function of differentiation . MUC5AC and MUC5B mRNA were mainly expressed between the 7th and the 14th day after confluence with relatively weak MUC8 and lysozyme expression . By the 28th day after confluence however, as the MUC5AC mRNA expression became weaker, MUC5B, MUC8, and lysozyme mRNA expression became stronger . In conclusion, we speculate that in in vitro studies with NHNE cells, the time point of treatment should vary according to the purpose of the study . In addition, the MUC5B and MUC8 gene may play an important role in mucin secretion in fully differentiated human nasal epithelial cells. APMIS, 2002 May, 110(5), 410 - 4 Synergistic effects of 1,25-Dihydroxyvitamin D3 and TGF-beta1 on the production of insulin-like growth factor binding protein 3 in human bone marrow stromal cell cultures; Kveiborg M et al.; 1,25-Dihydroxyvitamin D3 (calcitriol), transforming growth factor-beta (TGF-beta), and insulin-like growth factors (IGFs) are all important bone regulatory factors known to affect proliferation and differentiation of human bone-forming cells (osteoblasts) . We have previously shown that TGF-beta1 increased IGF-I and IGF-binding protein (IGFBP)-3 production in human bone marrow stromal (hMS) osteoblast progenitors and calcitriol stimulated IGFBP-3 and IGFBP-4 production . As interaction between signaling pathways of these factors has been reported, the present study aimed at examining the concerted actions on components of the IGF-system . We report that co-treatment with TGF-beta1 and calcitriol resulted in a synergistic increase in IGFBP-3 production, thereby suggesting that the effects of these factors on hMS osteoblast differentiation may involve the observed increase in IGFBP-3. Biotechnol Appl Biochem, 2002 Jun, 35(Pt 3), 171 - 80 Effect of medium properties and additives on antibody stability and accumulation in suspended plant cell cultures; Tsoi BM et al.; Factors affecting antibody accumulation and stability were investigated in transgenic plant cell cultures . Whereas IgG(1) antibody was stably maintained in media used for animal cell culture, there was a rapid loss over a period of 1-2 h of antibody added to sterile plant culture media . Antibody stability in Gamborg's B5 medium was improved in the absence of Mn . Tobacco suspensions producing IgG(1) antibody were used to test various medium-based strategies for improving antibody accumulation in plant culture . Even though growth was suppressed, antibody levels in the biomass and medium were increased in media containing mannitol at osmolalities up to approximately 450 mOsm.(kg of water)(-1) . Adding gibberellic acid and haemin to the cultures was also beneficial, but the effects were not as great as those obtained under hyperosmolar conditions . Moderate increases in antibody accumulation were found by culturing plant cells in B5 medium without Mn. J Biomech Eng, 2002 Jun, 124(3), 308 - 14 Effects of cyclic pressure on bone marrow cell cultures; Nagatomi J et al.; The present in-vitro study used bone marrow cell cultures and investigated the effects of cyclic pressure on osteoclastic bone resorption . Compared to control (cells maintained under static conditions), the number of tartrate resistant acid phosphatase (TRAP)-positive, osteoclastic cells was significantly (p<0.05) lower when, immediately upon harvesting, bone marrow cells were exposed to cyclic pressure (10-40 kPa at 1.0 Hz) . In contrast, once precursors in bone marrow cells differentiated into osteoclastic cells under static culture conditions for 7 days, subsequent exposure to the cyclic pressure of interest to the present study did not affect the number of osteoclastic cells . Most important, exposure of bone marrow cells to cyclic pressure for 1 h daily for 7 consecutive days resulted in significantly (p<0.05) lower osteoclastic bone resorption and in lowered mRNA expression for interleukin-1 (IL-1) and tumor necrosisfactor-a (TNF-a), cytokines that are known activators of osteoclast function . In addition to unique contributions to osteoclast physiology, the present study provided new evidence of a correlation between mechanical loading and bone homeostasis as well as insight into the molecular mechanisms of bone adaptation to mechanical loading, namely cytokine-mediated control of osteoclast functions. Glycobiology, 2002 May, 12(5), 353 - 60 Analyses of dolichol pyrophosphate-linked oligosaccharides in cell cultures and tissues by fluorophore-assisted carbohydrate electrophoresis; Gao N et al.; Lipid-linked oligosaccharides (LLOs) are the precursors of asparagine (N)-linked glycans, which are essential information carriers in many biological systems, and defects in LLO synthesis cause Type I congenital disorders of glycosylation . Due to the low abundance of LLOs and the limitations of the chemical and physical methods previously used to detect them, simple and sensitive nonradioactive methods for LLO analysis are lacking . Thus, almost all studies of LLO synthesis have relied on metabolic labeling of the oligosaccharides with radioactive sugar precursors . We report that LLOs in cell cultures and tissues can be easily detected and quantified with a sensitivity of 1-2 pmol by fluorophore-assisted carbohydrate electrophoresis (FACE) . These analyses required efficient removal of contaminants, most likely trace quantities of glycogen breakdown products, that interfered with FACE . Studies with CHO-K1 cells showed that LLOs detected by FACE and by metabolic labeling had similar turnover rates . Glc(3)Man(9)GlcNAc(2)-P-P-dolichol was the most prominent LLO detected by FACE in normal cultured cells and mouse tissues . However, the relative amounts of Glc(0-2)Man(5-9)GlcNAc(2)-P-P-dolichol intermediates in tissues, such as liver and kidney, were unexpectedly greater than for cultured cells . IV injection of D-mannose, raising the circulatory concentration by three- to fourfold, did not affect LLO composition . Thus, the relative accumulation of LLO intermediates in mouse liver and kidney is not likely due to inadequate D-mannose in the circulation . In summary, FACE is a facile, accurate, and sensitive method for LLO analysis, permitting investigations not feasible by metabolic labeling. BMC Microbiol . 2002 Jun 09;2(1):12. Detection and subtyping of Herpes simplex virus in clinical samples by LightCycler PCR, enzyme immunoassay and cell culture; Burrows J et al.; BACKGROUND: Prompt laboratory diagnosis of Herpes simplex virus (HSV) infection facilitates patient management and possible initiation of antiviral therapy . In our laboratory, which receives various specimen types for detection of HSV, we use enzyme immunoassay (EIA) for rapid detection and culture of this virus . The culture of HSV has traditionally been accepted as the diagnostic 'gold standard' . In this study, we compared the use of real time PCR (LightCycler) for amplification, detection and subtyping of specific DNA with our in-house developed rapid and culture tests for HSV . RESULTS: The LightCycler PCR (LC-PCR) detected and subtyped HSV in 99% (66/67) of HSV positive specimens, compared to 81% (54/67) by rapid antigen EIA or 57% (36/63) by culture . A specimen was considered positive when two or more tests yielded HSV identifications or was culture positive . Discordant results were confirmed with an in-house developed PCR-ELISA or DNA sequence analysis . The typing results obtained with the LC-PCR and by culture amplified test were completely concordant . CONCLUSIONS: This study showed that the LC-PCR provided a highly sensitive test for simultaneous detection and subtyping of HSV in a single reaction tube . In addition to increased sensitivity, the LightCycler PCR provided reduced turn-around-times (2 hours) when compared to enzyme immunoassay (4 hours) or culture (4 days). J Cell Mol Med, 2001 Jan-Mar, 5(1), 60 - 73 Tri-dimensional prostate cell cultures in simulated microgravity and induced changes in lipid second messengers and signal transduction; Clejan S et al.; The high aspect rotating-wall vessel (HARV) was designed to cultivate cells in an environment that simulate microgravity . We studied previously the effects of HARV cultivation on DU-145 human prostate carcinoma cells . We determined that HARV cultivation produced a less aggressive, slower growing, less proliferative, more differentiated and less pliant cell than other cell cultivation methods . The result was a 3-dimensional (3D) growth model of prostate cancer which mimics in vivo tissue growth . This work examines the signal transduction-second messenger pathways existing temporarily in these HARV cells and correlates these features with the special properties in growth and 3D spheroid formation . We found an initial very active ceramide, a diacylglycerol increase together with increases in PI-PLC and PLA(2) a central defect in PLD (no phosphatic acid or phosphatidylethanol at any time during 15 days of HARV cultivation) . There is a cross-talk between ceramide and PI3K pathways with activation of PI3K, after 6 days of HARV growth concomitant with down-regulation of ceramide . At this time, there is also an increase of cAMP (seen by increases in arachidonic acid) . Taken together these results can explain the 3D organoid-like growth . We therefore developed a model for growth in HARV prostate cancer cells which involve temporal "switches" between second messengers, activation and cross-talk between multiplicity of signaling pathways and a central defect in PLD pathways . Essential to the late slow growth, and 3D organotypic formation are the apoptotic, anti-survival, anti-proliferation and differentiation pathways in the first days of HARV, with growth of "new" different types of prostate cancer cells which set-up for later "switch" in ceramide-PI3K to survival and proliferation. J Neurochem, 2002 May, 81(3), 414 - 21 The cytotoxicity of dopamine may be an artefact of cell culture; Clement MV et al.; Administration of L-DOPA is commonly used to treat Parkinson's disease, yet controversy continues as to whether the dopamine arising from it aggravates neuronal loss . Several authors have reported cytotoxic effects of L-DOPA and dopamine on cultured cells, but others have not . In this report using the rat pheochromocytoma cell line PC12 and the M14 human melanoma cell line we show that dopamine-mediated cell death is not specific for neuronal cells . Moreover, our data show that both L-DOPA and dopamine interact with commonly used cell culture media, undergoing oxidation to generate hydrogen peroxide and dopamine semiquinones/quinones . Catalase and reduced glutathione could protect against cytotoxicity . These results suggest that caution needs to be employed when using cell culture studies to predict effects of L-DOPA and/or dopamine in vivo because of the extracellular generation of reactive species in the culture media. Cell Mol Biol (Noisy-le-grand), 2002 Jun, 48(4), 351 - 8 Cell culture of bivalves: tool for the study of the effects of environmental stressors; Pennec JP et al.; Spontaneous beating cells can be isolated from the heart of the oyster (Crassostrea gigas) and cultured for more than two months . They form adherent contractile networks in culture conditions . They show muscarinic and beta-adrenergic reactivity thus showing that they are functional cardio-myocytes: Acetylcholine induced a dose dependent decrease in spontaneous beating rate via an increase in potassium conductance, this effect being blocked by atropine . Epinephrine induced a dramatic increase in calcium conductance which was blocked by high concentrations of propranolol but not by sotalol and reversed by verapamil . Tributyltin and cadmium induced a dose and time dependent decrease mainly in inward ionic conductances, leading to a decrease or even a total suppression of the beating rate . Present study indicates that this model could be used as a sensitive test to study the effects of some marine pollutants at the cellular level in molluscs. Cryobiology, 2002 Feb, 44(1), 38 - 45 Cryopreservation of mantle dissociated cells from Haliotis tuberculata (Gastropoda) and postthawed primary cell cultures; Poncet JM et al.; Dissociated mantle cells from the gastropod mollusc Haliotis tuberculata were cultured after a freezing-thawing procedure using either 10% dimethyl sulfoxide (Me(2)SO) or 10% glycerol (Gly) as a cryoprotector . The survival rate of 2-day-old cultured cryopreserved cells after thawing, based on analysis of DNA and protein contents, was nearly 80% in comparison with 2-day-old cultured fresh cells . Cells thawed after cryopreservation exhibited the maintenance of all tested physiological activities . Metabolic activity (measured by the MTT test) and the activity of alkaline phosphatase (a plasma membrane-bound enzyme) were not decreased in comparison to those in cultured fresh cells . In addition, cryopreserved cultured cells maintained a physiological stimulation ability in response to treatment with growth factors . These results taken together represent one of the most convincing demonstrations of the survival and of the recovery of intact functional activities of molluscan cells after a freeze-thawing procedure . Our results suggest that in the future primary cultures of cryopreserved mantle cells will be able to be used for fundamental research, in toxicity tests, or in the field of biotechnology . (c) 2002 Elsevier Science (USA). Arch Biochem Biophys, 2002 Jul 1, 403(1), 111 - 20 Phosphoinositide 3-kinase mediates protein kinase C beta II mRNA destabilization in rat A10 smooth muscle cell cultures exposed to high glucose; Patel NA et al.; High-glucose exposure down-regulates protein kinaseC beta II posttranscriptionally in rat and human vascular smooth muscle cells and contributes to increased cell proliferation . High-glucose-induced mRNA destabilization is specific for PKC beta II mRNA, while PKC beta I and other PKC mRNA are not affected . This study focused on whether glucose metabolism was required . The effect was blocked by cytochalasin B, suggesting a requirement for glucose uptake . Glucosamine did not mimic the effect, indicating that metabolism via hexosamine pathway was not involved . The effect was hexokinase-independent since 3-O-methylglucose, in a dose-dependent manner, mimicked high-glucose effects . Cycloheximide did not block the effect excluding dependency on new protein synthesis . Wortmannin and LY294002, phosphoinositide 3-kinase (PI3-kinase) inhibitors, blocked glucose effects in the presence of 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole . Glucose and 3-O-methylglucose activated PI3-kinase, and LY294002 blocked glucose effects on Akt phosphorylation . In these cells, high-glucose concentrations activated a metabolically linked signaling pathway independent of glucose metabolism to regulate mRNA processing. Biotechnol Prog, 2002 May-Jun, 18(3), 617 - 22 Production of retrovirus and adenovirus vectors for gene therapy: a comparative study using microcarrier and stationary cell culture; Wu SC et al.; In gene therapy, retrovirus and adenovirus vectors are extensively used as gene-delivery vehicles and further large-scale processing of these viral vectors will be increasingly important . This study examined stationary and microcarrier cell culture systems with respect to the production of a retrovirus vector (encoding a monounit hammerhead ribozyme gene with an intron) and an adenovirus vector (encoding a reporter lacZ gene) . Cytodex 1 and Cytodex 3 solid microcarriers were found to be able to provide good cell growth and high-titer vector production in suspension cultures . Porous microcarriers such as Cytopore 2 gave slightly lower but still efficient growth but produced significantly lower titers of retrovirus and adenovirus vector from the producer cells . The specific retrovirus production was not proportionally related to the specific growth rate of the producer cells . High MOI infection was essential for high-titer production of adenovirus vector in 293 cells . Hydrodynamic shear forces on microcarrier-grown cells increased the production yield for retrovirus vector but decreased for adenovirus vector . The cellular productivity was much more efficient for adenovirus vector produced in 293 cells as compared to the retrovirus vector produced in PA317-RCM1 cells . These findings can provide further insight into the feasibility of applying microcarrier cell culture technology to produce gene-therapy virus vectors. Cytotherapy, 2000, 2(4), 267 - 80 Cell density-dependent proliferation in frequently-fed peripheral blood mononuclear cell cultures; Patel SD et al.; BACKGROUND: Our goal was to produce granulocyte progenitor (CFU-G) and post-progenitor (CD15(+)CD11b(+/-)) cells for subsequent transplantation . We hypothesized that increasing the feeding frequency and maintaining constant densities may overcome inhibitory growth conditions (i.e . low pH) in high-density cultures . METHODS: To study the effect of cell density on total cell expansion, differentiation and lactate production, 50% daily medium exchanges were used in cultures of peripheral blood mononuclear cells (PB MNC) maintained at constant densities (ranging from 5 x 10(4)cells/mL to 2.5 x 10(6)cells/mL) . RESULTS: We observed a significant increase in total cell expansion when the density was increased from 5 x 10(4) cells/mL to 1 x 10(6) cells/mL, but a further increase to 2.5 x 10(6)cells/mL resulted in a decline in cell expansion . Increasing feeding to 90% daily exchange in cultures with 2.5 x 10(6) cells/mL did not enhance cell expansion; nor did reducing the extent of feeding in cultures with 5 x 10(4) cells/mL to 10% daily exchange . We did not observe a relationship between cell density and the percentage of granulocyte progenitor and post-progenitor (CD15(+)CD11b(-/+)) cells . While specific lactate production (q(lac)) in cultures with 2.5 x 10(6) cells/mL was approximately 60% of those observed in lower density cultures by Day 13, this difference was largely eliminated by increasing the extent of feeding in cultures with 2.5 x 10(6) cells/mL . DISCUSSION: Our results suggest that feeding rates must be adjusted according to cell density to maximize culture performance . They also suggest that cellular crowding on the culture surface can limit expansion in suspension (nonadherent) cultures. Ukr Biokhim Zh, 2001 Sep-Oct, 73(5), 108 - 13 {Induction of active forms of oxygen by biotic elicitors in tomato cell cultures}; Perkovskaia GIu et al.; The elicitor-induced generation of two oxygen species in tomato cell culture as well as their involvement into hypersensitive reaction was investigated . Generation of superoxide O2.- was measured by a lucigenin-related chemiluminescence . Accumulation of hydrogen peroxide H2O2 was measured by a fluorescent probe pyranin . Xylanase and chitosan were used as biotic elicitors with different mode of action . It was found that both O2.- and H2O2 had been accumulated in elicitor-treated tomato cells . The results obtained show that reactive oxidants are important signal transduction elements for activation of hypersensitive response in tomato cells. J Agric Food Chem, 2002 Jun 5, 50(12), 3586 - 91 Comparison of iron bioavailability from 15 rice genotypes: studies using an in vitro digestion/caco-2 cell culture model; Glahn RP et al.; An in vitro digestion/Caco-2 model was used to compare iron bioavailability from 15 selected Fe-dense and normal genotypes of unpolished rice from the International Rice Research Institute . Iron uptake was determined using Caco-2 cell ferritin formation in response to exposure to a digest of the cooked rice . Iron bioavailabilities from all rice genotypes were ranked as a percent relative to a control variety (Nishiki) . Iron concentration in the rice samples ranged from 14 to 39 microg/g . No correlation was observed between Fe uptake and grain-Fe concentration . Furthermore, phytic acid levels were not correlated with Fe bioavailability . Genotypes with low Fe bioavailability (Tong Lan Mo Mi, Zuchein, Heibao, and Xua Bue Nuo) were noticeably more brown to purple in color . The results suggest that certain unknown compounds related to rice grain color may be a major factor limiting Fe bioavailability from unpolished rice. Anal Chem, 2002 Apr 1, 74(7), 1695 - 701 Automated in-tube solid-phase microextraction coupled with HPLC for the determination of N-nitrosamines in cell cultures; Mullett WM et al.; An automated in-tube solid-phase microextraction (SPME) HPLC analysis method for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and several metabolites has been developed . NNK is one of the tobacco-specific N-nitrosamines (TSNA), which has been linked to cancers associated with the use of or exposure to tobacco products . In-tube SPME is an on-line extraction technique in which analytes are extracted and concentrated from the sample directly into a coated capillary by repeated draw/eject steps . In this study, a tailor-made polypyrrole (PPY)-coated capillary and several commercially available capillaries (capillary GC columns) were used to evaluate their extraction efficiencies for NNK and several metabolites in cell cultures . Compared with commercial capillaries that were currently used for in-tube SPME, the PPY-coated capillary showed better extraction efficiency for all of the compounds studied . After optimization of the extraction conditions, NNK and five metabolite compounds were analyzed in spiked cell cultures, confirming the applicability of the developed method . Excellent linearity was observed for all compounds (av R2 = 0.9942) and detection limits that ranged from 20 to 250 ng/mL . The average within-day and between day variations (% RSD) were 2.9 and 3.6%, respectively . This automated extraction and analysis method simplified the determination of the TSNA, requiring a total sample analysis time of only approximately 30 min. Gene Ther, 2002 Jun, 9(11), 700 - 2 Optimising gene repair strategies in cell culture; Thorpe P et al.; Gene repair, the precise modification of the genome, offers a number of advantages over replacement gene therapy . In practice, gene targeting strategies are limited by the inefficiency of homologous recombination in mammalian cells . A number of strategies, including RNA-DNA oligonucleotides (RDOs) and short DNA fragments (SDFs), show promise in improving the efficiency of gene correction . We are using GFP as a reporter for gene repair in living cells . A single base substitution was introduced into GFP to create a nonsense mutation (STOP codon, W399X) . RDOs and SDFs are used to repair this mutation episomally in transient transfections and restore green fluorescence . The correction efficiency is determined by FACS analysis . SDFs appear to correct GFP W399X in a number of different cell lines (COS7, A549, HT1080, HuH-7), although all at a similar low frequency ( approximately 0.6% of transfected cells) . RDOs correct only one of our cell lines significantly (HT1080-RAD51), these cells overexpress the human RAD51 gene; the bacterial RecA homologue . The GFP W399X reporter is a fusion gene with hygromycin (at the 5' end), this has allowed us to make stable cell lines (A549, HT1080) to study genomic correction . Initial studies using our correction molecules show only low efficiencies of genomic repair ( approximately 10(-4)) . Polyethylenimine (PEI) is used to deliver RDOs and SDFs into mammalian cells in culture for our study . We have used fluorescently labelled RDOs and SDFs to study the effectiveness of this process . FACS analysis of transfected nuclei implied efficient delivery (>90%) both with SDFs and RDOs . However, confocal fluorescence microscopy suggests that a large proportion of the complexed RDO/SDF appears to remain outside the nucleus (or attached to the nuclear membrane) . On the basis of these data we are assessing new delivery methods and factors that may alter recombination status to optimise gene repair. Biochim Biophys Acta, 2002 May 8, 1589(3), 261 - 72 An acidic fibroblast growth factor-like factor secreted into the brain cell culture medium upregulates apoE synthesis, HDL secretion and cholesterol metabolism in rat astrocytes; Ueno S et al.; Production and release of apolipoprotein (apo) E and cholesterol were highly upregulated in the astrocytes prepared by 1-week secondary culture after 1-month primary culture of rat fetal brain cells (M/W cells) in comparison to the cells prepared by a conventional method of 1-week primary and 1-week secondary culture (W/W cells) . Both cell preparations were mostly composed of astrocytes with small population of other glial cells, except that type-2 astrocyte-like cells accounted for 5-15% of M/W cells indicating more activated and/or matured status . The conditioned medium of the 1-month primary culture stimulated W/W cells to increase the release of apoE and cholesterol into the medium . The treatment of W/W cells by acidic fibroblast growth factor (aFGF) similarly upregulated biosyntheses and release of apoE and cholesterol . The effect of the conditioned medium was completely inhibited by pretreatment with an anti-aFGF antibody . The increase of the aFGF message was demonstrated in the brain cells after 1-month primary culture . The findings suggested that an aFGF-like trophic factor upregulates biosynthesis and secretion of apoE-high density lipoprotein (HDL) in astrocytes probably by autocrine stimulation in this culture system . Since this cytokine is highly expressed in the development or post-injury period of the brain, it putatively activates intercellular cholesterol transport to support construction or recovery of the brain. Acta Pol Pharm, 2002 Jan-Feb, 59(1), 31 - 5 Influence of vitamins C and E on cytotoxic activity of adriamycin in chosen cell cultures; Wozniak G et al.; Influence of different concentrations of ascorbic acid (vitamin C) and dl-alpha-tocopherol acetate (vitamin E) on in vitro cytotoxic adriamycin activity, in: embryonic human fibroblasts (CLV102), human melanoma cells (ME18) and adriamycin-resistant subline cells (ME18/R), was studied . IC50 value for each compound (compound concentration in the culture medium, for which 50% of cells survive) was determined . Cells' survival after the used agent was examined with trypan blue test . The relationship between different concentrations of vitamin C and toxicity of adriamycin, used at appropriate IC50 concentrations, was expressed for all the examined cells as their survival decrease, being in direct proportion to the concentration increase of this vitamin in the medium . In the case of influence of vitamin E on adriamycin cytotoxicity, the protective effect of this vitamin was observed in the concentration range: from 5 to 300 microg/ml (p < or = 0.0001), as an increase of the examined cell survival for ME18, ME18/R as well as for CLV102, comparing to the control (p=0.05) without this vitamin . Parallelly, a statistically significant survival decrease was observed, if the concentration of vitamin E in the culture medium exceeded 500 microg/ml . Received results showed different, in defined concentration range, effects of the vitamin C or vitamin E activity for adriamycin cytotoxicity . These effects were similar for all the examined cells. J Gerontol A Biol Sci Med Sci, 2002 Jun, 57(6), B239 - 46 Relationship between in vivo age and in vitro aging: assessment of 669 cell cultures derived from members of the Baltimore Longitudinal Study of Aging; Smith JR et al.; We examined the in vitro proliferative potential of 669 cell cultures established from skin biopsies of members of the Baltimore Longitudinal Study of Aging . The colony size distribution was used to estimate the proliferative life span of the cultures . A significant decline in proliferative potential with donor age was observed for female but not male donors . For both male and female donors, the proliferative potential was significantly greater for donors under the age of 30 years compared with all donors over the age of 30 years . In an attempt to reduce genetic heterogeneity, we examined the proliferative potential of cultures derived at different ages from the same donor . These studies revealed a trend (approaching statistical significance) toward low proliferative potential as donors aged . Interestingly, samples obtained from donors who had a history of skin cancer at the time of biopsy had a significantly lower doubling potential than those from donors who did not . The implications of these results for the use of cells derived from donors of different ages for aging research are discussed. Antimicrob Agents Chemother, 2002 Jun, 46(6), 2049 - 51 Effects of chemical and physical agents on viability and infectivity of Encephalitozoon intestinalis determined by cell culture and flow cytometry; Santillana-Hayat M et al.; We combined tissue culture and flow cytometry to assess the activities of various temperatures, chemicals, and disinfectants on the viability and infectivity of spores of Encephalitozoon intestinalis . Surfanios and benzalkonium chloride, disinfectants currently used in the hospital, were remarkably efficient in destroying spore viability and infectivity. J Endod, 2002 Mar, 28(3), 177 - 80 Interleukin-10 inhibits expression of interleukin-6 and -8 mRNA in human dental pulp cell cultures via nuclear factor-kappaB deactivation; Tokuda M et al.; The effects of interleukin (IL)-10 on the expression of IL-6 and IL-8 mRNA in human dental pulp cell cultures were investigated by using the Northern blot analysis . On stimulation with Prevotella intermedia lipopolysaccharide (PiLPS), IL-10 was produced in peripheral blood but was not detected in human dental pulp cell culture supernatants . IL-10 inhibited IL-8 mRNA expression, which is normally stimulated by PiLPS, IL-1alpha, and tumor necrosis factor-alpha and inhibited IL-6 mRNA expression, which is normally stimulated by IL-1alpha . In addition, IL-10 inhibited the activation of nuclear factor-kappaB, which is normally induced by PiLPS . We conclude that IL-10 inhibits expression of IL-6 and IL-8 mRNA in dental pulp cell cultures by inhibiting the activation of nuclear factor-kappaB. Rev Neurol, 2002 Feb 1-15, 34(3), 208 - 11 {Protective effects of growth hormone en cell cultures of the central nervous system}; Isla A et al.; OBJECTIVE: We examine a possible protective effect from growth hormone (GH) against radiation in cell cultures of rat embryon brain cortex . MATERIALS AND METHODS: Brain cortexes were removed from rat embryos of 17 days development and prepared for cell culture . The culture medium of half the plates was enriched with 500 ng/ml GH . All plates received a radiation dose of 3 Gy per plate and were again incubated for 24 hours . The TUNEL technique was employed to verify cell apoptosis in the irradiated plates and compare its level in plates with and without GH . CONCLUSIONS: We observed that irradiated cultures with GH had significantly less cell apoptosis than those without GH and concluded that this hormone excercised a protective effect on the cell cultures . The present study demonstrated the protective effect from GH in rat embryonary cells and suggests the need for future in vitro and in vivo studies using central nervous system cells. Endocrinology, 2002 Jun, 143(6), 2250 - 8 Paracrine regulation of FSH by follistatin in folliculostellate cell-enriched primate pituitary cell cultures; Kawakami S et al.; Primary pituitary cell cultures are an important tool for understanding pituitary hormone gene expression . In the course of study of pituitary cell cultures from nonhuman adult male primates, pituitary secretory cells were noted to be rapidly overgrown by epithelioid cells with the morphological, immunocytochemical, and proliferative characteristics of folliculostellate cells . Using competitive RT-PCR assays, follistatin mRNA levels were found to increase 4-fold as folliculostellate cells proliferated with time in culture, whereas FSH-beta mRNA and FSH secretion were suppressed . Follistatin gene expression was stimulated by activin-A and pituitary adenylate cyclase-activating polypeptide but not by {D-Trp(6)}-GnRH ethylamide . Testosterone (T) also increased follistatin mRNA levels and follistatin protein secretion . FSH-beta mRNA was stimulated by {D-Trp(6)}-GnRH ethylamide and activin but was suppressed by T . The reciprocal relationship between follistatin and FSH-beta mRNA levels as folliculostellate cells proliferate with time in culture implies a role for folliculostellate cells in the follistatin-activin system in primates . The actions of GnRH and T on follistatin and FSH-beta mRNA levels in these cultures were opposite to effects observed in pituitary cultures from rats and identify species differences in the control of FSH production that may be folliculostellate cell-related. Toxicol In Vitro, 2002 Jun, 16(3), 253 - 8 Genotoxic and anti-genotoxic properties of Calendula officinalis extracts in rat liver cell cultures treated with diethylnitrosamine; Perez-Carreon JI et al.; Calendula officinalis flower extracts are used to cure inflammatory and infectious diseases, for wound healing and even cancer with partial objective evidence of its therapeutic properties or toxic effects, many of which can be attributed to the presence of flavonols . We studied whether C . officinalis extracts induce unscheduled DNA synthesis (UDS) in rat liver cell cultures, and if these extracts can reverse diethylnitrosamine (DEN)-induced UDS . Four different flower extracts were prepared: aqueous (AE), aqueous-ethanol (AEE), ethanol (EE) and chloroform (CE) . AE and AEE were evaporated to 6.72 and 4.54 mg of solid material per ml, respectively and final ethanol concentration in AEE was 0.8% . EE and CE were dried and resuspended in dimethyl sulfoxide (DMSO) to 19.2 and 10 mg of solid material per ml . Ethanol residue of EE was 0.34% . In the UDS assay in liver cell cultures, DEN at 1.25 microM produced a maximal increase of 40% (3)H-thymidine ((3)HdTT) incorporation, and both, AE and AEE showed complete reversion of the DEN effect at around 50 ng/ml and between 0.4 to 16 ng/ml, respectively . In the absence of DEN, these two polar extracts induced UDS at concentrations of 25 microg for AE and 3.7 microg/ml for AEE to 100 microg/ml in rat liver cell cultures . Concentrations producing genotoxic damage were three orders of magnitude above concentrations that conferred total protection against the DEN effect . Thus, at the lower end, ng/ml concentrations of the two polar extracts AE and AEE conferred total protection against the DEN effect and at the higher end, g/ml concentrations produced genotoxic effects . These results justify the study of C . officinalis flower extracts to obtain products with biological activity and to define their genotoxic or chemopreventive properties. J Nutr Biochem, 2002 May, 13(5), 273 - 281 Ferulic acid antioxidant protection against hydroxyl and peroxyl radical oxidation in synaptosomal and neuronal cell culture systems in vitro: structure-activity studies; Kanski J et al.; In this study, free radical scavenging abilities of ferulic acid in relation to its structural characteristics were evaluated in solution, cultured neurons, and synaptosomal systems exposed to hydroxyl and peroxyl radicals . Cultured neuronal cells exposed to the peroxyl radical initiator AAPH die in a dose-response manner and show elevated levels of protein carbonyls . The presence of ferulic acid or similar phenolic compounds, however, greatly reduces free radical damage in neuronal cell systems without causing cell death by themselves . In addition, synaptosomal membrane systems exposed to oxidative stress by hydroxyl and peroxyl radical generators show elevated levels of oxidation as indexed by protein oxidation, lipid peroxidation, and ROS measurement . Ferulic acid greatly attenuates these changes, and its effects are far more potent than those obtained for vanillic, coumaric, and cinnamic acid treatments . Moreover, ferulic acid protects against free radical mediated changes in conformation of synaptosomal membrane proteins as monitored by EPR spin labeling techniques . The results presented in this study suggest the importance of naturally occurring antioxidants such as ferulic acid in therapeutic intervention methodology against neurodegenerative disorders such as Alzheimer's disease in which oxidative stress is implicated. Microsc Res Tech, 2002 May 15, 57(4), 212 - 6 Polygonal epithelial cells in glomerular cell culture: Podocyte or parietal epithelial origin? Yaoita E, Yoshida Y. Podocytes are unique cells with regard to morphology, their inability to proliferate in situ, and their apparent essential function for glomerular filtration and permselectivity . Upon transfer into culture conditions, polygonal epithelial cells grow out from isolated glomeruli and these cells have been postulated to represent dedifferentiated podocytes, even though they are negative for podocyte-specific markers and undergo cell division . However, it is controversial whether they originate from podocytes or parietal epithelial cells (PECs) of Bowman's capsule, because isolated glomeruli contain more or less Bowman's capsule . In this review, we shall summarize recent progress in the identification of the origin of the polygonal cells and the characterization of podocytes in culture . Chemotherapy, 2002 May, 48(2), 88 - 93 Combination treatment of influenza A virus infections in cell culture and in mice with the cyclopentane neuraminidase inhibitor RWJ-270201 and ribavirin; Smee DF et al.; Treatment of virus infections with compounds acting by different mechanisms may lead to more potent effects when these agents are used in combination . Under this premise, two known active influenza virus inhibitors, ribavirin and the novel cyclopentane influenza virus neuraminidase inhibitor (1S,2S,3R,4R)-3-{(1S)-(acetylamino)-2-ethylbutyl}-4-{(aminoiminomethyl)amino}-2-hydroxy-cyclopentanecarboxylic acid (RWJ-270201, BCX-1812) were studied . Experiments in cell culture demonstrated that RWJ-270201 plus ribavirin synergistically reduced extracellular influenza A/NWS/33 (H1N1) virus yields at low concentrations of each inhibitor . Mice were treated with ribavirin at 20 and 6.25 mg/kg/day combined with RWJ-270201 at 1, 0.32, or 0.1 mg/kg/day, or used alone . Treatments were twice daily for 5 days starting 4 h before exposure to influenza A/NWS virus . Only RWJ-270201 alone at 1 mg/kg/day significantly prevented mortality . In contrast, most drug combinations increased survival significantly compared to the placebo group . Doses of the two compounds used in combination delayed the mean day of death, and improved arterial oxygen saturation levels, as measured on day 11 of the infection . The combination of the two inhibitors produced additive to synergistic interactions in these mouse experiments with no enhancement of host toxicity . Treatment of influenza infections in the clinical setting may benefit by these two agents in combination . J Agric Food Chem, 2002 May 22, 50(11), 3156 - 60 Can apple antioxidants inhibit tumor cell proliferation? Generation of H(2)O(2) during interaction of phenolic compounds with cell culture media; Lapidot T et al.; It has recently been suggested that the ability of apple extracts to inhibit proliferation of tumor cells in vitro may be due to phenolic/flavonoid antioxidants . Our study demonstrates that this inhibition is caused indirectly by H(2)O(2) generated through interaction of the phenolics with the cell culture media . The results indicate that many previously reported effects of flavonoids and phenolic compounds on cultured cells may result from similar artifactual generation of oxidative stress . We suggest that in order to prevent such artifacts, the use of catalase and/or metmyoglobin in the presence of reducing agents should be considered as a method to decompose H(2)O(2) and prevent generation of other reactive oxygen species, which could affect cell proliferation . The use of tumor cells and "nontumor cells" in a bioassay to measure antioxidant activity, in this context, is potentially misleading and should be applied with caution. J Biomed Mater Res, 2002 Aug, 61(2), 197 - 202 Application of frog (Rana tigerina Daudin) skin collagen as a novel substrate in cell culture; Kumar KV et al.; Collagen and collagen-based materials have extensive application in biomedical devices and tissue engineering . The current paper pertains to the application of frog (Rana tigerina Daudin) skin collagen as a novel substrate in cell culture . The study deals with the behavior, morphology, and physiology of keratinocytes and fibroblasts over dry and reconstituted collagen substratum, which are the key cells involved in wound repair . The advantage of using frog skin collagen as a substratum lies in the ease with which the reconstituted gel can be formed . Further, frog skin collagen is highly hydrophilic, which may be attributed to the fact that amphibians, as the first vertebrates connecting water and land, must have evolved certain physiologic specializations . These studies also contribute to the hypothesis that part of the healing efficacy of frog skin may be due to the collagen since proliferation, migration, and differentiation of epithelial cells are prime requisites for a normal healing mechanism . J Ocul Pharmacol Ther, 2002 Apr, 18(2), 163 - 75 Comparison of an immortalized human corneal epithelial cell line and rabbit corneal epithelial cell culture in cytotoxicity testing; Huhtala A et al.; The cytotoxicity of benzalkonium chloride (BAC) and disodium edetate (EDTA) was evaluated in vitro in rabbit corneal epithelial primary cells and in the immortalized human corneal epithelial cell line SV40 . Cell injury was assessed by lactate dehydrogenase (LDH) leakage and by reduction of the tetrazolium salt WST-1 to formazan by mitochondrial metabolic activity . Cell cultures were exposed to test compounds both in serum-free and in serum-containing medium . Although WST-1 and LDH tests measured different physiological endpoints, they yielded comparable results . However, the LDH test seemed less reliable due to great variation . The use of serum was found to result in lower toxicity of the compounds in both tests . The rabbit primary cell culture and the human corneal cell line were quite similar in their responses to BAC and EDTA . The human cell line is a promising in vitro alternative in oculotoxicity testing. Biotechnol Bioeng, 2002 Jun 30, 78(7), 806 - 14 Controlled shear affinity filtration (CSAF): a new technology for integration of cell separation and protein isolation from mammalian cell cultures; Vogel JH et al.; Controlled shear affinity filtration (CSAF) integrates animal cell separation and product isolation in a single unit operation through the use of a specifically designed rotating disk filter with incorporated membrane chromatography column . Because of the decoupling of shear force and pressure generation and the specific hydrodynamics of the system, shear rates can be easily optimized and precisely controlled to maximize filtration performance while viability of the shear sensitive animal cells is maintained . In this study, the general methodology is demonstrated using the integration of Chinese hamster ovary cell separation and isolation of recombinant tissue plasminogen activator (t-PA) as a model example . Direct capture of t-PA from cell culture broth was realized by using custom-made affinity membranes with lysine as a robust, small molecular weight affinity ligand . Small-scale t-PA adsorption experiments, as well as microfiltration experiments, were used to design the integrated CSAF process . A Chinese hamster ovary batch culture was processed with a lab-scale prototype, yielding 86% of the t-PA in the concentrated, particle-free eluate, whereas 95% of the bulk protein was removed . Because the viability of the cells is not significantly affected and high specific flux rates can be achieved, the CSAF technology should also be well suited for continuous perfusion with integrated product isolation . A truly continuous operation could be realized with two systems in tandem configuration . Pharmazie, 2002 Apr, 57(4), 270 - 4 The usefulness of a biosensor controlled perfusion cell culture for the investigation of new drugs demonstrated with the marine fungus Kirschsteiniothelia maritima; von Woedtke T et al.; The investigation of new biological active substances from limited sources for example from marine organisms needs sensitive and evident test systems . Such a system is the glucose biosensor controlled perfusion cell culture . The glucose consumption of cells is a very sensitive parameter which allows the continuous measurement of external effects of test substances on the cells under in vivo-like conditions . Cytotoxic concentrations of active substances as well as a virus infection lowers the glucose consumption of continuously perfused cells . This effect can be monitored using a glucose biosensor . The influence of drugs and the virus infection can be observed simultaneously in the same system continuously over several days . With two substances isolated from the marine fungus Kirschsteiniothelia maritima investigations for cytotoxic and for antiviral effects are demonstrated. Apoptosis, 2002 Jun, 7(3), 231 - 9 Role of vitamins in determining apoptosis and extent of suppression by bcl-2 during hybridoma cell culture; Ishaque A et al.; The identification of cell culture media components that may instigate apoptosis in cell lines used for the production of commercial antibodies and recombinant proteins, is crucial to aid the development of improved media for reduced cell death and to understand the role of nutrient components in cell survival and maintenance . Here we determine the impact of depriving all or individual B-group media vitamins either, D-CaPantothenate (DCaP), choline chloride (CC), riboflavin (Rb), i-inositol, nicotinamide (NAM), pyridoxal hydrochloride (PyrHCl), folic acid (FA), or thiamine hydrochloride (ThHCl) on hybridoma cell growth and viability using fluorescence microscopy techniques . Cultivation in media deprived of all these vitamins prevented cell proliferation from reaching maximum capacity while increasing cell death rate, predominantly via apoptosis . Deletion of either DCaP, CC, or Rb showed that these components were most likely responsible for the development of apoptosis . Exclusion of either i-inositol, NAM or PyrHCl failed to inhibit cell growth and viability, while marginal improvements in viability were noted by ThHCl deprivation and more so by FA exclusion . Over-expression of the anti-apoptotic gene bcl-2 suppressed cell death initiated by all or single vitamin (either DCaP, CC or Rb) deprivation . The involvement of bcl-2 activity, established a close association between small vitamin molecules particularly DCaP, CC or Rb and the biochemical activation of apoptosis. Exp Mol Med, 2002 Mar 31, 34(1), 75 - 82 Expression of integrins, cyclooxygenases and matrix metalloproteinases in three-dimensional human endometrial cell culture system; Yang H et al.; The objective of this investigation was to establish a three-dimensionally cultured human endometrium which could be used as a tissue model for the mechanism study of implantation in vitro . By using human endometrial stromal (ES) and epithelial cells (EE) from hysterectomy specimens, reconstruction of endometrium in culture was established by first layering a collagen gel containing ES cells, then overlaying with the Matrigel containing endometrial epithelial (EE) cells . Ultrastructural examination of the 48 h-endometrial cell culture revealed monolayered columnar EE cells with microvilli on the collagen layer containing ES cells and appearance of the tight junctions and desmosomes between EE cells, a cell layer closely resembling the native endometrium . Immunohistochemical characterization of the reconstructed endometrium showed a strong immunoreactivity for cytokeratin, integrin alpha1, alpha4 and beta3 subunits, cyclooxygenases-1 and -2, matrix metalloproteinases-1, -2, -3 and -9, and tissue inhibitor of metalloproteinases-1 and -2 in the EE cells comparable to the native endometrial epithelium . ES cells also showed stronger immunoreactivity for cyclooxygenases, integrins and MMPs, but less for cytokeratin . Gelatin zymographic analyses of the media obtained from the reconstructed endometrium model showed gelatinase activity bands at 57, 60, 72, 92 and 97 kDa molecular weight, respectively . The present study provides a possibility that our three-dimensionally cultured endometrium model could mimic the morphological and functional characteristics of the native endometrium . The model could be used to clarify the roles of various molecules involved in the human implantation. Can J Vet Res, 2002 Apr, 66(2), 117 - 21 Comparison of embryonated chicken eggs with MDCK cell culture for the isolation of swine influenza virus; Clavijo A et al.; Embryonated chicken eggs (ECE) and the Madin-Darby canine kidney (MDCK) cell line were compared for isolation of swine influenza virus (SIV) from nasal swabs and tissue samples . Samples originated from 30 pigs experimentally inoculated with 2 x 106 to 2 x 10(7) embryo infectious dose 50% (EID50)/mL of swine influenza strain A/Swine/Indiana/1726/88 (H1N1) . The results were analyzed with McNemar's chi-squared test for symmetry . The results indicated that more samples were SIV-positive with ECE than with tissue culture (P < 0.001), suggesting that ECE remains the system of choice for isolation of SIV . It is recommend that routine use of both SIV isolation systems will increase the sensitivity of detection of virus shedding by considering the differences in growth and tropism of diverse SIV strains. Sheng Wu Gong Cheng Xue Bao, 2002 Jan, 18(1), 30 - 4 {Expression of human interleukin-11 in cell culture and larvae of silkworm}; Guo XJ et al.; A recombinant transfer vector, pBacIL-11, containing hIL-11 cDNA of 546 nucleotides lacking leader sequence was constructed and co-transfected into BmN cells with linearized BmBacPAK(modified BmNPV) DNA for construction of a recombinant baculovirus carrying the hIL-11 gene . Southern hybridization analysis suggested that the recombinant baculovirus DNA contained hIL-11 cDNA fragment . RNA dot blotting demonstrated that the hIL-11 gene was transcribed . The recombinant baculovirus has a strong infectivity to BmN cell line and to silkworm larvae and pupae . Specific hIL-11 bands were detected from all the samples of cell extract, culture supernatant, haemolymph of larvae and pupae by SDS-PAGE analysis . Biological activity of the expressed product was determined with IL-11 dependent B9-11 cell line and by MTT colorimetric assay, which indicated that biologically active rhIL-11 protein was overexpressed in BmN cell line and in silkworm larvae and pupae. Appl Environ Microbiol, 2002 May, 68(5), 2576 - 9 Efficacy of common laboratory disinfectants on the infectivity of Cryptosporidium parvum oocysts in cell culture; Weir SC et al.; Nine liquid disinfectants were tested for their ability to reduce infectivity of Cryptosporidium parvum oocysts in cell culture . A 4-min exposure to 6% hydrogen peroxide and a 13-min exposure to ammonium hydroxide-amended windshield washer fluid reduced infectivity 1,000-fold . Other disinfectants tested (70% ethanol, 37% methanol, 6% sodium hypochlorite, 70% isopropanol, and three commercial disinfectants) did not reduce the infectivity after a 33-min exposure . The results indicate that hydrogen peroxide and windshield washer fluid or ammonium hydroxide disinfectant may be suitable laboratory disinfectants against C . parvum oocysts. J Neurol, 2002 Apr, 249(4), 424 - 31 Effects of nonenzymatic glycosylation of extracellular matrix components on cell survival and sensory neurite extension in cell culture; Luo ZJ et al.; Diabetic sensory polyneuropathy is characterized by a distal axonopathy of dying-back type . It is accompanied by a failure of axonal regeneration, in which nonenzymatic glycosylation (glycation) of the extracellular matrix may be involved . In the present study, the effects of glycation of collagen IV and laminin, major components of basal lamina, on neuron survival and neurite extension were investigated in tissue culture . Fast glycation of laminin was achieved by incubation with glycolaldehyde and glycation of collagen IV by incubation with glucose . The degree of glycation was estimated by fluorescence analysis . Glycated or nonglycated laminin or collagen IV were used as substrates for culture of dorsal root ganglion (DRG) neurons from neonatal rats . Cultures were assessed for the proportion of cells attaching to the substrate, surviving and bearing neurites . Cell attachment and the proportion bearing neurites were significantly reduced on collagen IV glycated for 2 weeks, but survival was only affected by glycation for 4 or 5 weeks . All 3 parameters were significantly reduced on glycated compared with unglycated laminin . Glycation of both laminin and collagen IV produced considerable morphological differences in the cultured neurons on scanning electron microscopy . Dissociated DRG neurons from adult animals with streptozotocin-induced diabetes cultured on nonglycated substrates survived less well and produced fewer neurites . Glycation of collagen IV and laminin thus affects neuronal survival, neurite production and cell morphology, and diabetes affects both the survival of sensory neurons in culture and their ability to extend neurites. Curr Opin Infect Dis, 2001 Dec, 14(6), 743 - 7 Hepatitis C virus cell culture replication systems: their potential use for the development of antiviral therapies; Randall G et al.; Hepatitis C virus is a significant public health problem . Current drug regimens have low efficacy against some hepatitis C virus genotypes, while no vaccine is available . The absence of an efficient cell culture system and an accessible small animal model to study hepatitis C virus replication and pathogenesis are major obstacles to the development of effective antiviral therapies . Studies of surrogate model systems, either related viruses or chimeric viruses containing part of the hepatitis C virus genome, have given insight into hepatitis C virus replication, in addition to being a powerful tool for drug discovery . The recent development of an efficient system for the initiation of replication in cell culture provides a viable screen for inhibitors of hepatitis C virus replication . It also brings us much closer to the ultimate goal of an infectious cell culture system for hepatitis C virus. Biotechniques . 2002 Apr;32(4):876, 878, 880 passim. Quantification of fibronectin adsorption to silicone-rubber cell culture substrates; Cunningham JJ et al.; As the role of mechanical force in cellular signaling gained recognition, investigators designed a number of devices to deliver controlled regimens of mechanical force to cultured cells . One type of device uses thin silicone-rubber membranes to support monolayer cell adhesion and to transmit mechanical force in the form of biaxial strain . We have observed that cell attachment and spreading are impaired on these membranes compared to polystyrene, even when both are passively coated with identical amounts of extracellular matrix . The purpose of these studies was to quantify the efficiency and stability of passive matrix adsorption onto commercially available elastic culture substrates . A theoretically saturating density (1 microg/cm2) of fibronectin was added to each well, and the initial efficiency of adsorption to the walls and elastic membranes was found to be 31 +/- 2% of the protein added . Strikingly, when the protein adsorbed specifically to the membranes was quantified after seven days, only 10-26 ng/cm2 fibronectin were present, revealing that most of the adsorption is to the sides of the wells . These results indicate that the adsorption of matrix proteins to silicone-rubber substrates is relatively inefficient and that investigators who use these systems must be aware of this fact and design their experiments accordingly. Nutr Cancer, 2001, 40(2), 173 - 9 Potential cancer-chemopreventive activities of wine stilbenoids and flavans extracted from grape (Vitis vinifera) cell cultures; Waffo-Teguo P et al.; Moderate consumption of wine is associated with a reduced risk of cancer . Grape plant cell cultures were used to purify 12 phenols: the stilbenoids trans-astringin, trans-piceid (2), trans-resveratroloside, trans-resveratrol, trans-piceatannol, cis-resveratroloside, cis-piceid, and cis-resveratrol; the flavans (+)-catechin, (-)-epicatechin, and epicatechin 3-O-gallate; and the flavan dimer procyanidin B2 3'-O-gallate . These compounds were evaluated for potential to inhibit cyclooxygenases and preneoplastic lesion formation in carcinogen-treated mouse mammary glands in organ culture . At 10 micrograms/ml, trans-astringin and trans-piceatannol inhibited development of 7,12-dimethylbenz{a}anthracene-induced preneoplastic lesions in mouse mammary glands with 68.8% and 76.9% inhibition, respectively, compared with untreated glands . The latter compound was the most potent of the 12 compounds tested in this assay, with the exception of trans-resveratrol (87.5% inhibition) . In the cyclooxygenase (COX)-1 assay, trans isomers of the stilbenoids appear to be more active than cis isomers: trans-resveratrol {50% inhibitory concentration (IC50) = 14.9 microM, 96%} vs . cis-resveratrol (IC50 = 55.4 microM) . In the COX-2 assay, among the compounds tested, only trans- and cis-resveratrol exhibited significant inhibitory activity (IC50 = 32.2 and 50.2 microM, respectively) . This is the first report showing the potential cancer-chemopreventive activity of trans-astringin, a plant stilbenoid recently found in wine . trans-Astringin and its aglycone trans-piceatannol were active in the mouse mammary gland organ culture assay but did not exhibit activity in COX-1 and COX-2 assays . trans-Resveratrol was active in all three of the bioassays used in this investigation . These findings suggest that trans-astringin and trans-piceatannol may function as potential cancer-chemopreventive agents by a mechanism different from that of trans-resveratrol. J Biomech, 2002 May, 35(5), 579 - 84 A cell-culture system for long-term maintenance of elevated hydrostatic pressure with the option of additional tension; Hasel C et al.; In cell stress research, there is still a need to apply long-term hydrostatic pressure without changing any other environmental condition . We present here a new, open, pressurized chamber system allowing long-term sustained and dynamic application of hydrostatic pressure with the option of additional tension . Based on the computer-controlled Flexcell Strain Unit, we designed a pressurized chamber with a dynamic airflow and a defined membrane extension, which can be regulated by spacers . During operation up to 26.6kPa, O(2) partial pressures and pH in the cell-culture medium do not change compared to control cultures kept at normal atmosphere. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2002 Jan, 19(1), 60 - 3 {Three-dimensional finite element analysis on cell culture membrane under mechanical load}; Guo X et al.; A three-dimensional finite element model of the cell culture membrane was developed in the culture device under tension state made by us . The magnitude of tension and the displacement distribution in the membrane made of silicon rubber under different hydrostatic load were obtained by use of FEM analysis . A comparative study was made between the numerical and the experimental results . These results can serve as guides to the related cellular mechanical research. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2002 Jan, 19(1), 20 - 4 {A tissue-engineered strain scaffold for three-dimensional cell cultures}; Qin T et al.; This article introduces a three-dimensional scaffold which is used to perform three-dimensional cell culture under mechanical stretch from the point of construction of tissue-engineered tissue . The composition, structure, surface characteristics, mechanical property, and cell compatibility of the scaffold have been studied by using surface chemistry and material mechanics testing methods . The results indicate that the polyvinyl alcohol (PVA) sponge, which is water-tolerant, coated with Poly-DL-lactic-co-glycolic acid (PLGA) possesses a good nature in appropriate surface feature, porosity, elastic recoil, and cell compatibility . These features provide wide options for using this scaffold to study the effects of mechanical stretch on cells maintained in three-dimensional culture to provide a three-dimensional matrix. Neurosurgery, 2002 May, 50(5), 1094 - 102 Human brain tumor cell culture characterization after immunostimulatory gene transfer; Parney IF et al.; OBJECTIVE: Immunogene therapy is a novel cancer treatment strategy based on vaccination with irradiated autologous tumor cells transduced with immunostimulatory genes . To characterize such cells before clinical applications, we studied a human glioma cell line (D54 MG) and early passage human glioma (Ed147.BT, Ed149.BT) and melanoma (Ed141.MEL) cultures after immunostimulatory gene transfer . METHODS: Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-12 (IL-12), and B7-2 genes were retrovirally transferred to tumor cells . Gene expression before and after irradiation (200 Gy) was assessed by enzyme-linked immunosorbent assay (GM-CSF, IL-12) and flow cytometry (B7-2) . Viability and clonogenicity were determined via trypan blue staining before and after irradiation . Growth rates were determined by serial cell counts . RESULTS: GM-CSF expression was high in GM-CSF-transduced (10.36-162.10 ng/10(6) cells/d preirradiation and 10.22-122.02 ng/10(6) cells/d postirradiation) but lower in B7-2/GM-CSF-transduced cultures (1.41-2.90 ng/10(6) cells/d preirradiation, 1.96-5.02 ng/10(6) cells/d postirradiation) . IL-12 expression also was lower (1.30-2.10 ng/10(6) cells/d preirradiation, 0.47-1.70 ng/10(6) cells/d postirradiation) . B7-2 expression was high (one- to two-logarithm increase in fluorescence) and unaffected by radiation . Postirradiation viability was initially high (94.20 +/- 8.46%, Day 1) but decreased rapidly (28.13 +/- 4.64%, Day 10) . No cultures demonstrated evidence of clonogenicity (i.e., cell division) after 200-Gy irradiation . Growth rates were similar in wild-type and gene-transduced Ed141.MEL, Ed147.BT, and Ed149.BT . However, D54MG-IL-12 growth was slower than that of wild-type D54MG . CONCLUSION: GM-CSF, IL-12, and B7-2 genes can be transferred to human glioma and melanoma cell cultures efficiently by use of our retroviral vectors . Irradiation (200 Gy) does not significantly alter therapeutic gene expression . Irradiated cells remain viable for several days but cannot undergo further cell division . Early passage culture growth rates are not altered by therapeutic gene expression but are decreased by IL-12 in an immortalized cell line (D54MG) . These results suggest that it is feasible to create vaccines with irradiated, autologous, genetically modified brain tumor cells. Brain Res Dev Brain Res, 2002 Mar 31, 134(1-2), 149 - 54 Evaluation of progenitor cell cultures from human embryos for neurotransplantation; Poltavtseva RA et al.; Human neural stem cells (HNSCs) are used in studies of neural development and differentiation, and are regarded as an alternative source of tissue for neural transplantation in degenerative diseases . Selection and standardization of HNSC samples is an important task in research and clinical approaches . We evaluated embryonal brain matter obtained from human 8-12-week-old fetuses by means of flow cytometry on a panel including: nestin; vimentin; NeuN; GFAP; beta-tubulin III; CD56; N-Cad; OB-Cad; HLA-ABC; HLA-DR; CD34, and annexin . Samples from embryos of even the same gestation differ dramatically regarding neural cell development, their phenotype and viability . The samples containing the highest proportion of stem cells and multipotent progenitors of neural types, and the least of definitive cells and antigens of histocompatibility, were selected for further expansion in serum-free medium . Secondary phenotyping 14 days later revealed again a marked heterogeneity of the cultures . For the final culturing for 24 h in a serum-containing medium we selected only samples having following phenotype: nestin+, and vimentin+ no less than 25%; HLA-DR+ and CD34+ no more than 5%; GFAP+ no more than 10%; beta-tubulin+ no more than 20%; CD56+, N-Cad+, OB-Cad+, HLA-A,B,C+, and annexin+ no more than 15%; cell viability no less than 60% . Immunocytochemical study of selected samples proved that numerous neural stem cells, and neuro- and glioblasts necessary for transplantation were present . Our results demonstrate that the flow cytometry phenotyping allows the screening and standardization of HNSC samples for further expansion and transplantation. FEBS Lett, 1971 Jan 12, 12(3), 143 - 147 Aflatoxin B(1): Cytotoxic mode of action evaluated by mammalian cell cultures; Scaife JF; Aflaxton B(1) rapidly inhibits RNA synthesis in rat liver cells, slices or liver in vivo . Established human cells lines (kidney T-cells, HeLa S(3), Chang liver) and mouse fibroblast 3t3 are more slowly affected . Prolonged exposure of synchronized cell cultures to the agent show that cells are retarded in their passage through the S-phase and exhibit a decreased rate of DNA synthesis . Consequent to this, mitosis is also inhibited . Liver cells appear to convert aflatoxin B(1) to a more potent cytotoxin which can then affect normally non-susceptible cells . This may explain the susceptibility of liver to tumorogenesis by this carcinogen. Southeast Asian J Trop Med Public Health, 2001 Sep, 32(3), 470 - 1 Increase in the sensitivity of dengue diagnosis by combination of reverse transcriptase-polymerase chain reaction and passage on cell cultures; Yamada K et al.; We passaged 52 serum samples from dengue patients on C6/36 cells for 7 days and checked the culture fluids by RT-PCR . Two serum samples, which were negative by direct RT-PCR, became positive . One sample was collected on fever day 1 and the other on fever day 2 . Results indicate that combination of reverse transcriptase-polymerase chain reaction (RT-PCR) with passage of serum samples on C6/36 cells increases the sensitivity of dengue diagnosis. Life Sci Space Res, 1971, 9, 99 - 103 Experiments with micro-organisms and human cell cultures in the Zond 5 and Zond 7 flights; Zhukov-Verezhnikov NN et al.; Lysogenic strains of Escherichia coli were exposed to space conditions aboard the flight of Zond 5 and Zond 7 . Space flight factors appeared to affect the state of episome systems of bacteria, as judged by data obtained with F-Lac+ donor cells which also carried genetic markers for threonine and leucine . Observations on phage induction are discussed and compared with results obtained aboard Biosatellite 2 . A number of monolayer cultures of human cells (HeLa cells, fibroblasts, and A-1 cells) were repeatedly exposed to the space environment . In one instance, HeLa 19 cells increased in size after exposure to space conditions, a change which appeared to be genetically stable . HeLa 19 cells which were carried on six separate space flights showed a higher viability than corresponding cultures which were exposed only once aboard Zond 5. Arch Physiol Biochem, 2001 Dec, 109(5), 404 - 9 Angiotensin II-induced growth effects in vascular smooth muscle in cell culture and in the aortic tunica media in organ culture; Mangiarua EI et al.; Several different studies have investigated the growth effects of angiotensin II on vascular smooth muscle cells in culture . However, smooth muscle cells change their phenotype when placed in culture . The objective of the present study was t