Biol Proced Online, 2002 Nov 11, 4, 70 - 80 PCR-based detection of a rare linear DNA in cell culture; Saveliev SV; The described method allows for detection of rare linear DNA fragments generated during genomic deletions . The predicted limit of the detection is one DNA molecule per 10(7) or more cells . The method is based on anchor PCR and involves gel separation of the linear DNA fragment and chromosomal DNA before amplification . The detailed chemical structure of the ends of the linear DNA can be defined with the use of additional PCR-based protocols . The method was applied to study the short-lived linear DNA generated during programmed genomic deletions in a ciliate . It can be useful in studies of spontaneous DNA deletions in cell culture or for tracking intracellular modifications at the ends of transfected DNA during gene therapy trials. Am J Reprod Immunol, 2003 Jan, 49(1), 14 - 20 The herbal medicine Unkei-to stimulates the secretion of a cytokine-induced neutrophil chemoattractant, CINC/gro, in the rat ovarian cell culture; Yasui T et al.; PROBLEM AND METHOD OF STUDY: We investigated the ovulation-inducing effects of Unkei-to, a Japanese herbal medicine, in relation to the production of sex steroid hormones (17beta-estradiol and progesterone), cytokine-induced neutrophil chemoattractant (CINC/gro), interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) in the rat ovarian cell culture . RESULTS: Unkei-to at a concentration of 100 microg/mL significantly stimulated the secretions of 17beta-estradiol and progesterone (P < 0.01) in cultured whole ovarian dispersates . Unkei-to also enhanced the secretion of CINC/gro in a dose-dependent manner, and the secretions of CINC/gro increased significantly at concentrations of 10 and 100 microg/mL (P < 0.01) . These stimulatory effects of Unkei-to on steroidgenesis and CINC/gro production are very similar to those of another Japanese herbal medicine, Toki-Shakuyaku-san . In addition, Unkei-to significantly (P < 0.01) enhanced the secretions of both IL-1beta and TNF-alpha, which are known to stimulate the secretion of CINC/gro in the ovulatory process, at concentrations of 10 and 100 microg/mL . The stimulatory effect of Unkei-to at a concentration of 100 microg/mL on IL-1beta/was significantly (P < 0.01) lower than that of Toki-Shakuyaku-san, while the stimulatory effects of these two herbal medicines at a concentration of 100 microg/mL on TNF-alpha were similar . CONCLUSIONS: These results show that Unkei-to can stimulate ovarian steroidgenesis and the ovulatory process by inducing the secretion of CINC/gro with IL-1beta and TNF-alpha in vitro . Unkei-to has stimulatory effects on both steroidgenesis and the ovulatory process in the ovary as well as a stimulatory effect on the hypothalamus-pituitary axis, and it may be useful for treating patients with ovulatory disorders. Pediatr Surg Int, 2003 Jul, 19(5), 321 - 5 Epub 2003 May 06. Interleukin-6 changes tight junction permeability and intracellular phospholipid content in a human enterocyte cell culture model; Tazuke Y et al.; Proinflammatory cytokines and secretory phospholipase A(2) (sPLA(2)) are elevated in patients with inflammatory bowel disease (IBD) . We previously reported that the proinflammatory cytokine IL-6 increased the expression of sPLA(2) (a hydrolyzer of phosphatidylcholine) and decreased membrane integrity in an intestinal epithelial cell culture model . To determine the physiological effects of the IL-6 mediated increase in sPLA(2) on decreased epithelial layer integrity, we investigated alterations of intracellular/secretory phospholipid (PL) composition in a cell culture model . In addition, since other PLs may also mediate epithelial membrane activity, we investigated the effect of IL-6 on PL activity in a Caco-2 enterocyte culture model . Caco-2 cells were incubated for 72 h with IL-6 or media alone (control) . Both media and cell lysate were analyzed for PL composition using thin-layer chromatography . The PL composition in the media did not show any differences between the two groups ( p>0.1) . Total intracellular PL contents were also unchanged; however, IL-6 led to significant changes in PL composition including an increase in phosphatidylethanolamine (PE) and sphingomyelin (SM) and a decrease in phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) ( p<0.05) . Both PE and SM are known as inflammatory signaling factors involved in human IBD . Our study suggests that the decreased membrane integrity seen with IL-6 application may occur via intracellular PL alterations, rather than through the direct effects of sPLA(2). Pediatr Surg Int, 2003 Jul, 19(5), 316 - 20 Epub 2003 May 06. The effect of hypoxia on permeability and bacterial translocation in Caco-2 adult and I-407 fetal enterocyte cell culture models; Tazuke Y et al.; Hypoxia has been implicated in the breakdown of the intestinal epithelial barrier in animals, leading to bacterial translocation (BT); however, the mechanism of this hypoxic insult is unknown . To determine the effects of hypoxic injury in vitro on epithelial membrane integrity, transepithelial electrical resistance (TEER), mannitol permeability (Ma-Pm), and BT were measured in both an adult (Caco-2) and fetal (I-407) intestinal epithelial cell culture model . Caco-2 adult and I-407 fetal epithelial cell monolayers were treated with or without bacteria (1 x 10(7) Escherichia coli . C-25), and then incubated under either normoxic (5% CO(2) in room air) or hypoxic (5% CO(2) and 95% N(2)) conditions at 37 degrees C for 6 h . Hypoxia caused a 10% increase in Ma-Pm in the I-407 fetal cell model independent of the bacterial challenge . In contrast, a bacterial challenge in the Caco-2 adult model caused a 485% increase in Ma-Pm independent of hypoxia . Neither hypoxia, nor C-25 bacteria, for 6 h caused BT in either cell culture model . In the adult cell culture model, bacteria appear to mediate changes in epithelial barrier function, with hypoxia having no effect . On the other hand, hypoxia is the major factor in the loss of epithelial barrier function in fetal epithelium, but has no effect on adult epithelium . The data suggest that the breakdown of barrier function caused by a hypoxic insult is the primary stimulus for subsequent BT in neonates. Appl Environ Microbiol, 2003 May, 69(5), 2505 - 11 Cell culture-Taqman PCR assay for evaluation of Cryptosporidium parvum disinfection; Keegan AR et al.; Cryptosporidium parvum represents a challenge to the water industry and a threat to public health . In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of C . parvum with disinfectants . The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine . The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst . Effective oocyst inactivation was achieved (>2 log(10) units) with LP-UV (20 mJ/cm(2)) or 2 mg of ozone/liter (for 10 min) . MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with <0.1 log(10) unit being inactivated . These results demonstrate the inability of MIOX to inactivate Cryptosporidium . The assay is a valuable tool for the evaluation of disinfection systems for drinking water and recycled water. J Exp Bot, 2003 Jun, 54(387), 1565 - 75 Epub 2003 Apr 28. Sugars regulate cold-induced gene expression and freezing-tolerance in barley cell cultures; Tabaei-Aghdaei SR et al.; The hypothesis that the extracellular concentration of sugars helps regulate the acclimation of plant cells to cold was tested in this work . Suspension cultures were used to control the concentration of sugars in the medium supplied to barley cell cultures (Hordeum vulgare L . cv . Igri), replacing the medium daily to help maintain the concentration . Freezing tolerance and the levels of mRNA expression of the stress-response genes blt4.9 (coding for a non- specific lipid transfer protein) and dhn1 (coding for a dehydrin) were measured . Similar levels of freezing-tolerance and gene expression were obtained in the experiments as occur during cold-acclimation in the crown of the whole plant . In the cell cultures, cold (6/2 degrees C) did not induce an increase in freezing tolerance or in the expression of detectable levels of blt4.9 or dhn1 mRNAs when only 1 g l-1 sucrose was supplied . However, the cells in this low sucrose medium in the cold were not sugar-starved, indicating that this did not explain the failure of the cells to acclimate when grown in the cold environment . Ten g l-1 sucrose supplied to cells grown in the warm (25 degrees C) induced acclimation to freezing and up-regulation of expression of blt4.9 and dhn1 mRNAs . Osmolality of the medium did not explain this . Thirty g l-1 sucrose induced yet higher levels of freezing tolerance and of blt4.9 and dhn1 mRNAs in cultures grown in either the cold or the warm environment . The results implicate sugars in the regulation of cold acclimation Biosci Biotechnol Biochem, 2003 Feb, 67(2), 244 - 9 Effects of corticosterone on Ca2+ uptake and myofibrillar disassembly in primary muscle cell culture; Machida K et al.; This study was done to examine the effects of corticosterone, a glucocorticoid, on Ca2+ uptake, proteolysis, and Ca2+ channels in primary cultures of chick muscle cells, to clarify the mechanism of glucocorticoid action on muscle proteolysis . Chick muscle cells were incubated for 24 h in a medium containing corticosterone (30 ng/ml) when the cells were confluent (6 days) . To examine the contribution of Ca2+ channels, nifedipine, a Ca2+ channels antagonist, was used . Ca2+ uptake measured with 45CaCl2 was increased three-fold by corticosterone, with a peak at 12 h after the treatment started . The growth of the cells estimated from the protein content and creatine kinase activity was not affected by corticosterone . Proteolysis, evaluated with {3H}tyrosine as a label of the protein and Ntau-methylhistidine release, was unchanged by corticosterone . However, the amount of easily releasable myofilament as a measure of myofibrillar disassembly in the muscle cells was increased by corticosterone, and prevented by nifedipine . These results show that corticosterone increases Ca2+ uptake and starts myofibrillar protein breakdown. Planta, 2003 May, 217(1), 96 - 101 Epub 2003 Jan 18. Cinnamic acid 4-hydroxylase from cell cultures of the hornwort Anthoceros agrestis; Petersen M; Cinnamic acid 4-hydroxylase (EC 1.14.13.11), a cytochrome P450-dependent hydroxylase was for the first time characterized from a hornwort, Anthoceros agrestis Paton (Anthocerotaceae) . In suspension cultures of A . agrestis up to 5% of the dry weight was accumulated as rosmarinic acid, a natural product commonly known from higher plants (e.g . species of the Lamiaceae and Boraginaceae) . Cinnamic acid 4-hydroxylase is involved in the biosynthesis of rosmarinic acid . The participation of cytochrome P450 was demonstrated by the inhibition of hydroxylase activity by cytochrome c and the inhibition of cinnamic acid hydroxylation in a CO-containing atmosphere, which is partially released by illumination with blue light . The apparent K(m) values were determined to be at 60 microM and 5 microM for NADPH and cinnamic acid, respectively . A comparatively high hydroxylation activity was seen with NADH as electron donor . While the hydroxylase activity with NADPH was strongly inhibited by the competitive electron acceptor cytochrome c, the activity with NADH was less susceptible, indicating the possibility of different electron-transfer pathways. J Androl, 2003 May-Jun, 24(3), 401 - 7 Hormonal regulation of bovine secretory proteins derived from caput and cauda epididymal epithelial cell cultures; De Pauw IM et al.; The goal of this study was to investigate the effect of hormones (testosterone, dihydrotestosterone {DHT}, and hydrocortisone) on the protein secretion of caput and cauda epididymal epithelial cells cultured in principal cell medium (PCM) . A confluent monolayer of caput and cauda epididymal epithelial cells was obtained from serum-containing PCM in the presence or absence of hormones after 7 days of culture at 38.5 degrees C (5% CO(2) in air) . The protein secretion of epididymal epithelial monolayers incubated in serum-free PCM for 3 days was examined . The secreted proteins were separated by 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE) . A comparison of the different protein patterns showed 61 spots, of which 11 were secreted only in the presence of hormones, 3 appeared to show hormone-related changes, and 25 were region-specific . Most of these secreted proteins were low-molecular-weight acidic proteins . To obtain evidence of the epididymal origin of the secreted proteins, proteins present in caput and cauda epididymal plasma were analyzed . In conclusion, our data indicate that hormones influence the synthesis of a number of caput and cauda epididymal proteins . Some of these proteins could be important for improving our understanding of spermatozoa maturation and storage and their acquisition of fertilizing ability. Anal Chem, 2003 May 1, 75(9), 2154 - 8 Scanning electrochemical microscopy-based drug sensitivity test for a cell culture integrated in silicon microstructures; Torisawa YS et al.; The respiratory activity of collagen-embedded living cells was imaged by scanning electrochemical microscopy (SECM) with the objective to study anticancer drug sensitivity . Two kinds of cancer cells, the human erythroleukemia cell line (K562) and its adriamycin-resistant subline (K562/ADM), were immobilized at the array of microholes micromachined on a silicon wafer for comparative characterization of their sensitivity to the anticancer drug, ADM . The results obtained by the SECM method showed correspondence to a conventional colorimetric assay (SDI assay) . Furthermore, since the SECM assay is based on the noninvasive measurement of the respiration activity, continuous monitoring of a dose response was possible. Nucl Med Commun, 2003 May, 24(5), 597 - 606 Accumulation of technetium-99m glucarate: in vitro cell cultures and in vivo tumour models; Ballinger JR et al.; 99mTc-glucarate is an investigational radiopharmaceutical which has been shown to accumulate in acute cerebral and myocardial injuries and in some tumours . In the present work, a survey of possible factors affecting the cellular accumulation of 99mTc-glucarate was carried out in cell lines and strains in vitro and in murine tumours in vivo . Accumulation was enhanced under hypoxic conditions in 12 of the 16 human and murine cell lines and strains studied, and inhibited in the presence of nitroimidazoles . At temperatures lower than 37 degrees C, accumulation was reduced, but a hypoxic/aerobic differential was maintained . Aerobic accumulation of 99mTc-glucarate was enhanced by cyanide . In transplanted tumours in mice, 99mTc-glucarate showed high tumour/muscle and tumour/blood ratios at early times after injection . Pharmacological enhancement of the extent of hypoxia by the administration of hydralazine or nitro-L-arginine resulted in significantly increased accumulation of 99mTc-glucarate in the tumour . The in vitro and in vivo properties of 99mTc-glucarate suggest that it may be useful for tumour imaging in the clinic, although the exact mechanism(s) by which it localizes in tumours remains unknown. J Proteome Res, 2003 Mar-Apr, 2(2), 173 - 81 Properties of 13C-substituted arginine in stable isotope labeling by amino acids in cell culture (SILAC); Ong SE et al.; We have recently described a method, stable isotope labeling by amino acids in cell culture (SILAC) for the accurate quantitation of relative protein abundances . Cells were metabolically labeled with deuterated leucine, leading to complete incorporation within about five cell doublings . Here, we investigate fully substituted 13C-labeled arginine in the SILAC method . After tryptic digestion, there is a single label at the C-terminal position in half of the peptides . Labeled and unlabeled peptides coelute in liquid chromatography-mass spectrometric analysis, eliminating quantitation error due to unequal sampling of ion profiles . Tandem mass spectrum interpretation and database identification are aided by the predictable shift of the y-ions in the labeled form . The quantitation of mixtures of total cell lysates in known ratios resolved on a one-dimensional SDS-PAGE gel produced consistent and reproducible results with relative standard deviations better than five percent under optimal conditions. Eur J Clin Invest, 2003 May, 33(5), 434 - 42 Measles virus induces apoptosis in uninfected bystander T cells and leads to granzyme B and caspase activation in peripheral blood mononuclear cell cultures; Vuorinen T et al.; BACKGROUND: Measles causes lymphopenia and depresses cell-mediated immunity, but the mechanisms of immunosuppression and cell loss are poorly known . METHODS: We have used an in vitro model of measles virus (MV)-infected peripheral blood mononuclear cells (PBMCs) and phytohaemagglutinin-stimulated PBMCs in order to assess MV-leucocyte interactions . Cell population undergoing apoptosis was measured by flow cytometry and Annexin-V-fluos staining . The expression of Fas, FasL, TNRF1, and Bcl-2 was analyzed by flow cytometry and Western blotting, and activation of caspase cascade was measured using a colourimetric caspase substrate set . The effects of caspase inhibitors were detected by flow cytometry . RESULTS: Measles virus was able to infect monocytes, but interestingly induced apoptosis in uninfected T cells, indicating that induction of apoptosis in T cells is mediated by MV-infected adherent cells . Only 1% of T cells contained MV antigen day 3 p.i . Interestingly the percentage of early apoptotic T cells at the same time was 35%, showing that apoptosis was not the result of MV infection in T cells . Measles virus-induced Fas but not FasL or TNFR1 expression on PMBC, as well as activation of granzyme B and caspase cascade . Simultaneously, overexpression of Bcl-2 protein was detected . Caspase inhibitor decreased the amount of apoptotic T cells . CONCLUSION: Measles virus-infected monocytes induce apoptosis in uninfected T cells, suggesting that infected monocytes probably interact via cell-surface molecules with uninfected T cells and induce apoptosis by indirect mechanisms . Apoptosis of the lymphocytes may contribute to the pathogenesis of MV-induced immunosuppression and cell loss. Biotech Histochem, 2003 Feb, 78(1), 17 - 21 The use of dimethylsulfoxide as a vehicle in cell culture experiments using ovarian carcinoma cell lines; Rodriguez-Burford C et al.; Dimethylsulfoxide (DMSO) is a well-known solvent that is commonly used in the laboratory . We selected DMSO as the vehicle for an experiment designed to determine if several nonsteroidal anti-inflammatory agents inhibit the growth of Caov-3, OVCAR-3, and SK-OV-3 ovarian carcinoma cell lines . Using the tetrazolium conversion assay, however, we observed some variability in the number of cells present in each ovarian carcinoma cell line with varying concentrations of DMSO (10(-6)-10(-2) M) compared to medium alone . Similarly, when Caov-3, OVCAR-3, and SK-OV-3 cells were treated with 10(-4) M DMSO plus medium (Dulbecco's Modified Eagle Medium with 10% fetal bovine serum) and plated on coverslips, the total number of cells present in 60 random fields increased significantly (P < 0.0001) for each ovarian carcinoma cell line treated with DMSO compared to medium alone . Ethanol did not demonstrate such prominent effects on cellular growth . Our observations are important to consider when selecting an appropriate solvent, especially for growth inhibition studies using Caov-3, OVCAR-3, and SK-OV-3 cell lines. J Biomater Sci Polym Ed, 2003, 14(3), 199 - 211 Uses of thermoresponsive and RGD/insulin-modified poly(vinyl ether)-based hydrogels in cell cultures; Gumusderelioglu M et al.; Thermoresponsive hydrogels were synthesized by radiation copolymerization of ethylene glycol vinyl ether (1) and butyl vinyl ether (2) in the presence of cross-linking agent diethylene glycol divinyl ether . The comonomer ratio (monomer 1/monomer 2) and the cross-linker concentration were kept constant at 60:40 (mole percentage in the monomeric mixture) and 4% (mole basis), respectively . The hydrogels showed a volume-phase transition in the temperature range 10-25 degrees C and their swelling behaviour was reversible . The gels were modified by a cell adhesion factor, the RGD sequence of fibronectin, and a cell growth factor, insulin . However, they lost their thermoresponsive character after modification . The use of the gels in cell culture was investigated without using a proteolytic enzyme or serum . Cell culture studies realized by human skin fibroblasts (HS An1) showed that the cells can attach and proliferate on the surface of a thermoresponsive polymer . 80% of the cultured cells were readily detached from the polymer surface by lowering the incubation temperature from 37 degrees C to 10 degrees C for 30 min . In the studies carried out with RGD or insulin-modified hydrogels in serum-free cultures, higher values of cell proliferation (9 x 10(5) cells/ml) were obtained on the insulin-modified hydrogels, whereas higher values of cell attachment were obtained on the RGD-immobilized surfaces. Ophthalmic Res, 2003 May-Jun, 35(3), 126 - 36 Multilayer primary epithelial cell culture from bovine conjunctiva as a model for in vitro toxicity tests; Civiale C et al.; OBJECTIVE: The purpose of this study was to obtain a primary cell culture of bovine origin similar to the conjunctiva in terms of morphology and cell types, which could be of use for in vitro toxicity studies . METHODS: After separation from the stroma by enzymatic treatment, conjunctival epithelial cells were dissociated and plated onto collagen-coated Transwell filters (1.13 cm(2) area) . One group of plates was maintained in immersion and another was cultured under air-lifted conditions . Anti-epithelial keratin antibodies (AE1/AE3, K4) and antidesmoplakin 1 and 2 were used to characterize the cells by indirect immunofluorescence . The cell layer was examined after histological processing of the Transwell filter . Ultrastructural analysis was carried out by scanning electron microscopy (SEM) . The bioelectric parameters transepithelial electrical resistance (TEER), potential difference (PD), short circuit current and paracellular permeability profile of carboxyfluorescein were monitored as indices of the functional characteristics of these cultures . Cytotoxicity was evaluated on morphological and functional (TEER) grounds after treating the cultures with several test substances . RESULTS: Morphological studies showed pure and homogeneous cell cultures . In the SEM analysis, we observed contiguous polygonal cells with numerous short microvilli, a characteristic proportion of light, medium and dark cells and a sparse population of rounded PAS-positive cells, i.e . resembling goblet cells . Air-lifted cultures also showed a tissue-like cellular organization (8-9 layers) . Immersion cultures reached a maximum TEER value of around 2.95 kOmega x cm(2) 7 days after plating while in air-lifted cultures TEER peaked up to 5.59 komega x cm(2) 11 days after plating . With regard to the use of bovine conjunctival epithelial cells (BCECs) for cytotoxicity screening, the system responded finely to the insults and yielded morphological and functional results in accordance with data obtained in vivo . CONCLUSIONS: BCECs reproduce cell morphology and differentiation of the original tissue and should prove a useful tool for initial studies of drug toxicity . J Cell Biochem, 2003 May 15, 89(2), 364 - 72 Proliferation and differentiation of osteoblasts and adipocytes in rat bone marrow stromal cell cultures: effects of dexamethasone and calcitriol; Atmani H et al.; During bone loss, osteoblast population can be replaced by adipose tissue . This apparent reciprocal relationship between decreased bone density and increased fat formation can be explained by an imbalance in the production of bone-forming and fat-forming cells in the marrow cavity . Thus, osteoblast and adipocyte pathways seem more closely and inversely related . In the present study, we investigated the effects of dexamethasone (dex) and calcitriol {1,25(OH)(2)D(3)} on proliferation and differentiation of osteoblasts and adipocytes in rat bone marrow stromal cell cultures . Stromal cells were grown in primoculture in presence of dex and subcultivated in presence of dex and/or 1,25(OH)(2)D(3) . Total cell proliferation, osteoblast and adipocyte-cells number, and -mRNA specific markers were used to study the effects of hormonal treatment on stromal cells . Total cell proliferation was stimulated by dex and inhibited by 1,25(OH)(2)D(3) . Dex increased osteoblast and adipocyte cell population whereas calcitriol decreased bone-forming cell number and increased fat cell population . The presence of both hormones led to a strong decrease in osteoblastic cells and to a strong increase in adipocytic cell number . Dex induced mRNA osteoblastic markers expression like bone sialoprotein (BSP) and osteocalcin (OC) and an adipocyte marker expression, the fatty acid binding protein aP2 . Calcitriol decreased the dex-induced BSP expression but stimulated slightly OC and aP2 mRNA . The effects of both hormones was to increase strongly OC and aP2 mRNA . These results support that, in rat bone marrow, adipocyte proliferation and differentiation are stimulated by glucocorticoids and calcitriol which act synergically, whereas osteoblastic cell proliferation and differentiation are increased by dex and inhibited by 1,25(OH)(2)D(3) . J Histochem Cytochem, 2003 May, 51(5), 633 - 41 Early expression of bone matrix proteins in osteogenic cell cultures; de Oliveira PT et al.; Osteogenic cells express some matrix proteins at early culture intervals . The aim of this study was to determine if, and in what proportion, cells used for plating contain bone sialoprotein (BSP) and osteopontin (OPN), two matrix proteins associated with initial events in bone formation . Their pattern of expression, as well as that of fibronectin (FN) and type I pro-collagen, was also examined at 6 hr and at 1 and 3 days . The cells were obtained by enzymatic digestion of newborn rat calvariae, and grown on glass coverslips . Cytocentrifuge preparations of isolated cells and coverslips were processed for single or dual immunolabeling with monoclonal and/or polyclonal primary antibodies, followed by fluorochrome-conjugated antibodies . The cell labeling was mainly associated with perinuclear elements . OPN was also distinctively found at peripheral cytoplasmic sites . About 31% of isolated cells were OPN-positive and 18% were BSP-positive . After 1 day, almost 50% of cells were immunoreactive for OPN and for type I pro-collagen, and still less than 20% reacted for BSP . Approximately 7% exhibited peripheral staining for OPN . Almost all cells were associated with extracellular FN . However, only 15% showed intracellular labeling . These results indicate that an important proportion of cells used for plating contain BSP and OPN, a situation that should be taken into consideration in experimental analyses of osteoblast activity in vitro. Cancer Genet Cytogenet, 2003 Apr 15, 142(2), 87 - 91 Cytogenetic analysis of melanoma cell lines: subclone selection in long-term melanoma cell cultures; Lotem M et al.; We employed G-banding cytogenetic analysis to follow the clonal constitution of short-term cultures of metastatic malignant melanoma compared to their long-term cultures . Eight metastatic melanoma cell lines were analyzed . No long-term culture was found to be identical to its line of origin . In all cultures there was a selection of one subclone and emergence of its own subclones . In the majority of cultured tumors (5/8), this process was associated with a decrease in the number of subclones composing the line . We suggest that subclone selection in long-term tumor cultures can be associated with a change in phenotype . Therefore, caution is required when employing long-term cultures for research and therapy. Cell Mol Biol (Noisy-le-grand), 2002 Dec, 48(8), 903 - 9 Two course illuminating scheme improves aminolevulinic acid photodynamic therapy in cell cultures; Hinnen P et al.; Photodynamic therapy with the pro-drug 5-aminolaevulinic acid (ALA-PDT) is being used for the treatment of Barrett's oesophagus . We postulated that a first early course of ALA-PDT would increase protoporphyrin IX (PPIX) accumulation and thus the efficacy of a second course of ALA-PDT, by manipulating ferrochelatase (FC) and porphobilinogen deaminase (PBG-d) activity . Human EBV-transformed lymphoblastoid cells were used as a model of human tumour cells for the ability to form haem is present in all cells . After a single course of illumination (633 nm, 100 mW/cm2) the FC activity decreased significantly whereas the PBG-d activity did not change . During continued incubation with ALA following the first illumination, cells accumulated up to four times more PPIX than non-illuminated controls {220% +/- 30% versus (vs) 55% +/- 5%; p<0.001} . Two illuminations resulted in more cell death than one illumination (97% +/- 1% vs 80% +/- 2%; p<0.001) . Since a second course of ALA-PDT within 3 hr after the first course resulted in a four fold increase in PPIX accumulation and significantly more cell death, we propose that a two course ALA-PDT scheme might improve the efficacy of this treatment for Barrett's oesophagus. Cell Cycle, 2003 Jan-Feb, 2(1), 42 - 5 Synchrony in human, mouse and bacterial cell cultures--a comparison; Helmstetter CE et al.; Growth characteristics of synchronous human MOLT-4, human U-937 and mouse L1210 cultures produced with a new minimally-disturbing technology were compared to each other and to synchronous Escherichia coli B/r . Based on measurements of cell concentrations during synchronous growth, synchrony persisted in similar fashion for all cells . Cell size and DNA distributions in the mammalian cultures also progressed synchronously and reproducibly for multiple cell cycles . The results demonstrate that unambiguous multi-cycle synchrony, critical for verifying the absence of significant growth imbalances induced by the synchronization procedure, is feasible with these cell lines, and possibly others. J Gen Virol, 2003 May, 84(Pt 5), 1269 - 74 The envelope glycoprotein E2 is a determinant of cell culture tropism in ruminant pestiviruses; Liang D et al.; Bovine viral diarrhoea virus (BVDV) isolates infect cultured Madin-Darby bovine kidney (MDBK) cells as efficiently as sheep kidney cells . In contrast, border disease virus (BDV) propagates poorly in MDBK cells but infects sheep cells very efficiently . The envelope glycoprotein E2 has been shown to be essential for virus infectivity . To explore the potential role of E2 in pestivirus host range in cell cultures, we engineered a chimeric BVDV with the E2 coding region from BDV . As expected, the BVDV-E2(bdv) chimera retained the ability of BDV to multiply in sheep cells but experienced a remarkable reduction in its ability to propagate and form plaques in MDBK, a phenotype that is characteristic of the E2 donor, BDV31 virus . Control chimeric BVDV bearing a type II E2 demonstrated that the heterologous E2 does not impair replication in MDBK or lamb cells . These results establish a role for E2 in determining the tropism of a pestivirus in cell culture. J Mol Cell Cardiol, 2003 Apr, 35(4), 421 - 5 FGF-1 enhanced cardiogenesis in differentiating embryonal carcinoma cell cultures, which was opposite to the effect of FGF-2; Hidai C et al.; To investigate the effect of fibroblast growth factors (FGFs) on cellular differentiation, we employed a multipotent mouse embryonal carcinoma cell line, P19, which differentiates into cardiac muscle, skeletal muscle and neural cells in the presence of the appropriate concentrations of retinoic acid (RA) . Under conditions appropriate for cardiac muscle differentiation, the expression of FGF-1 was significantly enhanced before any tissue-specific gene was induced . In contrast, up-regulation of the FGF-2 gene was observed with skeletal muscle-inducing concentrations of RA . Exogenous FGF-1, under skeletal muscle-inducing conditions, suppressed the expression of marker genes for skeletal muscle and induced cardiac alpha myosin heavy chain (alphaMHC) gene with up-regulation of bone morphogenetic protein-4 (BMP-4) and GATA-4 . Unlike FGF-1, exogenous FGF-2 promoted skeletal muscle differentiation . These results indicate that FGF-1 and FGF-2 play different roles in P19 cell differentiation induced by RA. FEBS Lett, 2003 Apr 10, 540(1-3), 3 - 6 Oxidative stress in cell culture: an under-appreciated problem? Halliwell B. Cell culture studies have given much valuable information about mechanisms of metabolism and signal transduction and of regulation of gene expression, proliferation, senescence, and death . However, cells in culture may behave differently from cells in vivo in many ways . One of these is that cell culture imposes a state of oxidative stress on cells . I argue that cells that survive and grow in culture might use ROS-dependent signal transduction pathways that rarely or never operate in vivo . A further problem is that cell culture media can catalyse the oxidation of compounds added to them, resulting in apparent cellular effects that are in fact due to oxidation products such as ROS . Such artefacts may have affected many studies on the effects of ascorbate, thiols, flavonoids and other polyphenolic compounds on cells in culture . J Vet Diagn Invest, 2002 Jan, 14(1), 73 - 6 Failure of porcine reproductive and respiratory syndrome virus to replicate in porcine endothelial cell cultures; Howerth EW et al.; Clinical, gross, and microscopic pathologic and immunohistochemical findings in pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV) suggest that PRRSV may replicate in endothelial cells . Endothelial cell cultures from porcine aorta and pulmonary artery were tested for susceptibility to various strains of PRRSV . Cultures were identified as endothelium by light microscopy and immunohistochemical staining for P-selectin and von Willebrand factor . Five strains of PRRSV, i.e., the National Veterinary Services Laboratories, Ames, IA PRRSV strain 130-PDV and 4 field strains isolated from pneumonic lungs, failed to replicate in these porcine large-vessel endothelial cell cultures. Eur J Histochem, 2003, 47(1), 17 - 28 Muscle biopsy and cell cultures: potential diagnostic tools in hereditary skeletal muscle channelopathies; Meola G et al.; Hereditary muscle channelopathies are caused by dominant mutations in the genes encoding for subunits of muscle voltage-gated ion channels . Point mutations on the human skeletal muscle Na+ channel (Nav1.4) give rise to hyperkalemic periodic paralysis, potassium aggravated myotonia, paramyotonia congenita and hypokalemic periodic paralysis type 2 . Point mutations on the human skeletal muscle Ca2+ channel give rise to hypokalemic periodic paralysis and malignant hyperthermia . Point mutations in the human skeletal chloride channel CIC-1 give rise to myotonia congenita . Point mutations in the inwardly rectifying K+ channel Kir2.1 give rise to a syndrome characterized by periodic paralysis, severe cardiac arrhythmias and skeletal alterations (Andersen's syndrome) . Involvement of the same ion channel can thus give rise to different phenotypes . In addition, the same mutation can lead to different phenotypes or similar phenotypes can be caused by different mutations on the same or on different channel subtypes . Bearing in mind, the complexity of this field, the growing number of potential channelopathies (such as the myotonic dystrophies), and the time and cost of the genetic procedures, before a biomolecular approach is addressed, it is mandatory to apply strict diagnostic protocols to screen the patients . In this study we propose a protocol to be applied in the diagnosis of the hereditary muscle channelopathies and we demonstrate that muscle biopsy studies and muscle cell cultures may significantly contribute towards the correct diagnosis of the channel involved . DNA-based diagnosis is now a reality for many of the channelopathies . This has obvious genetic counselling, prognostic and therapeutic implications. Tsitologiia, 2003, 45(1), 51 - 8 {Effect of some polycyclic aromatic hydrocarbons on gap junction intercellular communication in hepatoma Hep G2 cell culture}; Sharovskaia IuIu et al.; Systems regulating tissue homeostasis are gap junction intercellular communications (GJIC) . It is accepted that the down-regulation of GJIP has been due to tumor promoting properties of carcinogens . In this study, effects of some carcinogenic and noncarcinogenic polycyclic aromatic hydrocarbons (PAH) on GJIC were investigated . Noncarcinogenic PAHs do not influence GJIC function . In dose 5 microg/ml carcinogenic PAHs down-regulated GJIC by 70-100% after a 24 h treatment . Dependent on the structure of PAHs, down-regulation was observed after a 1 h treatment . The methyl group in PAH structure decreased down-regulation of GJIC in 1 h experiments, whereas after a 24 h treatment the down-regulation caused by methyl group either contained or not contained PAH was nearly the same . To clarify the role of Ah-receptor in PAH action on GJIC, the effect of 2,3,7,8-tetrachlorodibezdioxin, a specific ligand of Ah-receptor was studied, which appeared to be insignificant . Benzo/a/pyrene does not influence the functioning of gramicidine channels formed in the phospholipid membrane . This result indicates that PAH action on GJIC is not associated with non-specific destruction of the membrane . Thus, two steps are there in PAH action on GJIC: one is fast and caused by specific interaction of unchanged PAH molecule, the other develops in time and is presumably associated with the formation of active metabolites. Neurosci Lett, 2003 Apr 24, 341(1), 61 - 4 Chronic treatment with ionotropic glutamate receptor antagonist kynurenate affects GABAergic synaptic transmission in rat hippocampal cell cultures; Ivanova SY et al.; It is well documented that prolonged treatment with antagonists of ionotropic glutamate receptors activates a number of homeostatic mechanisms including alteration of glutamatergic transmission . We studied whether this treatment can also affect GABAergic transmission . Using whole-cell voltage clamp recording and local extracellular stimulation we investigated evoked inhibitory postsynaptic currents (IPSCs) in cultured rat hippocampal neurons grown in the presence of ionotropic glutamate receptor antagonist kynurenate (1 mM) and in control conditions . Chronic kynurenate treatment did not significantly affect the amplitude of evoked IPSCs and IPSC reversal potentials . In contrast we found that the paired-pulse depression was increased by 67% in cultures treated with kynurenic acid . We conclude that additional mechanism(s), alteration of GABAergic synaptic transmission, may contribute to homeostatic plasticity induced by chronic block of ionotropic glutamate receptors. Biotechnol Prog, 2003 Mar-Apr, 19(2), 624 - 30 Use of flow cytometry to monitor infection and recombinant human alpha-1,3/4 fucosyltransferase production in baculovirus infected Sf9 cell cultures; Deparis V et al.; This paper describes the setup and the use of a flow cytometric method for monitoring Sf9 insect cell infection by a recombinant baculovirus expressing the human alpha1,3/4 fucosyltransferase Fuc-TIII . Using side scattered light coupled to green fluorescence detection after immunolabeling of the recombinant protein, this method made it possible to monitor baculovirus infection of Sf9 cells grown in batch cultures and infected at different cell densities and multiplicities of infection . The method was able to precisely assess the extent of infection of the insect cells from 60 h postinfection . In asynchronously infected Sf9 cell cultures, the two-step infection process (primary and secondary infection) was well-characterized using this technique . Finally, a reduced sensitivity to baculovirus infection was observed for cells infected at the end of the growth phase compared to the cells infected during exponential growth phase. Biotechnol Prog, 2003 Mar-Apr, 19(2), 243 - 53 Integration of cell culture and microfabrication technology; Park TH et al.; Recent progress in cell culture and microfabrication technologies has contributed to the development of cell-based biosensors for the functional characterization and detection of drugs, pathogens, toxicants, and odorants . The cell-based biosensors are composed of two transducers, where the primary transducer is cellular and the secondary transducer is typically electrical . Advances in gene manipulation and cell culture techniques have contributed to the development of the cell as a transducer, while microfabrication techniques have been applied to the development of integrating the cell with the second transducer . Cellular patterning using microfabrication techniques is essential for cell-based biosensors, cell culture analogues, tissue engineering, and fundamental studies of cell biology . The photolithographic technique is highly developed and has been widely used for patterning cells . Recently, a set of alternative techniques, largely based on soft lithoghraphy, has been developed for biological applications . Those techniques include microcontact printing, microfluidic patterning using microchannels, and laminar flow patterning . A classical metallic stencil patterning method has been improved by employing a rubber-like stencil . These cellular micropatterning techniques have been usefully employed to understand questions in fundamental cell biology, especially cellular interactions with various materials and other cells . Using these micropatterning tecchniques and insights into the interaction of cellular biology with surfaces, a wide array of biosensors have been developed . In this manuscript examples of cell-based biosensors are described . Neurons have a great potential for use in a cell-based biosensor because they are electrically excitable cells, from which electrical signals are generated with the binding of detecting molecules . Consequently, the electrical signals generated in the cell can be determined in a noninvasive manner . A microphysiometer is a device to detect functional responses from cells by measuring the change of extracellular pH . The main application of the microphysiometer is the analysis of functional responses of cells upon receptor stimulation . Development of a microscale cell culture analogue system, an in vitro animal or human surrogate, is another promising area using cell culture and microfabrication technologies . Such devices are potentially very useful in the fields of toxicology and drug testing because they may increase the accuracy of in vitro predictions, simplify testing procedures, and reduce the cost of such tests, allowing many more tests to be done with a limited set of resources. Sheng Wu Gong Cheng Xue Bao, 2002 Nov, 18(6), 709 - 12 {Biocompatibility of SA/CS-CaCl2/PMCG Microcapsule in cell culture}; Zhang LY et al.; A novel multi-components microcapsule--SA/CS-CaCl2/PMCG system was introduced . The effects of PMCG and SA/CS-CaCl2/PMCG microcapsules on the growth of free E . coli and Saccharomyces cerevisiae were studied respectively . In addition, the growth of immobilized E . coli and Saccharomyces cerevisiae were also investigated . The results showed that: Just like other synthetic polycations, PMCG above certain concentration (0.5%) strongly inhibited the growth of free E . coli and Saccharomyces cerevisiae, but SA/CS-CaCl2/PMCG microcapsules almost had no effects on their growth and on the consumption of glucose concentration by Saccharomyces cerevisiae . What's more, immobilized E . coli and Saccharomyces cerevisiae grew almost as normally as free cultivation . As a whole, SA/CS-CaCl2/PMCG microcapsules had good biocompatiability and can be used as a new immobilization system. Cells Tissues Organs, 2003, 173(3), 129 - 37 Dural cell culture . A new approach to study duraplasty; Schick B et al.; Fistulas of the cerebrospinal fluid are often repaired by insertion of grafts of various kinds . However, current knowledge of wound healing after graft insertion is limited, and only a few animal studies are available . The objective of this study is to test whether an in vitro model is suited to analyze cellular healing aspects after duraplasty and to assess dura substitutes in such conditions in regard to their surface attractiveness for cellular migration from the dura margins . Harvested dura pieces from minipigs were perforated to mimic central dura lesions, placed on various coated surfaces (collagen, laminin, poly-L-lysine) or grafts, and investigated in a cell culture for cellular closure of the perforation . Cellular migration from the dura into the central perforation was noted on collagen-coated surfaces and when defects were filled with collagen gels, but there was no cell growth on surfaces with poly-L-lysine or laminin coating . Immunocytochemistry identified the migrating cells mainly as fibroblasts with some intermingled epithelial cells . Scanning electron microscopy proved cellular closure of defects after dura placement on allogenic non-crosslinked collagen transplants . Less cellular migration was observed on poly-P-dioxanon sheets, while no cells migrated into the central dura perforation after placement on a cartilage substitute . Cell counting indicated enhanced cellular closure of the dura opening after introduction of insulin or fibroblast growth factor (sign test for both: 0.031) . Our study succeeded in establishing a cell culture model for duraplasty and indicated cellular migration from the dura borders at the site of the defect during the wound healing process . The cell culture model presented in this report shows that collagen grafts are best suited for duraplasty . In accordance with the immunocytological finding of fibroblast migration from the dura borders additional application of fibroblast-stimulating growth factors accelerated cellular defect closure . Biol Pharm Bull, 2003 Apr, 26(4), 544 - 6 Quercetin attenuates oxygen-glucose deprivation- and excitotoxin-induced neurotoxicity in primary cortical cell cultures; Ha HJ et al.; The possible role of quercetin, a naturally occurring plant flavonoid, in protecting against oxygen-glucose deprivation (OGD)-, excitotoxins-, and free radical-induced neuronal injury in mouse cortical cell cultures was investigated . Pre- and co-treatment with quercetin (100 microM) inhibited 50 min OGD-, 20 microM N-methyl-D-aspartate (NMDA)-, and 50 microM kainate-induced neurotoxicity by 36, 22, and 61%, respectively . Quercetin significantly ameliorated free radical-induced neuronal injury caused by buthionine sulfoximine, sodium nitroprusside, ZnCl(2), and FeCl(2) . These results suggest that quercetin may contribute a neuroprotective action against ischemic neural injury, partially via antioxidant actions. Int J Parasitol, 2003 Mar, 33(3), 229 - 34 A Hammondia-like parasite from the European fox (Vulpes vulpes) forms biologically viable tissue cysts in cell culture; Schares G et al.; Tissue cysts of parasites of the genus Hammondia are rarely described in naturally or experimentally infected intermediate hosts . However, ultrastructural examinations on tissue cyst stages of Hammondia sp . are needed, e.g . to compare these stages with those of Neospora caninum and other related parasites . We describe a cell culture system employed to examine the in vitro development of tissue cysts of a Hammondia sp.-like parasite (isolate FOX 2000/1) which uses the European fox as a definitive host . Cells of a diploid finite cell line from embryonal bovine heart (KH-R; CCLV, RIE 090) were infected by inoculation of sporozoites und cultivated for up to 3 months . Transmission electron microscopic examination of 17 day old cell culture material revealed the presence of cyst walls . Infected cell cultures cultivated for 2 months were used to feed a fox . Six to 13 days post infection the fox shed large numbers (n=1.2 x 10(7)) of Hammondia-sp . like oocysts which could not be distinguished from those used to infect the cell culture as determined by DNA sequencing of the internal transcribed spacer 1 and the D2/D3 domain of the large subunit ribosomal DNA . To find out the proportion of parasitophorous vacuoles that had developed into tissue cysts, the expression of bradyzoite markers was examined by probing infected cell cultures with mouse polyclonal antibodies against Toxoplasma gondii bradyzoite antigen 1 (anti-BAG1) and rat monoclonal antibodies against a cyst wall protein (mAbCC2) . Nineteen and 90 days post infection all parasitophorous vacuoles in the cell cultures were positive with anti-BAG1 and mAbCC2 . This shows that biologically viable (i.e . infectious) tissue cysts of a fox-derived Hammondia sp . isolate (FOX 2000/1) can be efficiently produced in this cell culture system . Since in vitro cystogenesis of dog-derived Hammondia heydorni has not been observed yet, in vitro cyst formation might be one trait to separate fox-derived Hammondia sp . from H . heydorni on a species level. Pharm Res, 2003 Mar, 20(3), 373 - 81 An improved cell culture model based on 2/4/A1 cell monolayers for studies of intestinal drug transport: characterization of transport routes; Tavelin S et al.; PURPOSE: To improve the viability of the 2/4/A1 cell culture model and to investigate different routes of drug transport in this cell line . METHODS: Two approaches were taken to decrease apoptosis . First, rat intestinal 2/4/A1 cells were transfected to overexpress the antiapoptotic protein Bcl-2 . Second . normal 2/4/A1 cells were cultivated under conditions that stimulate differentiation and limit apoptosis . The monolayer integrity was investigated by transepithelial electrical resistance, permeability, and microscopy . The expression of drug transporters was investigated by RT-PCR, and transport function was assessed using specific markers . RESULTS: Normal 2/4/A1 cells died by apoptosis at 39 degrees C . Bcl-2-expressing 2/4/A1 cells were viable but adopted a morphology of less-differentiated epithelial cells . Optimization of the culture conditions for 2/4/A1 cells inhibited cell death . The integrity was comparable to that of the human jejunum (50 omega x cm2), making this approach preferable to Bcl-2 overexpression . Transcriptional analysis showed that some (e.g., MDRI) . but not all (e.g., PepT1), transporters were found in 2/4/A1 cells . Studies using substrates for PepT1, P-gp . MRP2, and BCRP showed that none of the transporters were functional in 2/4/A1 . CONCLUSIONS: The improved culture procedure will facilitate the use of 2/4/A1 cells . 2/4/A1 lack several transporters, which makes them a promising alternative to Caco-2 cells and artificial membranes in studies of passive drug transport. J Endod, 2003 Mar, 29(3), 201 - 4 Proinflammatory cytokines induce cyclooxygenase-2 mRNA and protein expression in human pulp cell cultures; Chang YC et al.; The increased release of prostaglandins (PG) within pulpal tissues is considered to play a pathogenic role during pulpal disease progression . The rate-limiting step in the formation of PG from arachidonic acid is catalyzed by cyclooxygenase (COX) . COX-2 is an inducible enzyme believed to be responsible for PG synthesis at site of inflammation . The effect of proinflammatory cytokines on human pulp cells with special reference to COX-2 expression has not been reported earlier . The aim of the present study was to investigate the effects of interleukin (IL)-1alpha and tumor necrosis factor-alpha (TNF-alpha) on the expression of COX-2 mRNA gene and protein in cultured human pulp cells . Investigations of the time dependence of COX-2 mRNA expression in proinflammatory cytokines-treated human pulp cells revealed a rapid accumulation of the transcript, a significant signal first detectable 1 h after exposure . In addition, both IL-1alpha and TNF-alpha up-regulated COX-2 protein expression by human pulp cells . The kinetics of this response showed that COX-2 was detectable in cell lysates as early as 2 h post proinflammatory cytokines challenge and remained elevated throughout the 24-h incubation period . This suggests that one of the pathogenic mechanisms of pulpal inflammation in vivo may be the synthesis of COX-2 by resident cells in response to a proinflammatory cytokines challenge . COX-2 may play an important role in the regulation of prostanoid formation in the pathogenesis of pulpal inflammation . Taken together, we propose that the use of selective COX-2 inhibitors might provide a valuable tool in the control of pulpal inflammation. J Virol Methods, 2003 Apr, 109(1), 47 - 54 Comparison of cell culture-grown JC virus (primary human fetal glial cells and the JCI cell line) and recombinant JCV VP1 as antigen for the detection of anti-JCV antibody by haemagglutination inhibition; Knowles WA et al.; JC virus (JCV) is the causative agent of the demyelinating disease progressive multifocal leucoencephalopathy (PML), which can be diagnosed by detection in the cerebrospinal fluid (CSF) of both JCV DNA and intrathecally-produced anti-JCV antibody . However, the restricted in-vitro species and cell tropism shown by JCV has made antigen production difficult and limited serological investigations both in PML diagnosis and for JCV epidemiology . In this study antigen prepared as a crude cell lysate of JCV-infected primary human fetal glial (PHFG) cells was compared in a haemagglutination inhibition (HI) assay with antigen produced from the JCV carrier cell line, JCI, and yeast-expressed JCV VP1 . Forty-two sera were tested with each antigen and there was a high level of correlation between the assays: 96.5% between the HI assays with PHFG and JCI antigens and 98.1% between the HI assays with PHFG and recombinant VP1 (rVP1) antigens . The JCI antigen gave HI titres 19% lower than the PHFG antigen (P=0.022) . Titres with the rVP1 antigen were 2% higher than with the PHFG antigen (P=0.83) . When serum/CSF pairs from 11 PML patients were tested, the antibody index calculated in each case confirmed the production of intrathecal anti-JCV antibody . Antibody testing for JCV is no longer reliant on PHFG cells and JCV serological tests should be available more widely. Anim Health Res Rev, 2002 Dec, 3(2), 57 - 68 Applications of a cell culture system for studying the interaction of Anaplasma marginale with tick cells; Blouin EF et al.; A cell culture system for the tick-borne rickettsia Anaplasma marginale offers new opportunities for research on this economically important pathogen of cattle . A . marginale multiplies in membrane-bound inclusions in host cells . Whereas erythrocytes appear to be the only site of infection in cattle, A . marginale undergoes a complex developmental cycle in ticks and transmission occurs via the salivary glands during feeding . We recently developed a cell culture system for A . marginale using a cell line derived from embryos of Ixodes scapularis . Here we review the use of this cell culture system for studying the interaction of A . marginale with tick cells . Several assays were developed using the A . marginale/tick cell system . An adhesion assay was developed for the identification of proteins required by A . marginale for adhesion to tick cells . The effect of antibodies against selected major surface proteins in inhibiting A . marginale infection was tested in an assay that allowed further confirmation of the role of surface proteins in the infection of tick cells . A drug screening assay for A . marginale was developed and provides a method of initial drug selection without the use of cattle . The culture system was used to test for enhancing effects of tick saliva and saliva components on A . marginale infection . The tick cell culture system has proved to be a good model for studying A . marginale-tick interactions . Information gained from these studies may be applicable to other closely related tick-borne pathogens that have been propagated in the same tick cell line. Arch Ital Biol, 2003 Feb, 141(1), 1 - 10 Metabolism of {1-13C} glucose in extracts and in immobilized rat glioma C6 cell cultures: effects of hypoxia; Perrin A et al.; The metabolism of {1-13C} glucose was followed in C6 rat glioma cells immobilized on a gel thread and in perchloric extracts of the same cells in culture . The results showed that the main metabolite of {1-13C} glucose is {3-13C} lactate . The effects of hypoxia were followed in the perchloric acid extracts of C6 cells . In normoxic conditions, the main metabolites produced by the cells were {3-'3C} lactate, {3-13C} alanine, {2-13C}, {3-13C} and {4-13C} glutamate . Lactate newly synthesized from glucose appeared to be exported in the perfusion medium when living cells were immobilized in gel threads made of extracellular matrix . After 5 h of hypoxia, the lactate labelling measured in PCA cell extracts was increased that of glutamate decreased and the appearance of a spectral line at 66.01 ppm, identified as {1-13C} glycerol-3-phosphate, was observed . The data suggest that the synthesis of glycerol-3-phosphate in these cells might represent a sign of hypoxia. Biomed Mater Eng, 2003, 13(1), 1 - 9 Three-dimensional model of bone marrow stromal cell culture; Januszewski M et al.; In a hematopoietic microenvironment in vivo, spatial organisation of hematopoiesis is possible due to the existence of a three-dimensional framework, the main part of which is formed by a branching population of stromal cells . Most of the previous in vitro studies, concerning long-term bone marrow cultures, were based on a previously prepared, flat adherent layer of stromal cells . There are only few reports concerning the three-dimensional growth pattern of the bone marrow stroma . In the present study we used a new three-dimensional model of the stromal cell culture . The framework for the cultured stromal cells was a structure of a nonliving trabecular bone (Unilab Surgibone) . After a period of about four weeks the stromal cells created a spatial network which filled the intertrabecular spaces of the spongy bone. J Chromatogr B Analyt Technol Biomed Life Sci, 2003 Mar 25, 786(1-2), 161 - 76 Optimizing expression and purification from cell culture medium of trispecific recombinant antibody derivatives; Willems A et al.; Antibody fragments offer the possibility to build multifunctional manifolds tailored to meet a large variety of needs . We optimized the production of a manifold consisting of one (bibody) or two (tribody) single-chain variable fragments coupled to the C-terminus of Fab chains . Different strong mammalian promoters were compared and the influence of expression media on production and recovery was investigated . Since the physical and chemical nature of these molecules largely depends on the nature of the antibody building blocks incorporated, a generally applicable process for the purification of recombinant antibody derivatives from serum containing mammalian cell culture medium was designed . To this end we compared protein L, hydroxyapatite, immobilized metal affinity chromatography, cation-exchange chromatography and hydrophobic charge induction chromatography. J Chromatogr B Analyt Technol Biomed Life Sci, 2003 Apr 25, 787(2), 415 - 9 Simple liquid-chromatographic method for Nile Red quantification in cell culture in spite of photobleaching; Lamprecht A et al.; Nile Red fluorescent marker is widely-used for different purposes, such as staining cell structures and for the visualization and localization of colloidal drug carriers . However, when fluorescence-dependent imaging or quantification is performed, the risk of inexact results is increased due to photobleaching . The proposed, simple quantification method of using an HPLC-UV-Vis system allows the determination of Nile Red even in photobleached samples . The intra- and inter-assay accuracies for all analytes were found to be within 94.9 and 100.8%, respectively, of target values . When samples underwent photobleaching by laser, UV-Vis detection varied at around 99+/-5%, whereas fluorescence decreased down to 86% . Such results show this method to be interesting for approaches where quantification should be performed after analysis such as fluorescent imaging. J Med Dent Sci, 2002 Dec, 49(4), 109 - 20 High extracellular calcium affects osteoclastogenesis in mouse bone marrow cell culture; Takahashi E et al.; The effects of high extracellular calcium (high Ca) in the local microenvironment on osteoclasts, osteoclast progenitors and stromal cells are not fully understood . We examined high Ca effect on osteoclastogenesis in mouse bone marrow cell culture . Mouse bone marrow cells were cultured for up to 6 days in the medium supplemented with 1, 25(OH)2 vitamin D3 (D3) . High Ca treatment at the early stage of culture (the initial 24 hours) reduced the number of tartrate resistant acid phosphatase-positive multinuclear cells (TRAP(+)MNCs) . This treatment slightly up-regulated the mRNA expressions of receptor activator of NF-(B ligand (RANKL), RANK and osteoprotegerin (OPG) . This inhibitory effect on the formation of TRAP(+)MNCs was recovered by RANKL . In contrast, high Ca treatment at the later stage of osteoclastogenesis (the last 2 days of culture) stimulated the formation of TRAP(+)MNCs, increased RANKL and RANK mRNA expressions and decreased OPG mRNA . High Ca at neither the early nor the later stage of culture affected the total number of adherent cells and the mRNA expression of alkaline phosphatase and osteopontin . In conclusion, high Ca affects osteoclastogenesis in a manner depending on the stage of osteoclastogenesis, which is partly mediated via the RANKL-RANK-OPG regulatory system. Dermatol Online J . 2003 Feb;9(1):4. Pilot study of a novel treatment for androgenetic alopecia using enriched cell culture medium: clinical trials; Lindenbaum ES et al.; Androgenetic Alopecia (AA) afflicts a large part of the population and of the many treatments available today none is completely satisfactory . Testing the efficacy and safety of a novel topical treatment for AA which is based on cell culture medium supplemented with insulin, thyroxin and growth hormone (CCM) . The 48 participants classified as androgenetic alopecia Type II, III or IV on the Hamilton scale, concluded a randomized, vehicle-controlled, double-blind trial of 6 months duration . Under occlusive cover the gel was self applied for at least 3 hours daily . Evaluation was based on hair counts, investigator global assessment and participants self-administered questionnaire . Cessation of hair loss was reported by most participants within 28 weeks, and further confirmed by the hair count (HC) in ~80% of participants . Moreover, as early as 4 months after the start of the treatment, a time dependent increase of up to 50% in HC was observed . The average change in HC between the two groups differed significantly (p=0.007), with values of 4.1% for control and 13.8% for CCM . Following 4 months of treatment, a time dependent increase in HC (>10%) above minimal was observed in 55% of the CCM and 25% of the control and this trend continued . At 6 months 63% of the CCM and 33% of the control group exhibited increase of HC higher than 10% . The average increase in HC in the CCM and the control groups was 17.1% and 8.9% respectively (p=0.035) . Self evaluation questionnaires revealed a time dependent increase in satisfaction in the CCMusers compared to the control . While the average score at T2 was similar in CCM and control (2.7 and 2.6 respectively), the score at T6 in the CCM increased to 5.9 and decreased to -0.4 in the control (p=0.007) . Global-clinical evaluation following six months treatment revealed significantly (p=0.02) more hair loss in the control group (40%) compared to the CCM (7%) treated group . CCM was found effective in treating androgenetic alopecia in men . It induced cessation of hair loss, increased rate of hair growth and appearance of new hair . No side effects were reported or observed. Exp Anim, 2003 Jan, 52(1), 43 - 52 D-galactosamine induced hepatocyte apoptosis is inhibited in vivo and in cell culture by a calcium calmodulin antagonist, chlorpromazine, and a calcium channel blocker, verapamil; Tsutsui S et al.; Studies were conducted in C57BL/6N Crj male mice and in cultured hepatocytes to clarify the relationship between galactosamine (GaIN) induced apoptosis and {Ca2+}i kinetics . Chlorpromazine (CPZ), a Ca(2+)-calmodulin antagonist, and verapamil (VR), a Ca(2+)-channel blocker each inhibited GaIN-induced DNA fragmentation and the appearance of apoptotic bodies . The kinetics of calcium uptake were evaluated using a calcium analyzer with the acetoxymethyl ester of fura-PE3 (fura-PE3/AM, 2.5 microM) as the calcium reporter . An increase in {Ca2+}i was detected in the cultured hepatocytes within 3 hours after treatment with 20 mM GaIN; this increase was inhibited by pretreatment with either 20 microM CPZ or 30 microM VR . Ca2+ imaging by confocal laser scanning microscopy showed that increase in {Ca2+}i after treatment with GaIN was initially localized around nuclei, while {Ca2+}i signals were later diffuse and observed throughout the cytoplasm . The activities of lactate dehydrogenase (LDH) and serum glutamate-pyruvate transaminase (sGPT), used as indicators of plasma membrane damage and leakage, however, were not reduced by pretreatment with CPZ or VR . From these findings, we infer that the DNA fragmentation in GaIN-induced hepatocyte apoptosis is associated with an elevation in the perinuclear concentration of Ca2+, but GaIN-induced necrotic cell death is triggered through pathway(s) that are insensitive to blockage of Ca2+ influx and therefore appear to occur independently of elevation in {Ca2+}i . These results help to clarify the role of calcium flux in hepatocyte apoptosis and necrosis induced by exposure to hepatotoxins in vivo and in vitro. Am J Physiol Cell Physiol, 2003 Jul, 285(1), C48 - 55 Epub 2003 Mar 12. Posttranslational inactivation of human xanthine oxidoreductase by oxygen under standard cell culture conditions; Linder N et al.; Xanthine oxidoreductase (XOR) catalyzes the final reactions of purine catabolism and may account for cell damage by producing reactive oxygen metabolites in cells reoxygenated after hypoxia . We found a three- to eightfold higher XOR activity in cultured human bronchial epithelial cells exposed to hypoxia (0.5-3% O2) compared with cells grown in normoxia (21% O2) but no difference in XOR protein or mRNA . XOR promoter constructs failed to respond to hypoxia . The cellular XOR activity at 3% O2 returned to basal levels when the cells were returned to 21% O2, and hyperoxia (95% O2) abolished enzyme activity with no change in XOR protein . Our data suggest reversible enzyme inactivation by oxygen or its metabolites . NADH was normally oxidized by the oxygen-inactivated enzyme, which rules out damage to the flavin adenine dinucleotide cofactor . Hydrogen peroxide partially inactivated the molybdenum center of XOR, as shown by a parallel decrease in XOR-catalyzed xanthine oxidation and dichlorophenolindophenol reduction . We conclude that the transcription or translation of XOR is not influenced by hypoxia or hyperoxia . Instead, the molybdenum center of XOR is posttranslationally inactivated by oxygen metabolites in "normal" (21% O2) cell culture atmosphere . This inactivation is reversed in hypoxia and accounts for the apparent induction. J Virol, 2003 Apr, 77(7), 4401 - 8 Cytoskeletal requirements for hepatitis C virus (HCV) RNA synthesis in the HCV replicon cell culture system; Bost AG et al.; Hepatitis C virus (HCV) induces microtubule aggregates in infected hepatocytes . To determine if cytoskeletal elements are important for HCV RNA synthesis, we examined the effect of cytoskeleton inhibitors on HCV replicon transcription in Huh7 cells . The data demonstrate that HCV replication complex-mediated RNA synthesis requires microtubule and actin polymerization. J Biomed Mater Res B Appl Biomater, 2003 Apr 15, 65(1), 157 - 62 IGF-I and TGF-beta 1 incorporated in a poly(D,L-lactide) implant coating stimulates osteoblast differentiation and collagen-1 production but reduces osteoblast proliferation in cell culture; Schmidmaier G et al.; Previous in vivo studies revealed a stimulating effect of locally applied IGF-I and TGF-beta1 released from poly(D,L-lactide)-coated titanium implants on rat and porcine fracture healing . The purpose of the present study was to evaluate the effect of IGF-I (5% w/w) and TGF-beta1 (1% w/w) and the carrier PDLLA on osteoblasts in cell culture to improve the understanding of these growth factors . The well-characterized human osteoblast cell line hFOB 1.19 was used in the study . The implants and cells were cocultured in a noncontact manner . The cells were incubated for 10 days in total, and the implants (n = 6 each group and time point) were added for 1 h, 12 h, 24 h, 2 d, 4 d, or 10 d . To analyze a possible effect of the growth factors or the coating, cell proliferation, metabolism, and differentiation were investigated . As an indicator for differentiation the production of collagen I was chosen . All experimental groups showed comparable cell vitality . No change in the pH of the medium was detectable between the analyzed groups . When the effect of the titanium implant and the PDLLA coating were compared with the control culture, no differences in proliferation, metabolic activity, and collagen I production were detectable . The osteoblasts treated with IGF-I and TGF-beta1 released from PDLLA revealed a significantly enhanced collagen I production with a decrease in proliferation and metabolic activity compared to the other groups . No significant differences in collagen I production were seen due to the incubation time points . None of the experimental groups evoked an immunological response on mouse macrophages . In conclusion, the PDLLA-carrier showed no negative effect on osteoblasts, whereas the incorporated growth factors stimulated osteoblast differentiation . J Endocrinol, 2003 Mar, 176(3), 339 - 48 Vitamin K stimulates osteoblastogenesis and inhibits osteoclastogenesis in human bone marrow cell culture; Koshihara Y et al.; Accumulating evidence indicates that menaquinone-4 (MK-4), a vitamin K(2) with four isoprene units, inhibits osteoclastogenesis in murine bone marrow culture, but the reason for this inhibition is not yet clear, especially in human bone marrow culture . To clarify the inhibitory mechanism, we investigated the differentiation of colony-forming-unit fibroblasts (CFU-Fs) and osteoclasts in human bone marrow culture, to learn whether the enhancement of the differentiation of CFU-Fs from progenitor cells might relate to inhibition of osteoclast formation . Human bone marrow cells were grown in alpha-minimal essential medium with horse serum in the presence of MK-4 until adherent cells formed colonies (CFU-Fs) . Colonies that stained positive for alkaline phosphatase activity (CFU-F/ALP(+)) were considered to have osteogenic potential . MK-4 stimulated the number of CFU-F/ALP(+) colonies in the presence or absence of dexamethasone . The stimulation was also seen in vitamin K(1) treatment . These cells had the ability to mineralize in the presence of alpha-glycerophosphate . In contrast, both MK-4 and vitamin K(1) inhibited 1,25 dihydroxyvitamin D(3)-induced osteoclast formation and increased stromal cell formation in human bone marrow culture . These stromal cells expressed ALP and Cbfa1 . Moreover, both types of vitamin K treatment decreased the expression of receptor activator of nuclear factor kappaB ligand/osteoclast differentiation factor (RANKL/ODF) and enhanced the expression of osteoprotegerin/osteoclast inhibitory factor (OPG/OCIF) in the stromal cells . The effective concentrations were 1.0 microM and 10 microM for the expression of RANKL/ODF and OPG/OCIF respectively . Vitamin K might stimulate osteoblastogenesis in bone marrow cells, regulating osteoclastogenesis through the expression of RANKL/ODF more than through that of OPG/OCIF. Arch Androl, 2003 Mar-Apr, 49(2), 95 - 105 Glutathione-related enzymes in cell cultures from different regions of human epididymis; Montiel EE et al.; Protection of maturing sperm from potential endogenous or exogenous harmful substances during their transit throughout the epididymis is a critical event . The authors studied the activity of gamma-glutamyl transpeptidase (GGT) and glutathione S-transferase (GST), and glutathione (GSH) levels in epithelial cell cultures from human caput, corpus, and cauda epididymides . Tissue was obtained from patients undergoing therapeutic orchidectomy for prostatic cancer . Enzymatic activity was measured in conditioned media and cellular fractions . Androgen influence was also evaluated . Both enzymatic activities were found in cellular homogenates and conditioned media from cultures of all epididymal regions . GGT activity was highest in cultures from cauda epididymis, both in conditioned media and cell fractions, while GST activity did not show regional differences in conditioned media, but exhibited higher activity in cell homogenates from cauda cultures than those obtained from corpus and caput epididymis . GSH level showed no regional difference in cell homogenates and it could not be detected in conditioned media by the method used . Presence of different concentrations of dihydrotestosterone (DHT) had no influence neither on the enzymatic activities nor GSH concentration . The results indicate that GGT and GST are present along the human epididymis and a fraction or isoform of these enzymes might be secreted to the luminal fluid to play a detoxificative role in sperm maturation. Phytochemistry, 2003 Feb, 62(3), 483 - 9 Thalictrum minus cell cultures and ABC-like transporter; Terasaka K et al.; Cultured Thalictrum minus cells produce a benzylisoquinoline alkaloid, berberine, in the presence of benzyladenine, and excrete it into the culture medium . T . minus cells excluded berberine, even if berberine was exogenously added to the medium, without benzyladenine treatment . Similarly, T . minus cells excluded a heterocyclic dye (neutral red) and calcein AM, which is used as a fluorescent probe to detect the drug efflux pump activity by ABC transporters . The addition of several inhibitors of P-glycoprotein, a representative ABC transporter, induced the accumulation in of both berberine and calcein AM ATP-dependent manner . The expression of P-glycoprotein-like ABC transporter genes was also demonstrated . The involvement of ABC transporter in the secretion of berberine in T . minus cells is discussed. Cancer Lett, 2003 Mar 10, 191(2), 165 - 70 Resistance to the anti-proliferative activity of recombinant arginine deiminase in cell culture correlates with the endogenous enzyme, argininosuccinate synthetase; Shen LJ et al.; Recombinant mycoplasma enzyme, arginine deiminase (rADI), has been proposed as a possible cancer treatment via arginine depletion . However, many cell lines are resistant to rADI-treatment, even though most require arginine for proliferation . We compared eight different cell lines for sensitivity in cell proliferation to the effect of either rADI or arginine deprivation . The activity of argininosuccinate synthetase (AS), the rate-limiting enzyme for converting citrulline to arginine, was also measured . Our results indicate that resistance to rADI-treatment may correlate with cellular AS activity, either constitutive or inducible, allowing cell survival by conversion of the product of the rADI reaction, i.e . citrulline to arginine . J Biol Chem, 2003 May 9, 278(19), 16741 - 6 Epub 2003 Feb 28. Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase; Grobler JA et al.; Efficient replication of hepatitis C virus (HCV) replicons in cell culture is associated with specific sequences not generally observed in vivo . These cell culture adaptive mutations dramatically increase the frequency with which replication is established in vitro . However, replicons derived from HCV isolates that have been shown to replicate in chimpanzees do not replicate in cell culture even when these adaptive mutations are introduced . To better understand this apparent paradox, we performed a gain-of-function screen to identify sequences that could confer cell culture replication competence to replicons derived from chimpanzee infectious HCV isolates . We found that residue 470 in domain II of the NS3 helicase is a critical determinant in cell culture adaptation . Substitutions in residue 470 when combined with the NS5A-S232I adaptive mutation are both necessary and sufficient to confer cell culture replication to otherwise inactive replicons, including those derived from genotype 1b HCV-BK and genotype 1a HCV-H77 isolates . The specific substitution at residue 470 required for replication is context-dependent, with R470M and P470L being optimal for the activity of HCV-BK and HCV-H77 replicons, respectively . Together these data indicate that mutations in the NS3 helicase domain II act in concert with previously identified adaptive mutations and predict that introduction of compatible residues at these positions can confer cell culture replication activity to diverse HCV isolates. J Biotechnol, 2003 Mar 20, 101(3), 275 - 87 Evaluation of phenylboronate agarose for industrial-scale purification of erythropoietin from mammalian cell cultures; Zanette D et al.; The search for novel, cost-effective ways to produce erythropoietin (Epo), the world top-selling biopharmaceutical, is a major challenge for today's biotechnology industry . However, Epo's high glycosylation content (almost 40% of total mass) and the requirement for sialic acid for optimal in vivo activity still make mammalian cells the expression system of choice . In contrast to the abundance of reports on Epo production, robust, cost-effective methods for large-scale Epo purification can hardly be found in literature . To fill this gap, we describe here a process specifically studied for industrial-scale purification of the protein . Our method is based on the ability of phenylboronate agarose (PBA) to form reversible complexes with 1,2-cis-diol-containing molecules, like sugars in glycoproteins . Finding that additional factors (i.e., ionic and hydrophobic interactions) contribute to the Epo-PBA binding reaction, chromatography conditions have been optimized in scale-down experiments to improve selectivity and yield . As a result, the high performance of affinity chromatography has been achieved using a support possessing the robustness, chemical stability and low cost of a small synthetic ligand . By adding an anion exchange chromatography step and gel filtration for polishing, a pure and active product can easily be obtained by an integrated, start-to-end process optimized for industrial-scale operations. Vopr Virusol, 2003 Jan-Feb, 48(1), 26 - 30 {Antiviral effect of alpha-interferon and cytokine mRNA level in cell cultures infected with a cytopathogenic variant of the hepatitis C virus}; Vershinina MIu et al.; An experimental model of the viral C-hepatitis (VCH) infection was worked out in vitro and it was found suitable to study the influence of interferon (IFN) preparations produced on the infection caused by an HCV cytopathogenic variations, i.e . the SW-13 human adrenocarcinoma cellular culture sensitive to the anti-VCH action of alpha-IFN and the MT-4 human lymphoblastoid cellular culture non-sensitive to the anti-VCH action of alpha-IFN . The above cellular models were employed to study, by using the methods of reverse transcription and polymerize chain reaction (RT-PCR), the influence produced by alpha-IFN on the VCH infectious activity as well as to study the changes in the activity of the below cytokine mRNAs: alpha-IFN, gamma-IFN, IL-1 beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-18 and TNF-alpha . A double treatment of the SW-13 alpha-IFN cellular cultures 24 and 48 hours after the infection was found to essentially suppress (by 4 Ig) reproduction of the VCH cytopathogenic variant . It was detected that the VCH reproduction is mediated by the regulation of a number of cytokine genes . The study results can be a basis for a more effective use of the alpha-IFN preparations in the therapy of VCH-infections. Am J Med Genet A, 2003 Apr 1, 118(1), 43 - 8 Rapid detection of 17p11.2 rearrangements by FISH without cell culture (direct FISH, DFISH): a prospective study of 130 patients with inherited peripheral neuropathies; Ravise N et al.; Charcot-Marie-Tooth (CMT) disease and hereditary neuropathy with pressure palsies (HNPP) are two frequent hereditary motor and sensory neuropathies . CMT is characterized by slowly progressive weakness and atrophy, primarily in peroneal and distal leg muscles . The most frequent form, CMT1A, is due, in most cases, to the duplication of a 1.5 Mb region on chromosome 17p11.2 containing the peripheral myelin protein 22 gene (PMP22) . The phenotype seems to result from dosage of the PMP22 gene . This hypothesis is reinforced by the existence of HNPP, which is clinically characterized by various recurrent truncular palsies or sensory loss precipitated by minor trauma, which is caused by deletion of the same 1.5 Mb region in 17p11.2 . In clinical practice, the detection of the duplication or the deletion in 17p11.2, which permits a positive diagnosis, is still performed by time consuming methods (Southern blot or various combinations of molecular tools) . We developed a method for the rapid detection of 17p11.2 rearrangements, using "direct FISH" and PRINS analyses, which does not require cell culture . In a prospective study of 92 patients with CMT and 38 with suspected HNPP, we compared this new technique to classical strategies like Southern blot . The results demonstrate the high sensitivity and specificity of the new FISH technique for the diagnosis of CMT1A and HNPP . Moreover, because of its simplicity and rapidity, this technique provides a useful alternative to the molecular approaches that have been used to diagnose segmental aneusomies, especially in the case of duplications that often go undetected . Toxicol Sci, 2003 Mar, 72(1), 92 - 102 Role of residual additives in the cytotoxicity and cytokine release caused by polyvinyl chloride particles in pulmonary cell cultures; Xu H et al.; Occupational exposure to polyvinyl chloride (PVC) dust has been linked to pulmonary disease . The aim of the present study was to investigate, in vitro, the role of additives in the cytotoxicity and the release of inflammatory mediators caused by PVC particles in different cells . We compared two types of emulsion PVC particles (E3 and E8) with their washed (hence, "additive-free") counterparts (W3 and W8) . A positive control (crystalline SiO2, Min-U-Sil) and the pure additives, sodium lauryl sulfate (A3) and sodium alkylbenzenesulfonate (A8), were tested concurrently . Cytotoxicity (MTT assay) was assessed in primary cultures of rat alveolar macrophages, rat type II pneumocytes, and human alveolar macrophages (h-AM), and cultures of the A549 cell line (type II cell-derived) and the differentiated THP-1 cell line (macrophage-like) . Hemolytic potential was assessed after a 2-h incubation with human erythrocytes . Cytokine release (IL-8, IL-6, and TNF-alpha) by A549 cells, THP-1 cells, and h-AM, was measured by ELISA after 4, 16, 24 and/or 48 h of exposure . Cytotoxicity and hemolytic activity of the washed particles were abolished or markedly decreased compared with their nonwashed forms . In A549 cells, E3 and E8 (2.5 mg/ml) caused a 3-fold increase in IL-8 release and a more than 10-fold increase in IL-6 release, whereas W3 and W8 did not elicit any significant response at similar concentrations . Compared with Min-U-Sil (0.1, 0.5, and 2.5 mg/ml), the response to E3 and E8 occurred later and was slightly lower (IL-8) or much more pronounced (IL-6) . A3 and A8 exhibited similar responses to E3 and E8, at concentrations corresponding to those present in the particles . In conclusion, the in vitro cytotoxicity and inflammatory potential of some PVC particles appear to be mostly due to their residual additives. Rev Argent Microbiol, 2002 Oct-Dec, 34(4), 177 - 85 Detection of rubella-virus-induced apoptosis in Vero cell cultures with hematoxylin and eosin staining; Adamo MP et al.; In order to facilitate the detection of apoptotic cells (Apo C) in Rubella virus (RV) infected cultures in settings of low resources, we compared hematoxylin and eosin staining (H&E) with the conventional TUNEL technique, and confirmed our findings with DNA electrophoresis and transmission electron microscopy . H&E allowed to distinguish Apo C from non-apoptotic cells . The proportion of Apo C in infected cultures was proportional to the multiplicity of infection (MOI) . At a MOI of 10, the percent of Apo C at 3, 4 and 5 days post infection (pi) were 26, 45 and 47%, respectively, which were significantly reduced when the caspase inhibitor z-VAD-fmk was present in the supernatant . By the TUNEL assay, the percent of Apo C in RV-infected cultures were lower (0.8, 1.2 and 1.2% at 3, 4 and 5 days pi, respectively) . Our results have shown that H&E staining is an easy, rapid, economic and reproducible method to detect Apo C in RV infected Vero cells cultures . It is possible that H&E makes evident early stages of apoptosis, when an apoptotic cell shows chromatin condensation, nuclear and cytoplasmic contraction (but is still attached to the monolayer), while TUNEL detects later stages of apoptosis because it needs an extensive DNA fragmentation, when apoptotic cells are about to or have already detached from the substratum. Biosci Biotechnol Biochem, 2002 Dec, 66(12), 2673 - 6 Use of shark collagen for cell culture and zymography; Nomura Y et al.; The uses of shark collagen as a matrix for cell culture and as a substrate for zymography were investigated . Fibroblasts were cultured on a gel matrix of shark type I collagen at 30 degrees C . The collagen gel had contracted by 4 days of incubation . Individual fibroblasts were visible against the transparent background of the contracted collagen as long, lean star-shaped cells . The matrix metalloproteinases (MMPs) from fibroblasts secreted from the medium more easily digested shark gelatin than pig gelatin . MMP-2, -9, and that of potential form were recognizable in the zymographic gel of shark gelatin. J Pharm Belg, 2002 Nov-Dec, 57(6), 153 - 8 Implementation of the caco-2 cell culture model as a predictive tool for the oral absorption of drugs . In-house evaluation procedures; Ingels F et al.; The Caco-2 cell culture model is widely used during drug development and lead optimization as a predictive tool for the oral absorption of drugs . In order to improve the reliability and quality of the results of Caco-2 experiments and to ensure that the system being used is functionally and enzymatically representative for the intestinal mucosa, it is important to perform a validation of the implemented Caco-2 system . In this paper, we summarize evaluation techniques to guarantee the in-house validity of the model . Theophyllin and sodium fluorescein are used as model compounds to evaluate passive transcellular and passive paracellular transport, respectively . Phenylalanine serves as a substrate to demonstrate active carrier mechanisms . Aminopeptidase and dipeptidyl peptidase are two brush border enzymes present in an active form in the Caco-2 culture model . The presence of an active efflux carrier mechanism is demonstrated with cyclosporin A as a substrate. Br J Cancer, 2003 Feb 24, 88(4), 613 - 23 Arginine deprivation, growth inhibition and tumour cell death: 2 . Enzymatic degradation of arginine in normal and malignant cell cultures; Philip R et al.; Arginase added to culture medium reduced arginine to negligible levels within approximately 6 h, and enzyme activity persisted relatively undiminished for at least 3 days . Human and bovine arginase proved equally effective . The response of normal cells was to enter G1 (G0) arrest, from which most of the cells could be recovered weeks later . In contrast, malignant cell lines treated with unpegylated or pegylated enzyme resulted in cell death on a massive scale within 3 - 5 days, with a very low to negligible percentage of cells (<0.01%) being recoverable on restoration with arginine . Although pegylation resulted in a 40% drop in specific activity, arginase was considerably more stable and remained active for >>8 days . Arginine decarboxylase caused malignant cell arrest at the same units per millilitre as arginase . Its breakdown product, agmatine, was relatively nontoxic in the presence of arginine, but exacerbated cell death above millimolar concentration in its absence . Although ornithine failed to rescue cells from deprivation, citrulline recovered cells in all cases, although less well in fast-growing tumour cell populations, whereas readdition of arginine failed to work unless a complete medium change was given (because of the persistence of the enzymes in the medium catabolising its destruction) . The advantages and disadvantages of these two arginine-catabolising enzymes are discussed, and compared with arginine deiminase. Phytochemistry, 2003 Mar, 62(6), 851 - 8 Metabolomic analysis of the consequences of cadmium exposure in Silene cucubalus cell cultures via 1H NMR spectroscopy and chemometrics; Bailey NJ et al.; Several essential and non-essential metals (typically those from periods 4, 5 and 6 in groups 11-15 in the periodic table) are commonly detoxified in higher plants by complexation with phytochelatin . The genetic and gross metabolic basis of metal tolerance in plants is, however, poorly understood . Here, we have analyzed plant cell extracts using 1H NMR spectroscopy combined with multivariate statistical analysis of the data to investigate the biochemical consequences of Cd(2+) exposure in Silene cucubalus cell cultures . Principal components analysis of 1H NMR spectra showed clear discrimination between control and Cd(2+) dosed groups, demonstrating the metabolic effects of Cd(2+) and thus allowing the identification of increases in malic acid and acetate, and decreases in glutamine and branched chain amino acids as consequences of Cd(2+) exposure . This work shows the value of NMR-based metabolomic approaches to the determination of biochemical effects of pollutants in naturally selected populations. Cell Biol Educ, 2002 Spring, 1(1), 26 - 42 Biotechnology apprenticeship for secondary-level students: teaching advanced cell culture techniques for research; Lewis JR et al.; The purpose of this article is to discuss small-group apprenticeships (SGAs) as a method to instruct cell culture techniques to high school participants . The study aimed to teach cell culture practices and to introduce advanced imaging techniques to solve various biomedical engineering problems . Participants designed and completed experiments using both flow cytometry and laser scanning cytometry during the 1-month summer apprenticeship . In addition to effectively and efficiently teaching cell biology laboratory techniques, this course design provided an opportunity for research training, career exploration, and mentoring . Students participated in active research projects, working with a skilled interdisciplinary team of researchers in a large research institution with access to state-of-the-art instrumentation . The instructors, composed of graduate students, laboratory managers, and principal investigators, worked well together to present a real and worthwhile research experience . The students enjoyed learning cell culture techniques while contributing to active research projects . The institution's researchers were equally enthusiastic to instruct and serve as mentors . In this article, we clarify and illuminate the value of small-group laboratory apprenticeships to the institution and the students by presenting the results and experiences of seven middle and high school participants and their instructors. Plant Physiol, 2003 Feb, 131(2), 814 - 23 Rapid alkalinization factors in poplar cell cultures . Peptide isolation, cDNA cloning, and differential expression in leaves and methyl jasmonate-treated cells; Haruta M et al.; A family of peptides inducing rapid pH alkalinization in hybrid poplar (Populus trichocarpa x Populus deltoides) cell culture medium was isolated from hybrid poplar leaves . Five related approximately 5-kD peptides were purified by high-performance liquid chromatography and analyzed by matrix-assisted laser desorption ionization-mass spectrometry . The N-terminal sequence of one of the isolated peptides was very similar to a previously characterized peptide from tobacco (Nicotiana tabacum), rapid alkalinization factor (RALF), which causes a rapid increase in culture medium pH when added to tobacco cell cultures (G . Pearce, D.S . Moura, J . Stratmann, C.A . Ryan {2001} Proc Natl Acad Sci USA 98: 12843-12847) . Two unique poplar RALF cDNAs (PtdRALF1 and PtdRALF2) were isolated from a poplar cDNA library and used to study RALF expression in poplar saplings and cultured poplar cells . Both genes were found to be expressed constitutively in poplar saplings and cultured cells . However, PtdRALF2 was expressed in leaves at very low levels, and its expression in suspension culture cells was transiently suppressed by methyl jasmonate (MeJa) . Although the function of these novel peptides remains enigmatic, our experiments suggest their role may be developmental rather than stress related . Overall, our study confirms the presence of active RALF peptides in other plants, and provides new data on the complexity of the RALF gene family in poplar. J Virol, 2003 Mar, 77(5), 3269 - 80 Evaluation of genetically engineered derivatives of a Chinese strain of foot-and-mouth disease virus reveals a novel cell-binding site which functions in cell culture and in animals; Zhao Q et al.; Adaptation of field isolates of foot-and-mouth disease virus (FMDV) to grow in cells in culture can result in changes in viral properties that include acquisition of the ability to bind to cell surface heparan sulfate (HS) . After 13 passages on BHK cells to produce a vaccine, a Cathay topotype isolate of FMDV serotype O from China (O/CHA/90) extended its cell culture host range and bound to heparin-Sepharose, although it did not require cell surface HS as a receptor molecule . To understand these phenomena, we constructed chimeric viruses by using a type A(12) infectious cDNA and the capsid protein-coding regions of O/CHA/90 and its cell culture-adapted derivative (vac-O/CHA/90) . Using a set of viruses derived from these chimeras by exchanging portions of the capsid-coding regions, we discovered that a group of amino acid residues that surround the fivefold axis of the icosahedral virion determine host range in cell culture and influence pathogenicity in pigs . These residues included aromatic amino acids at positions 108 and 174 and positively charged residues at positions 83 and 172 in protein 1D . To test if these residues participated in non-integrin-dependent cell binding, the integrin-binding RGD sequence in protein 1D was changed to KGE in two different chimeras . Evaluation of these KGE viruses indicated that growth in cell culture was not dependent on HS . One of these viruses was tested in pigs, where it produced a mild disease and maintained its KGE sequence . These results are discussed in terms of receptor utilization and pathogenesis of this important pathogen. J Virol, 2003 Mar, 77(5), 3181 - 90 Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture; Blight KJ et al.; Hepatitis C virus (HCV) genotype 1 (subtypes 1a and 1b) is responsible for the majority of treatment-resistant liver disease worldwide . Thus far, efficient HCV RNA replication has been observed only for subgenomic and full-length RNAs derived from genotype 1b isolates . Here, we report the establishment of efficient RNA replication systems for genotype 1a strain H77 . Replication of subgenomic and full-length H77 1a RNAs required the highly permissive Huh-7.5 hepatoma subline and adaptive amino acid substitutions in both NS3 and NS5A . Replication could be detected by RNA quantification, fluorescence-activated cell sorting, and metabolic labeling of HCV-specific proteins . Replication efficiencies were similar for subgenomic and full-length RNAs and were most efficient for HCV RNAs lacking heterologous RNA elements . Interestingly, both subtype 1a and 1b NS3 adaptive mutations are surface exposed and present on only one face of the NS3 structure . The cell culture-adapted subtype 1a replicons should be useful for basic replication studies and for antiviral development . These results are also encouraging for the development of adapted replicons for the remaining HCV genotypes. J Virol, 2003 Mar, 77(5), 3007 - 19 Viral and cellular determinants of hepatitis C virus RNA replication in cell culture; Lohmann V et al.; Studies on the replication of hepatitis C virus (HCV) have been facilitated by the development of selectable subgenomic replicons replicating in the human hepatoma cell line Huh-7 at a surprisingly high level . Analysis of the replicon population in selected cells revealed the occurrence of cell culture-adaptive mutations that enhance RNA replication substantially . To gain a better understanding of HCV cell culture adaptation, we characterized conserved mutations identified by sequence analysis of 26 independent replicon cell clones for their effect on RNA replication . Mutations enhancing replication were found in nearly every nonstructural (NS) protein, and they could be subdivided into at least two groups by their effect on replication efficiency and cooperativity: (i) . mutations in NS3 with a low impact on replication but that enhanced replication cooperatively when combined with highly adaptive mutations and (ii) . mutations in NS4B, -5A, and -5B, causing a strong increase in replication but being incompatible with each other . In addition to adaptive mutations, we found that the host cell plays an equally important role for efficient RNA replication . We tested several passages of the same Huh-7 cell line and found up to 100-fold differences in their ability to support replicon amplification . These differences were not due to variations in internal ribosome entry site-dependent translation or RNA degradation . In a search for cellular factor(s) that might be responsible for the different levels of permissiveness of Huh-7 cells, we found that replication efficiency decreased with increasing amounts of transfected replicon RNA, indicating that viral RNA or proteins are cytopathic or that host cell factors in Huh-7 cells limit RNA amplification . In summary, these data show that the efficiency of HCV replication in cell culture is determined both by adaptation of the viral sequence and by the host cell itself. Zhongguo Yi Liao Qi Xie Za Zhi, 2000 Jul, 24(4), 187 - 90, 202 {The development of an intellectualized dynamic strain device for three-dimensional cell culture}; Qin TW et al.; The design principles of an intellectualized dynamic strain device for three-dimensional cell cultures based on microcomputer are mainly introduced here, which includes principles of hardware and software design of controlled unit, principle and structure of mechanical unit, and construction of three dimensional scaffold and culture unit . The main technical index and advantages of the device have been also discussed in the paper. J Neurosci Methods, 2003 Feb 15, 123(1), 11 - 22 The establishment of a reliable cytotoxic system with SK-N-SH neuroblastoma cell culture; Ba F et al.; A reliable in vitro cytotoxic system is essential in neurocytotoxic and neuroprotective research . The present study examined four cytotoxic insults with the SK-N-SH human neuroblastoma cell line . These were beta-amyloid protein (Abeta), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), high density culture, and serum deprivation induced neuronal death . These insults induced significant reduction in cell numbers after 96 h culture, in a concentration dependent manner . Among all the insults, MPTP, serum deprivation, and high density culture induced apoptosis after 96 h, while Abeta presumably induced necrotic neuronal death since apoptosis was not detectable . The p38 MAP kinase inhibitor, SB203580 (1 microM), and the PKC inhibitor, chelerythrine (5 microM) successfully inhibited the loss in viability caused by Abeta and the high density culture, respectively . Other kinase inhibitors, including the non-specific protein kinase inhibitor, H7, the PKA inhibitor 14-22 Amide, the PKG inhibitor, KT5823, and the protein tyrosine kinase inhibitor, AG18 had no effect on any of the four cytotoxic models . This system allows the study of neuroprotection under conditions where the different pathways and mechanisms of the neurons can be considered within one cellular system, removing variations which may be due to different cell type studied . The present studies describe an effective model system for screening potential neuroprotective agents. J Interferon Cytokine Res, 2002 Dec, 22(12), 1185 - 9 Thrombopoietin production in human hepatic cell cultures (HepG2) is resistant to IFN-alpha, IFN-beta, and IFN-gamma treatment; Wolber EM et al.; Thrombocytopenia is an important complication of interferon (IFN) therapy for chronic viral hepatitis . To study whether IFN interferes with hepatic thrombopoietin (TPO) synthesis, we used the human hepatoma cell line HepG2 . Our results show that IFN-alpha, IFN-beta, or IFN-gamma did not impair TPO mRNA expression, as determined by quantitative RT-PCR, even when high IFN doses (up to 5000 U/ml) or long-term incubations (up to 14 days) were applied . Neither was the rate of secretion of immunoreactive TPO reduced on IFN treatment . These findings support the concept that IFNs primarily mediate effects on megakaryocytic cells and platelets rather than on TPO-producing hepatocytes. Yan Ke Xue Bao, 1998 Jun, 14(2), 69 - 72 {Study on human eye ciliary muscule cell culture and biologic characteristics}; Huang W et al.; PURPOSE: We cultured human ciliary muscle {HCM} cells to study their growth, ultrastructure, immunohistochemistry and functional characters . METHODS: HCM cells from 10 young donor eyes were cultured with collagenase IV digestion procedures in vitro, the cells were identified by eletronmicroscope and immunohistochemistry assay, their function were studied by single-cell-contraction assay . RESULTS: The cells were passed and grew in Hill-Valley pattern after conflunet; abundant filaments were presented under electronmicroscope . In Desmin protein immunohistochemistry study, the cultured cells were stained positive; in tissue sections, HCM cells stained positive, vascular smooth muscle stained positive weakly, but fibroblast cells and endothelial cells stained negative . 10(-3) M Carbachol could induce the cultured cells contract, this effect was antagonized by 10(-3) M Atropine . CONCLUSION: We successfully cultured HCM cells, which were able to contract. Yan Ke Xue Bao, 1999 Jun, 15(2), 97 - 102 {Calf trabecular cell culture in vitro, morphological and cytoskeletal ultrastructures and kinetics investigation}; Zhong L et al.; OBJECTIVE: To establish the method of culturing calf trabecular cells (CTCs) in vitro, to understand the morphology and function of CTCs, to probe into the effect of resistance of aqueous outflow in the pathogenesis and mechanism about primary open angle glaucoma(POAG) . To direct the clinical using of drugs and to open up new antiglaucomatous medicine by pharmacological studies of CTCs . METHODS: Trabecular meshwork was collected from twenty eyeballs of calf donors after slaughter . The tiusse was primarily cultured and cells were subcultured . The growing characteritics and morphological features of cultured primary and passaged cells were observed by light and electron microscopes . Cell kinetics of the third and tenth passaged cells were analysed using autoradiography and flow cytometry . The influence of the antiglaucomatous drugs 0.25 mg.ml-1 epinephrine (EPI) and 0.025 mg.ml-1 dipivalyl epinephrine (DPE) on cell kinetics of the third passaged cells was studied . RESULTS: The growing characteritics and morphological features of cultured CTCs were as same as those of human trabecular cells . Growing types of CTCs included most of epitheial cell and few of fibroblast . The amount of cellular microfilaments was reduced, DNA synthesis time(Ts) and cell cycle time(Tc) were obviously prolonged with passaged increasing . Antiglaucomatous drugs-EPI (0.25 mg.ml-1) and DPE (0.025 mg.ml-1) made microfilaments dissolving, Ts and Tc obviously prolonging . CONCLUSION: Establishing the method of culturing CTCs in vitro and understanding their morphology, function and pharmacological effects provided an important information for studying human trabecular cells and probing into the effect of resistance of aqueous outflow in the pathogenesis and mechanism about POAG . These studies indicated that antiglaucomatous drugs-EPI (0.25 mg.ml-1) and DPE (0.025 mg.ml-1) influenced obviously microfilaments and cell kinetics of the third passaged cells and suggested that it is not to be ignored that 1% EPI and 0.1% DPE may make CTCs' microfilaments dissolving and may inhibit CTCs' division and proliferation when they are used in clinical therapy. Int J Oncol, 2003 Mar, 22(3), 509 - 15 Novel cell culture models for prevention of human breast cancer (Review); Katdare M et al.; Human breast cancer is a multifactorial, multistep disease wherein genetic, endocrine and dietary factors represent crucial regulators of initiation, promotion and progression . Preclinical investigations utilizing human breast carcinoma derived cell lines either in culture, or upon xenotransplantation, have provided valuable leads for molecular pathogenesis of cancer progression and also for novel therapeutic modalities . The mechanistic significance of genetic factors on early events of initiation/promotion, however, is dependent on extrapolation, and is therefore, equivocal . Human tissue derived explant culture/cell culture models utilizing non-involved target tissue at risk for carcinogenic transformation provide a novel approach that minimizes extrapolation for clinical relevance and thereby maximizes the translational impact . This report provides an overview of laboratory investigations focused on: i) development of the model, ii) optimization of mechanistic biomarker assays for carcinogenic transformation, and iii) validation of the model as a high throughput mechanistic screen for preclinical efficacy of natural phytochemicals. Clin Cancer Res, 2003 Feb, 9(2), 845 - 52 Establishment and characterization of cancer cell cultures and xenografts derived from primary or metastatic Mullerian cancers; Verschraegen CF et al.; PURPOSE:The purpose of this study was to characterize cell cultures and xenografts derived from patients with ovarian cancer . EXPERIMENTAL DESIGN: Ninety specimens from 67 patients were plated in RPMI 1640 or inoculated in nude mice . Growth characteristics of cell cultures and xenografts were determined . Expression of receptors for estrogen, progesterone, androgen, epithelial growth factor, fibroblast growth factor, HER-2/erbB-2/c-neu proto-oncogene, and the P53 expression were characterized by immunocytochemistry in 28 cell cultures . RESULTS: Forty-nine percent of samples were cultured successfully in vitro . Ascitic and pleural effusion specimens were more likely to produce a cell culture or a xenograft than solid tissue specimens (P < 0.005) . All of the cell cultures had an epithelial morphology, and 89% were aneuploid with a mean DNA index of 1.6 (range, 0.9-3.0) . Of 54 and 61 specimens inoculated into nude mice i.p . and s.c., 15 (28%) and 18 (30%) produced a xenograft, respectively, with two-thirds of these xenografts being reproducibly tumorigenic . The median time to first passage was 21 weeks for cell cultures and 8-12 weeks for xenografts . Expression of epithelial growth factor receptor, HER-2/erbB-2/c-neu proto-oncogene, fibroblast growth factor receptor, estrogen, progesterone, and androgen was seen in 24, 21, 31, 17, 43, and 18%, respectively . P53 was overexpressed in 62% of cell cultures analyzed . CONCLUSIONS: Ovarian cancer cells collected from effusions are easier to grow in vitro than in vivo . The only characteristic that may be associated with tumorigenicity was abnormal P53 expression . This panel of ovarian cancer materials provides useful models for biological or therapeutical studies. Biochim Biophys Acta, 2003 Feb 20, 1631(1), 35 - 41 Novel prostaglandin D(2)-derived activators of peroxisome proliferator-activated receptor-gamma are formed in macrophage cell cultures; Soderstrom M et al.; Incubation of RAW 264.7 murine macrophages with 9,15-dihydroxy-11-oxo-, (5Z,9alpha,13E,15(S))-Prosta-5,13-dien-1-oic acid {prostaglandin D(2) (PGD(2))} induced formation of considerable peroxisome proliferator-activated receptor-gamma (PPARgamma) activity {Nature 391 (1998) 79} . Because PGD(2) itself is a poor PPARgamma ligand, we incubated RAW 264.7 macrophage cultures with prostaglandin D(2) for 24 h and studied the ability of the metabolites formed to activate PPARgamma . PGD(2) products were extracted and fractionated by reverse phase high-performance liquid chromatography . Chemical identification was achieved by UV spectroscopy, gas-liquid chromatography/mass spectrometry and chemical syntheses of reference compounds . PGD(2) was converted to eight products, six of which were identified . Ligand-induced interaction of PPARgamma with steroid receptor coactivator-1 was determined by glutathione-S-transferase pull-down assays and PPARgamma activation was investigated by transient transfection of RAW 264.7 macrophages . In addition to the previously known ligand 11-oxo-(5Z,9,12E,14Z)-Prosta-5,9,12,14-tetraen-1-oic acid (15-deoxy-delta(12,14)-PGJ(2)), a novel PPARgamma ligand and activator viz . 9-hydroxy-11-oxo-, (5Z,9alpha,12E,14Z)-Prosta-5,12,14-trien-1-oic acid (15-deoxy-delta(12,14)-PGD(2)) was identified . The biological significance of these results is currently under investigation. Biotechnol Prog, 2003 Jan-Feb, 19(1), 14 - 20 Heat inactivation of mammalian cell cultures for biowaste kill system design; Gregoriades N et al.