Cell cultures

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.; A biowaste kill system was implemented to treat biological waste generated from a clinical manufacturing and R&D antibody facility . To confirm that design parameters of this continuous decontamination system are sufficient to inactivate mammalian cell culture waste, bench-scale experiments were conducted . The biowaste kill system heat inactivates mammalian cell cultures before they are piped to a neutralization tank and subsequently released to the sewage system . Heat inactivation of cells is accomplished by exposing cells to 80 degrees C for 1 min . Small-scale heat inactivation studies were performed on CHO, 293-HEK, and hybridoma cells . Cells at 1 x 10(6) cells/mL or 1 x 10(7) cells/mL were exposed to 37, 60, 70, or 80 degrees C for 0, 30, 60, and 120 s . Viability based on trypan blue exclusion method and ability to proliferate was assessed after exposure to heat . Data suggest that exposure of cells to 80 degrees C for 60 s is sufficient to inactivate these cultures before they are released to the sewage system.

Biometals, 2003 Mar, 16(1), 215 - 23
Iron-induced oxidative stress modify tau phosphorylation patterns in hippocampal cell cultures; Egana JT et al.; Oxidative stress phenomena have been related with the onset of neurodegenerative diseases . Particularly in Alzheimer Disease (AD), oxygen reactive species (ROS) and its derivatives can be found in brain samples of postmortem AD patients . However, the mechanisms by which oxygen reactive species can alter neuronal function are still not elucidated . There is a growing amount of evidence pointing to a role for mitochondrial damage as the source of free radicals involved in oxidative stress . Among the species that participate in the production of oxygen reactive radicals, transition metals are one of the most important . Several reports have implicated the involvement of redox-active metals with the onset of different neurodegenerative diseases such as Alzheimer's Disease (AD), Progressive Supranuclear Palsy (PSP), Amyotrophic Lateral Sclerosis (ALS) and Parkinson's Disease (PD) . On the other hand, our previous studies have indicated that A beta-induced deregulation of the protein kinase Cdk5 associated with tau protein hyperphosphorylation constitute a critical pathway toward neurodegeneration . In the current paper we have shown that iron induces an imbalance in the function of Cdk5/p25 system of hippocampal neurons, resulting in a marked decrease in tau phosphorylation at the typical Alzheimer's epitopes . The loss of phosphorylated tau epitopes correlated with an increase in 4-hydroxy-nonenal (HNE) adducts revealing damage by oxidative stress . This effects on tau phosphorylation patterns seems to be a consequence of a decrease in the Cdk5/p25 complex activity that appears to result from a depletion of the activator p25, a mechanism in which calcium transients could be implicated.

Ann Biomed Eng, 2003 Jan, 31(1), 65 - 79
Nitric oxide delivery system for cell culture studies; Wang C et al.; To investigate the toxicity and mutagenicity of NO, methods are needed to deliver it to cell cultures at known, constant rates . To permit continuous exposures over lengthy periods, we fabricated a simple apparatus utilizing gas-permeable polydimethylsiloxane (Silastic) tubing to supply both NO and O2 to a stirred, cylindrical vessel . Mass transfer in this system was characterized by measuring the delivery rates of NO or O2 alone, and of NO to air-saturated solutions . The concentrations of NO, O2, and NO2- (the end product of NO oxidation) were monitored continuously . The total flux of nitrogen species into the liquid (as determined from the sum of NO and NO2- accumulation) was 50%-90% greater in the presence of O2, depending on the NO partial pressure in the gas . Also, the simultaneously measured mass transfer coefficients for NO and O2 differed greatly from the corresponding unreactive values . An analysis of the data using diffusion-reaction models showed that NO oxidation in the aqueous boundary layer contributed very little to the nitrogen flux increase or to variations in the mass transfer coefficients . However, the unusually strong dependence of the delivery rates on chemical reactions could be explained by postulating that partial oxidation of NO to NO2 occurred within the membrane . The rate constant we estimated for polydimethylsiloxane, 4.4 x 10(5) M-2 s(-1) at 23 degrees C, is only about one-fifth of values reported previously for water and nonpolar solvents, but the high solubilities of NO and O2 in the polymer are sufficient to make NO2 formation significant . Although considerable NO2 is calculated to enter the liquid, its reaction with aqueous NO is rapid enough to keep this undesired compound at trace levels, except within a few microns of the tubing . Thus, cells will have little exposure to NO2

Hum Reprod, 2003 Feb, 18(2), 291 - 8
Characterization of epithelial cell culture from human hydrosalpinges and effects of its conditioned medium on embryo development and sperm motility; Ajonuma LC et al.; BACKGROUND: Recent studies have reported the negative impact of hydrosalpinx on IVF outcome . Toxic effects of hydrosalpinx fluid (HF) have been the main reason for the recommendation of functional surgery, salpingectomy, prior to IVF . The present study characterized hydrosalpinx epithelial cell culture and examined the effects of its conditioned medium (CM) on sperm motility, acrosome reaction and embryo development . METHODS: Normal Fallopian tubes (n = 6) and hydrosalpinges (n = 9) were used to prepare epithelial cell culture and CM . Epithelial cell characterization was confirmed using electron microscopy . Sperm motility and acrosome reaction were determined using computer-aided sperm analysis and acrobead assay respectively and embryo development by mouse embryo development assay . RESULTS: The percentage of human motile sperm incubated in hydrosalpinx CM was significantly different from those in normal Fallopian tube (NFT) CM and modified human tubal fluid medium (hTF) (control) (P < 0.05 at 3 h and P < 0.001 at 5 and 24 h), with alteration in movement characteristic, linearity, 24 h after incubation in hydrosalpinx CM (P < 0.05) . However, other sperm movement characteristics remained unchanged . Reduced acrosome reaction and poor mouse embryo development were also observed in hydrosalpinx CM but not in NFT CM and hTF . CONCLUSIONS: The results suggest that hydrosalpinx epithelial cells may be producing a fluid milieu hostile to sperm and early embryo development . The established epithelial cell culture system may provide a model to further investigate the mechanisms underlying the toxic effects of HF on embryo development and the adverse effects on IVF outcomes.

Appl Environ Microbiol, 2003 Feb, 69(2), 971 - 9
Comparison of method 1623 and cell culture-PCR for detection of Cryptosporidium spp . in source waters; LeChevallier MW et al.; Analysis of Cryptosporidium occurrence in six watersheds by method 1623 and the integrated cell culture-PCR (CC-PCR) technique provided an opportunity to evaluate these two methods . The average recovery efficiencies were 58.5% for the CC-PCR technique and 72% for method 1623, but the values were not significantly different (P = 0.06) . Cryptosporidium oocysts were detected in 60 of 593 samples (10.1%) by method 1623 . Infectious oocysts were detected in 22 of 560 samples (3.9%) by the CC-PCR technique . There was 87% agreement between the total numbers of samples positive as determined by method 1623 and CC-PCR for four of the sites . The other two sites had 16.3 and 24% correspondence between the methods . Infectious oocysts were detected in all of the watersheds . Overall, approximately 37% of the Cryptosporidium oocysts detected by the immunofluorescence method were viable and infectious . DNA sequence analysis of the Cryptosporidium parvum isolates detected by CC-PCR showed the presence of both the bovine and human genotypes . More than 90% of the C . parvum isolates were identified as having the bovine or bovine-like genotype . The estimates of the concentrations of infectious Cryptosporidium and the resulting daily and annual risks of infection compared well for the two methods . The results suggest that most surface water systems would require, on average, a 3-log reduction in source water Cryptosporidium levels to meet potable water goals.

Zhong Yao Cai, 1998 Oct, 21(10), 524 - 6
{Study on the Rheum palmatum volatile oil against HBV in cell culture in vitro}; Zhang B et al.; The antiHBV effect of Rheum palmatum Volatile oil was studied by using 2215 cell line transfected with HBV DNA . At the same time MTT method was applied for the detection of cytoxicity of drugs, selecting acyclovir(ACV) as control medicine . It turns out that the toxic concentration of Rheum palmatum Volatile oil for 50% cells was (CD50) > 1.25 x 10(-1) g/L . When concentration was below 0.625 x 10(-1) g/L, the survival rate of cells was over 90% . The maximum inhibitory rates for HBsAg and HBeAg were 70.71 +/- 5.4% and 30.99 +/- 5.3% respectively . This shows Rheum palmatum Volatile oil possesses the effect of antiHBV in vitro.

Arch Pharm Res, 2003 Jan, 26(1), 34 - 9
Induction of growth hormone by the roots of Astragalus membranaceus in pituitary cell culture; Kim C et al.; The traditional Asian medicinal herb, roots of Astragalus (A.) membranaceus (Leguminosae), is used for many purposes, some of which are purported to stimulate the release of growth hormone in vivo . Extracts of A . membranaceus were tested to determine whether they stimulate the release of growth hormone in rat pituitary cell culture . A . membranaceus was extracted sequentially with 80% ethanol (fraction A), n-hexane (fraction B); the test compound from the herbal extraction was isolated using silica gel column chromatography and was identified with spectral data . Test compound was also extracted by traditional boiling water methods . Induction of growth hormone in pituitary cell culture was conducted with isolated compounds and extracted fractions of A . Radix (dried roots of A . membranaceus) . The fraction A was not active in the rat pituitary cell culture, but the fraction B derived from the ethanol fraction stimulated the release of growth hormone in culture . Six compounds from fraction B (1-6) were isolated and identified previously . The compounds 1,2-benzendicarboxylic acid diisononylester (1), beta-sitosterol (2), and 3-O-beta-D-galactopyranosyl-beta-sitosterol (5) did not induce growth hormone release in the culture . Formononetin (3), 9Z,12Z-octadecadienoic acid (4), stigmast-4-en-6beta-ol-3-one (6) and 98-E, a mixture of 1'-9,12-octadecadienoic acid (Z,Z)-2',3'-dihydroxy-propylester (7) and 1'-hexadecanoic acid-2',3'-dihydroxy-propylester (8) stimulated the release of growth hormone in the rat pituitary cell culture significantly compared to the control . In conclusions, four compounds isolated from extracts of A . Radix induced growth hormone release in the rat pituitary cell culture . The 98-E isolate was the most active inducer of growth hormone release.

Blood Purif, 2003, 21(1), 58 - 63
Affinity hemodialysis for antiviral therapy . II . Removal of HIV-1 viral proteins from cell culture supernatants and whole blood; Tullis RH et al.; BACKGROUND: HIV-1 gp120 may play a role in the progression from HIV infection to AIDS . AIMS: We investigated affinity hemodialysis for removing gp120 from cell culture and whole blood . METHODS: Anti-gp120 antibodies covalently coupled to agarose beads were packed into columns or hollow-fiber hemodialysis cartridges . Supernatants from HIV-infected HL2/3 cells or gp120 containing whole blood were pumped over the columns and gp120 measured by ELISA . RESULTS: Anti-gp120 agarose removed approximately 90% of HIV-1 gp120 from HL2/3 cultures in 30-60 min . Capture was antibody-dependent (F105 > IDX 1121 > ABI 13-108) . Affinity hemodialysis also efficiently captured gp120 from buffer in a first-order, flow-rate-dependent fashion (t((1/2)) = 13 min at 0.9 ml/min) . Clearance was faster than calculated diffusion (t((1/2)) approximately 2.5 h) suggesting significant convective transport . gp120 removal from blood was slower (t((1/2)) = 1.4 h) . CONCLUSION: Affinity hemodialysis efficiently clears gp120 from cell culture fluids and blood and may be useful in slowing the progression to AIDS .

Placenta, 2003 Feb-Mar, 24(2-3), 258 - 69
A three-dimensional cell culture model for bovine endometrium: regeneration of a multicellular spheroid using ascorbate; Yamauchi N et al.; The development of a multicellular spheroid comprising bovine endometrial epithelial cells (BEE) and bovine endometrial stromal cells (BES) is described in this study . The BES were cultured to confluence in medium with L -ascorbic acid phosphate magnesium salt n -hydrate (AsA-P) which stimulates collagen synthesis in BES . The BEE were co-cultured on a BES cell-sheet for 24h before detachment of the cell-sheet to generate a hetero-spheroid . After EDTA treatment and agitating with pipette, the floating cell-sheet shrank and became an aggregated cell mass in a few days; it finally formed a round-shaped hetero-spheroid composed of BES and BEE . Histological examination found that hetero-spheroids were covered with BEE on the outer layer . When cell viability was examined with TUNEL (terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling), no positive signal was detected in the spheroid for up to 10 days . Immunofluorescence observations showed that spheroids contained abundant extracellular matrices, including type-I, -III, -IV collagen, fibronectin, and laminin . PGF(2alpha) produced by hetero-spheroids in response to oxytocin was significantly higher than those produced by monolayer cultured BEE (P< 0.05) . MMPs were not detected in media from spheroids cultured for 5 days after detachment of the cell sheet . These results indicate that bovine endometrial cells have the capacity to regenerate as a multicellular spheroid after treatment with ascorbate in vitro . The spheroid displays an endometrium-mimic feature . Thus, we conclude that spheroids formed by BES and BEE are a useful in vitro model of bovine endometrium.

Neuroscience, 2003, 116(2), 437 - 45
Protective effect of apolipoprotein E-mimetic peptides on N-methyl-D-aspartate excitotoxicity in primary rat neuronal-glial cell cultures; Aono M et al.; Apolipoprotein E (apoE) is a 34-kD protein with multiple biological properties . Recent clinical and preclinical observations implicate a role for apoE in modifying the response of the brain to focal and global ischemia . One mechanism by which apoE might exert these effects is by reducing glutamate-induced excitotoxic neuronal injury associated with ischemic insults . We demonstrate that human recombinant apoE confers a mild neuroprotective effect in primary neuronal-glial cultures exposed to 100 microM N-methyl-D-aspartate . Furthermore, a peptide derived from the receptor-binding region of apoE (residues 133-149) maintained a significant helical population as assessed by circular dichroism, and completely suppressed the neuronal cell death and calcium influx associated with N-methyl-D-aspartate exposure . Neuroprotection was greatest when the peptide was added concurrently with N-methyl-D-aspartate; however, a significant protection was observed when peptide was preincubated and washed off prior to N-methyl-D-aspartate exposure . These results suggest that one mechanism by which apoE may modify the CNS response to ischemia is by partially blocking glutamate excitotoxicity . Moreover, small peptide fragments derived from the receptor-binding region of apoE have enhanced bioactivity compared with the intact holoprotein, and may represent a novel therapeutic strategy for the treatment of brain ischemia .

Tsitol Genet, 2002 Nov-Dec, 36(6), 28 - 34
{The investigation of genomes of some species of the genus Gentiana in nature and in vitro cell culture}; Mel'nyk VM et al.; The comparative study of the genomes of intact plants-representatives of some species of the genus Gentiana L . as well as cultured cells of G . lutea and G . punctata was performed using restriction analysis . Species specificity of restriction fragment patterns for studied representatives of this genus was revealed . The differences between electrophoretic patterns of digested DNA purified from rhizome and leaves of G . lutea and G . punctata were found . The changes in genomes of G . lutea and G . punctata cells cultured in vitro compared with the genomes of intact plants were detected . The data obtained evidence that some of them may be of nonrandom character.

J Exp Bot, 2003 Feb, 54(383), 647 - 56
Multiple signalling pathways mediate fungal elicitor-induced beta-thujaplicin biosynthesis in Cupressus lusitanica cell cultures; Zhao J et al.; The biosynthesis of a phytoalexin, beta-thujaplicin, in Cupressus lusitanica cell cultures can be stimulated by a yeast elicitor, H(2)O(2), or methyl jasmonate . Lipoxygenase activity was also stimulated by these treatments, suggesting that the oxidative burst and jasmonate pathway may mediate the elicitor-induced accumulation of beta-thujaplicin . The elicitor signalling pathway involved in beta-thujaplicin induction was further investigated using pharmacological and biochemical approaches . Treatment of the cells with calcium ionophore A23187 alone stimulated the production of beta-thujaplicin . A23187 also enhanced the elicitor-induced production of beta-thujaplicin . EGTA, LaCl(3), and verapamil pretreatments partially blocked A23187- or yeast elicitor-induced accumulation of beta-thujaplicin . These results suggest that Ca(2+) influx is required for elicitor-induced production of beta-thujaplicin . Treatment of cell cultures with mastoparan, melittin or cholera toxin alone or in combination with the elicitor stimulated the production of beta-thujaplicin or enhanced the elicitor-induced production of beta-thujaplicin . The G-protein inhibitor suramin inhibited the elicitor-induced production of beta-thujaplicin, suggesting that receptor-coupled G-proteins are likely to be involved in the elicitor-induced biosynthesis of beta-thujaplicin . Indeed, both GTP-binding activity and GTPase activity of the plasma membrane were stimulated by elicitor, and suramin and cholera toxin affected G-protein activities . In addition, all inhibitors of G-proteins and Ca(2+) flux suppressed elicitor-induced increases in lipoxygenase activity whereas activators of G-proteins and the Ca(2+) signalling pathway increased lipoxygenase activity . These observations suggest that Ca(2+) and G-proteins may mediate elicitor signals to the jasmonate pathway, and the jasmonate signalling pathway may then lead to the production of beta-thujaplicin.

Drug Deliv, 2003 Jan-Mar, 10(1), 29 - 34
Cell culture studies of a carborane cholesteryl ester with conventional and PEG liposomes; Peacock GF et al.; A new cholesterol-carborane conjugate (BCH) has been synthesized as a potential targeting agent for boron neutron capture therapy (BNCT) of cancers . The compound is extremely water insoluble and was formulated in two liposomal formulations to determine if the compound could be adequately taken up by 9L rat glioma cells in cell culture . Several factors potentially affecting the cellular uptake were evaluated, such as concentration of BCH in the incubation medium, incubation time, cell confluence, and the addition of polyethylene glycol (PEG) phospholipids to the liposomal formulation . The studies indicated that the cellular uptakes of BCH in the conventional and PEG liposomal formulations were 49.1 and 45.9 microg boron/g cells, respectively . Therefore, this compound, formulated in both liposomal formulations, delivered sufficient levels of boron to cancer cells in vitro, indicating that BCH is a promising approach for use in BNCT . The uptake appeared to depend upon BCH concentration in the media as well as the confluence of the cells . The greater boron uptake by nonconfluent cells indicated that active growth of cells was a factor in the uptake of this compound.

J Endocrinol, 2003 Feb, 176(2), 227 - 35
Effect of recombinant porcine IGF-binding protein-3 on proliferation of embryonic porcine myogenic cell cultures in the presence and absence of IGF-I; Pampusch MS et al.; IGF-binding protein (IGFBP)-3 is produced by cultured porcine embryonic myogenic cell (PEMC) cultures and is secreted into the medium . Levels of secreted IGFBP-3 and IGFBP-3 mRNA are significantly reduced during differentiation and increase after differentiation is complete, suggesting that IGFBP-3 may play some role in myogenesis and/or in changes in myogenic cell proliferation that accompany differentiation . IGFBP-3 reportedly may either suppress or stimulate proliferation of cultured cells depending on cell type . Additionally, IGFBP-3 has been shown to affect proliferation via both IGF-dependent and IGF-independent mechanisms in some cell types but not all . Currently, the effect, if any, of IGFBP-3 on myogenic cell proliferation is not known . Consequently, the goal of this study was to assess the IGF-I-dependent and IGF-I-independent actions of recombinant porcine IGFBP-3 on proliferation of cultured porcine myogenic cells . To facilitate these investigations, we have expressed porcine IGFBP-3 in the baculovirus system, purified and characterized the expressed recombinant porcine IGFBP-3 (rpIGFBP-3), and produced and characterized an anti-porcine IGFBP-3 antibody that neutralizes the biological activity of porcine IGFBP-3 . rpIGFBP-3 suppressed IGF-I-stimulated proliferation of PEMCs in a concentration-dependent manner with equimolar concentrations of IGF-I and rpIGFBP-3, resulting in complete suppression of IGF-I-stimulated proliferation . rpIGFBP-3 also suppressed Long-R3-IGF-I-stimulated proliferation of PEMC, indicating that rpIGFBP-3 possesses IGF-independent activity in this cell system . These data have established that IGFBP-3 has the potential to affect proliferation of PEMCs during critical periods of muscle development that may impact ultimate muscle mass achievable postnatally.

Dis Aquat Organ, 2002 Dec 10, 52(3), 179 - 84
Influence of storage temperature on infectious hematopoietic necrosis virus detection by cell culture isolation and RT-PCR methods; Hostnik P et al.; The detection of infectious hematopoietic necrosis virus (IHNV) in infected rainbow trout Oncorhynchus mykiss and in cell culture supernatants stored under different conditions was studied . IHNV-positive fish visceral organ homogenates and cell culture supernatants were incubated at 4 and 25 degrees C . Virus titre was measured by virus isolation on epithelioma papulosum cyprini (EPC) cells and the IHNV RNA was detected by RT-PCR and semi-nested RT-PCR . The influence of repeated freezing and thawing on the virus isolation from organ homogenates and from cell culture supernatants was studied as well . It was possible to isolate the virus from IHNV-positive organ material during the 3 d of incubation at 4 degrees C but, only on the first day of incubation at 25 degrees C . Viral RNA could be amplified during the incubation period of 35 d at 4 degrees C but only during 8 d of incubation at 25 degrees C . In IHNV-infected cell culture supernatant stored at 4 degrees C, it was possible to detect virus for 36 and 16 d in supernatant stored at 25 degrees C . Viral RNA could be followed by using molecular methods during the entire experimental period of 123 d . Each cycle of freezing and thawing of samples resulted in a reduction of IHNV titre in the suspension of visceral organs, while the virus titre in cell culture supernatant remained almost the same following 33 freezing-thawing cycles . The present results show that rapid laboratory processing and storage of potentially virus-containing tissue samples as well as the use of different detection methods are very important for efficient IHNV diagnosis.

Histochem Cell Biol, 2003 Jan, 119(1), 77 - 90 Epub 2002 Dec 20.
Different apoptotic responses and patterns in adhering and floating neoplastic cell cultures: effects of microtubule antagonists; Sciola L et al.; The relationship between apoptotic progression and cell cycle perturbation induced by microtubule-destabilising (vinblastine, Colcemid) and -stabilising (taxol) drugs was studied in two mesenchyme-derived neoplastic cell lines, growing as suspension (Jurkat) and monolayer (SGS/3A) culture, by morphocytochemical and biochemical approaches . The same kind of drug induced different effects on the cell kinetics (proliferation, polyploidisation, death) of the two cell lines . In floating cells, the drugs appeared more effective during the S phase, while in adherent cells they were more effective during the G2/M phase . Moreover two distinct neoplasia-associated apoptotic phenotypes emerged: the first pattern was the typical one and was found in cells with a low transition through the S/G2 phase (Jurkat), and the second one was mainly characterised by a cell death derived from micronucleated and mitotic cells, as a consequence of a low transition through the M/G1 phase (SGS/3A) . Our data show that the machinery required for the trigger and progression of apoptosis is present in every cell cycle phase, also in conditions of karyological alterations (aneugenic micronucleations) . On the other hand, a different sensitivity of the two microtubular components (interphasic network and mitotic spindle) appears to be related to the anchorage-dependence or -independence during the cell growth disturbances after exposure to antimicrotubular drugs.

Pharmacol Res, 2003 Feb, 47(2), 119 - 26
Pravastatin inhibits pro-inflammatory effects of Alzheimer's peptide Abeta(1-42) in glioma cell culture in vitro; Sun YX et al.; Statins are known to exert a number of biological effects apart from reducing cholesterol synthesis . The results of recent studies indicate that patients treated with pravastatin have a lower prevalence of diagnosed Alzheimer's disease (AD) . These observations prompted us to examine the effects of pravastatin on Alzheimer's peptide (Abeta(1-42))-induced pro-inflammatory activation in the human glioma cell line in vitro . Cells alone or cells pre-treated with pravastatin (0.1mg x ml(-1)) for 24h were stimulated with 5 microM of freshly dissolved Abeta(1-42) for the next 24h . The pre-treatment of cells with pravastatin diminished the capacity of Abeta to induce metalloproteinases, cytokine IL-6 and free radical levels . Although both pravastatin and Abeta(1-42) separately increased PPARgamma activity, the combination of Abeta(1-42) and pravastatin resulted in no effect on PPARgamma expression . These data indicate that soluble forms of Abeta(1-42), which are a potent stimulus of pro-inflammatory activation of glioma cells in vitro, could be a good target for pravastatin.

Acta Otolaryngol, 2002 Dec, 122(8), 836 - 40
Osteoblast-like cell cultures from human stapes; Dost P et al.; OBJECTIVE: In order to develop new middle ear prostheses for ossicular reconstruction it is important to study how the recipient middle ear tissues, especially the stapes footplate and superstructure, react to the implanted biomaterial . In this respect, animal studies and cell cultures using non-specific cells are of limited value . MATERIAL AND METHODS: The morphology and growth pattern of cells cultured from human stapes were studied . Cultured cells were examined for the presence of alkaline phosphatase and were processed for immunocytochemistry in order to detect the presence of osteocalcine . Fibroblast cultures served as controls . RESULTS: Cultured stapes cells proliferated in a polygonal-cubic shape and without any regular pattern in the culture . These cells were shown to contain alkaline phosphatase and osteocalcine . Cultured fascia fibroblasts proliferated in a spindle-shaped form and in a pattern resembling a shoal of fish . Cultured fibroblasts did not contain alkaline phosphatase or osteocalcine . CONCLUSIONS: It is possible to culture osteoblasts from human stapes . These cells can be characterized as osteoblast-like cells by means of their external shape and the presence of alkaline phosphatase and osteocalcine . Using these cultures, specific in vitro investigations concerning the interaction of biomaterials and middle ear ossicles could be performed.

Water Environ Res, 2002 Nov-Dec, 74(6), 564 - 8
An improved filter elution and cell culture assay procedure for evaluating public groundwater systems for culturable enteroviruses; Dahling DR; Large-scale virus studies of groundwater systems require practical and sensitive procedures for both sample processing and viral assay . Filter adsorption-elution procedures have traditionally been used to process large-volume water samples for viruses . In this study, five filter elution procedures using cartridge filters were evaluated for their effectiveness in processing samples . Of the five procedures tested, the third method, which incorporated two separate beef extract elutions (one being an overnight filter immersion in beef extract), recovered 95% of seeded poliovirus compared with recoveries of 36 to 70% for the other methods . For viral enumeration, an expanded roller bottle quantal assay was evaluated using seeded poliovirus . This cytopathic-based method was considerably more sensitive than the standard plaque assay method . The roller bottle system was more economical than the plaque assay for the evaluation of comparable samples . Using roller bottles required less time and manipulation than the plaque procedure and greatly facilitated the examination of large numbers of samples . The combination of the improved filter elution procedure and the roller bottle assay for viral analysis makes large-scale virus studies of groundwater systems practical . This procedure was subsequently field tested during a groundwater study in which large-volume samples (exceeding 800 L) were processed through the filters.

Clin Cancer Res, 2003 Jan, 9(1), 243 - 9
Cell culture in esophageal squamous cell carcinoma and the association with molecular markers; Shimada Y et al.; PURPOSE: We reported previously that the patients in whom cancer cells could be cultured as continuous cell lines had a poor prognosis of esophageal squamous cell carcinoma (ESCC) patients . In this study, to evaluate additional evidence of prognostic significance and the genetic background of cell culture, we analyzed 203 ESCC patients . EXPERIMENTAL DESIGN: Culture samples were obtained from resected 203 primary ESCC (from 1986 to 1998; R0 resection) . The expression of six molecular markers was evaluated retrospectively in resected primary esophageal tumors by immunohistochemical analysis, and the capability of establishing cell lines was compared . RESULTS: Thirty-five cell lines (17.2%) were established from 203 ESCC patients: group 1 (n = 35), from whom cancer cells could be cultured as continuous cell lines, and group 2 (n = 168), from whom cell lines could not be established . The cumulative survival rate of patients in group 1 was significantly lower than that of those in group 2 (P = 0.0006) . Cox's proportional hazard model revealed that cell culture capability was an independent prognostic factor (risk ratio, 1.98; P = 0.007) . Univariate logistic regression analysis revealed that cell culture capability had associations with the following molecular biological factors: cyclin D1, p53, murine double minute 2, p27, and fragile histidine triad gene (P < 0.05) . However, multivariate logistic regression analysis revealed that p53 protein accumulation and MDM2 protein expression predict establishment of cell line in ESCC (odds ratio, 7.72 and 8.62, respectively) . CONCLUSIONS: Cell culture capability is a significant prognostic factor in ESCC . p53 and MDM2 may have a crucial role in the establishment of ESCC cell lines.

J Microsc, 2003 Jan, 209(Pt 1), 34 - 40
Automatic quantification of viability in epithelial cell cultures by texture analysis; Malpica N et al.; Quantification of live cells in phase contrast microscopy images allows in vivo assessment of the viability of cultured cells . An automatic screening procedure seems advisable because of the large number of cells that must be counted to achieve reasonable accuracy . This paper presents a method that quantifies necrosis in cell cultures by texture analysis of microscope images . The image is divided into regions of equal size that are classified by means of a segmentation algorithm based on texture analysis into three categories: live cells, necrotic cells and background . The classification uses three discriminant functions, built from parameters derived from the histogram and the co-occurrence matrix and calculated by performing an initial stepwise discriminant analysis on 21 sample images from a training set . The areas occupied by live and necrotic cells and number of live cells have been obtained for primary cellular cultures in intervals of 48 h during 2 weeks . The results have been compared with those obtained by an experienced observer, showing a very good correlation (Pearson's coefficient 0.95, kappa 0.87, N= 1600) . A method has been developed that provides an accuracy similar to that provided by an expert, while allowing a much higher number of fields to be counted.

Reproduction, 2002 Dec, 124(6), 791 - 9
Maintenance of adult mouse type A spermatogonia in vitro: influence of serum and growth factors and comparison with prepubertal spermatogonial cell culture; Creemers LB et al.; The culture of spermatogonial cells under well-defined conditions would be an important method for elucidating the mechanisms involved in spermatogenesis and in establishing tissue regeneration in vivo . In this study, a serum-free culture system was established, with type A spermatogonia isolated from adult vitamin A-deficient mice . At days 1, 3 and 7 of culture, the viability and proliferation of cells were monitored . The viability of the cells decreased by day 7 to 10% of the cells present . Proliferation occurred mainly during day 1, when 1% of the germ cells was proliferating . Co-labelling for a germ cell marker (heat shock protein-90alpha, Hsp90alpha), and a marker used to detect dividing cells (bromodeoxyuridine, BrdU), showed that this proliferation was restricted to germ cells . In an attempt to improve these parameters, medium containing fetal calf serum (FCS) was used . Viability was not influenced by serum, but proliferation was markedly enhanced . However, after day 7 of incubation with FCS, co-immunolocalization for Hsp90alpha and BrdU showed a preferential proliferation of somatic cells . Comparison of cultures of adult cells with cultures of prepubertal germ cells, commonly used in studies of spermatogenesis, showed that prepubertal germ cells are twice as viable . In addition, a different proliferation profile was observed, with a peak at day 3 . Here, a distinct proliferation of somatic cells was also noted . The results from the present study indicate that the origin of isolated germ cells partly determines culture outcome and that cultures of prepubertal germ cells may not be representative for adult spermatogenesis . Moreover, adding FCS to the culture medium invokes the risk of profound and undesirable effects on cell composition, also underlining the need for identification of germ cells during culture.

Vascul Pharmacol, 2002 Jun, 38(6), 355 - 64
The use of in vitro cell culture models for mechanistic studies and as permeability screens for the blood-brain barrier in the pharmaceutical industry--background and current status in the drug discovery process; Lundquist S et al.; Successful drug delivery to the central nervous system (CNS) is highly dependent on DMPK as well as physicochemical properties and it is therefore important to characterise these properties and take them into account when designing chemical lead series that act at CNS targets . Since the drug discovery/development process is becoming increasingly focused on reducing the time required to enter molecules into the market, industrial DMPK scientists have emerged from their traditional supportive role in drug development to provide valuable support in the drug discovery process, using novel methods to meet the demands of combinatorial chemistry and bioscience groups.

Di Yi Jun Yi Da Xue Xue Bao, 2003 Jan, 23(1), 89 - 90
{Application of nitrocellulose membrane in cell culture and pathological techniques}; Zhang H et al.; OBJECTIVE: To provide a new supporting medium for carrying out cell culture and histological staining . METHOD: LoVo cell line was cultured using nitrocellulose membrane as the culture medium, in parallel with the same cell culture on the coverglass which served as control . RESULTS: The cells cultured on nitrocellulose membrane showed no visible difference from those cultured on coverglasses in terms of cell growth, morphology, and the structure displayed after staining . No toxic effect was observed due to the application of nitrocellulose membrane . CONCLUSIONS: Nitrocellulose membrane after treatment has such merit as good lucidity and stability without toxicity on the cells . Having also good affinity with the cells, nitrocellulose membrane can offer an promising alternative for glass as cell culture medium.

Biomaterials, 2003 Mar, 24(7), 1223 - 32
Accelerated cell sheet recovery by co-grafting of PEG with PIPAAm onto porous cell culture membranes; Hyeong Kwon O et al.; Fabrication of functional tissue constructs from designed three-dimensional structures of cells using the layered method of cultured cell sheets could prove to be an attractive approach to tissue engineering . Rapid recovery of cell sheets is considered to be important as a basic technology for practical assembly of tissue-mimicking structures . To accelerate required culture substrate hydrophilic/hydrophobic functional changes according to the hydrated/dehydrated structural changes in response to culture temperature alteration, poly(N-isopropylacrylamide) (PIPAAm) was grafted with poly(ethylene glycol) (PEG) onto porous culture membranes by electron beam irradiation . Analyses by attenuated total reflection-Fourier transform infrared and electron spectroscopy for chemical analysis revealed that PIPAAm and PEG were successfully grafted to surfaces of porous membranes . PIPAAm-grafted porous membranes (PIPAAm-PM) were compared with porous membranes co-grafted with various amounts of PEG and PIPAAm (PIPAAm(PEG)-PM) for cell sheet detachment experiments . Approximately 35min incubation at 20 degrees C was required to completely detach cell sheets from PIPAAm-PM in a static condition, while only 19min to detach cell sheets from PIPAAm(PEG0.5%)-PM, which is co-grafted with PIPAAm and 0.5wt% of PEG . With porous membranes, water molecules were accessed by the PIPAAm molecules grafted on the surfaces from both underneath and peripheral to the attached cell sheet, resulting in more rapid hydration of grafted PIPAAm molecules and detachment of cell sheet than that for nonporous tissue culture polystyrene (TCPS) dish . With PIPAAm(PEG)-PMs, grafted PEG chains should accelerate the diffusion of water molecules to PIPAAm grafts, showing more rapid detachment of cell sheet compare to PIPAAm-PMs.

Int J Pharm, 2003 Jan 30, 251(1-2), 107 - 12
Effects of citicholine and dimethylsulfoxide on transepithelial transport of passively diffused drugs in the Caco-2 cell culture model; Demirbas S et al.; The objective of this study was to determine, using a Caco-2 cell monolayer model, the extent to which the paracellular and transcellular routes are altered by citicholine (CDP-Ch) and DMSO in the presence of human serum albumin (HSA) . The apparent permeability (Papp) of mannitol in the presence of 4% (w/v) HSA was investigated using 0, 0.5, 1.0, 2.5, 5.0, and 10.0% (v/v)) of DMSO . The Papp for mannitol ranged from 0.56 x 10(-6) to 0.89 x 10(-6) cm/s (mean 0.77 x 10(-6)) . Increasing the concentration of DMSO does not appear to have an effect on the paracellular transport of mannitol and on the transepithelial resistance (TEER) of the monolayer, (P>0.05) . The effect of citicholine (CDP-Ch) was investigated in confluent Caco-2 cell monolayers incubated in the presence of 2, 4, 10, 40, 60, 100 and 200 mM CDP-Ch at 37 degrees C in an atmosphere of 7% CO(2) and 95% relative humidity . Papp of mannitol and diltiazem in the presence of CDP-Ch ranged from 0.53 x 10(-6) to 8.52 x 10(-6) cm/s and from 1.30 x 10(-5) to 2.71 x 10(-5) cm/s, respectively . CDP-Ch may have an effect on the stability of the tight junction complex resulting in an increase in the apparent permeability of mannitol.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 1998 Sep, 12(3), 269 - 71
{Study on the anti-herpes simplex virus activity of a suppository or ointment form of Astragalus membranaceus combined with interferon alpha 2b in human diploid cell culture}; Zhang L et al.; A study on the anti-herpes simplex virus activity of the suppository or ointment form of Astragalus membranaceus(AM) combined with recombinant human interferon alpha 2b(IFN) was carried out in human diploid cell culture . AM is a Chinese herb medicine and have been used in China as a tonic for thousands of years and the IFN was produced from E . coli with 95% purity . Obtained results indicated that the placebo suppository and ointment(without AM and IFN) seemed not to decrease markedly the anti-viral activity of IFN in WISH/VSV assay system . The anti-herpes simplex virus activities of suppository and ointment forms of AM and IFN were shown to be significantly higher than that of IFN alone . It is well known that chronic cervicitis is closely related to papillomavirus, cytomegalovirus as well as herpes virus infections . The AM-IFN suppository is suggested to be used in the treatment of cervicitis and the ointment in the treatment of skin herpes.

Brain Res Dev Brain Res, 2003 Jan 10, 140(1), 15 - 28
FGF-2, NGF and IGF-1, but not BDNF, utilize a nitric oxide pathway to signal neurotrophic and neuroprotective effects against alcohol toxicity in cerebellar granule cell cultures; Bonthius DJ et al.; Neuronal death is a prominent neuropathological component of fetal alcohol syndrome (FAS) . Identification of molecular agents and pathways that can ameliorate alcohol-induced cell loss offers possible therapeutic strategies for FAS and potential insight into its pathogenesis . This study investigated the effects of growth factors on cellular survival in alcohol-exposed cerebellar granule cell (CGC) cultures and examined the role of the nitric oxide (NO)-cGMP-PKG (cGMP-dependent protein kinase) pathway in the cell survival-promoting effects of these growth factors . Primary CGC cultures were exposed to 0 or 400 mg/dl ethanol, accompanied by either no growth factor or 30 ng/ml fibroblast growth factor-2 (FGF-2), nerve growth factor (NGF), insulin-like growth factor-1 (IGF-1), brain-derived neurotrophic factor (BDNF) or epidermal growth factor (EGF) . Viable neurons were quantified after 1 day of exposure . Two distinct types of cell survival-promoting effects of growth factors were detectable: (1) a neurotrophic effect, in which the growth factors diminished the background death of neurons that occurred in alcohol-free cultures; and (2) a neuroprotective effect, in which the growth factors diminished alcohol-induced cell death . The various growth factors differed markedly in their patterns of cell survival promotion . While BDNF and FGF-2 exerted both a neurotrophic and a neuroprotective effect, IGF-1 had only a neurotrophic effect and did not protect against alcohol toxicity, and NGF had only a neuroprotective effect and did not diminish background cell death . EGF had neither a neurotrophic nor a neuroprotective effect . In order to determine the role of the NO-cGMP-PKG pathway in the cell survival-promoting effects mediated by growth factors, cultures were exposed to one of several pharmacological inhibitors of the pathway, including NAME, LY83583 and PKG inhibitor . The cell survival-promoting effects of FGF-2, NGF and IGF-1 were all substantially reduced by each of the pathway inhibitors . In contrast, neither the neurotrophic nor the neuroprotective effects of BDNF were altered by any of the pathway inhibitors . Thus, growth factors differ in their patterns of neurotrophic and neuroprotective effects, and they differ in their reliance on the NO-cGMP-PKG pathway . While FGF-2, NGF and IGF-1 all signal their survival-promoting effects through the NO-cGMP-PKG pathway, BDNF does not rely upon this pathway for signal transduction in CGC cultures.

Proc Natl Acad Sci U S A, 2003 Jan 7, 100(1), 235 - 40 Epub 2002 Dec 23.
Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs; Randall G et al.; RNA interference is a cellular process of gene silencing in which small duplexes of RNA specifically target a homologous sequence for cleavage by cellular ribonucleases . The introduction of approximately 22-nt small interfering RNAs (siRNAs) into mammalian cells can specifically silence cellular mRNAs without induction of the nonspecific IFN responses that are activated by longer RNA duplexes . We investigate in this article whether siRNAs can also silence the expression of the cytoplasmically replicating hepatitis C virus (HCV) RNAs by using a replicon system that supports robust HCV replication, but not the production of infectious virions . We report the efficient silencing of both cellular lamin AC and HCV RNAs in Huh-7 hepatoma cell lines supporting HCV replication . Silencing of HCV RNAs was dose dependent and specific, inasmuch as two HCV variants that differ by 3 nt within the target sequence were only silenced by the exact homologous sequence for each . siRNAs designed to target HCV RNA triggered an exponential decrease in HCV RNA, resulting in an 80-fold decrease in HCV RNA after 4 days . The introduction of siRNAs into cells with established HCV replication cured >98% of these cells of detectable HCV antigen and replication-competent HCV RNAs . These data support the principle of siRNA-based HCV antiviral therapy.

Biosci Rep, 2002 Jun-Aug, 22(3-4), 407 - 20
The 78 kDa glucose-regulated protein (GRP78/BIP) is expressed on the cell membrane, is released into cell culture medium and is also present in human peripheral circulation; Delpino A et al.; In human rabdomiosarcoma cells (TE671/RD) chronic exposure to 500 nM thapsigargin (a powerful inhibitor of the endoplasmic reticulum Ca2+-ATPases) resulted in the induction of the stress protein GRP78/BIP . Making use of the surface biotinylation method, followed by the isolation of the GRP78 using ATP-agarose affinity chromatography, it was found that a fraction of the thapsigargin-induced GRP78 is expressed on the cell surface . The presence of GRP78 on the membrane of thapsigargin-treated cells was confirmed by fractionation of cell lysates into a soluble and a membrane fraction, followed by Western blot analysis with an anti-GRP78 antibody . It was also found that conspicuous amounts of GRP78 are present in the culture medium collected from thapsigargin-treated cultures . This extracellular GRP78 originates mostly by an active release from intact cells and does not result solely from the leakage of proteins from dead cells . Moreover, small amounts of circulating, free GRP78 and naturally-occurring anti-GRP78 autoantibodies were detected in the peripheral circulation of healthy human individuals.

Rev Med Virol, 2003 Jan-Feb, 13(1), 17 - 20
The new cell culture smallpox vaccine should not be offered to the general population; Mortimer PP; Cell based smallpox vaccines are to be welcomed, but any decision to vaccinate whole populations must await firstly better intelligence about the gravity of the threat from bioterrorists, including their ability to release smallpox in such a way that wide dissemination could take place; secondly evidence that vaccines grown in cell culture are protective and safe; and thirdly that the vaccines would be generally acceptable and their introduction would not compromise the rest of national immunisation programmes . Smallpox vaccination should not be offered to the general population until these uncertainties have been resolved, by which time bioterrorism might possibly have been overcome or the development of antiviral treatment might have made renewed smallpox vaccination unnecessary . Meanwhile, preparations for rapid deployment of the historically well-tried containment measures at the epicentres of any smallpox release should proceed, their effectiveness should be tested, and their adequacy kept under review .

Rev Med Virol, 2003 Jan-Feb, 13(1), 5 - 15
The new cell culture smallpox vaccine should be offered to the general population; Bicknell W et al.; A series of major factors must be weighed in deciding whether or not, and to what extent, a particular country should consider pre-exposure vaccination for smallpox . These include the risk of a bioterrorist attack using smallpox, the risk of secondary spread from another country, the risks and benefits of vaccination, the effectivenes s of vaccination pre- and post-exposure, the prevalence of immunocompromised persons, the capacity of the medical care delivery system and the wealth of a nation . We review here the issues and variables relevant for policy making, propose a framework for country-specific decision making and suggest the World Health Organization has a key role to play, particularly with regard to lower-income countries . In doing so, we support the proposition .

Antibiot Khimioter, 2002, 47(8), 9 - 11
{Ani immunomodulator Hepon inhibits hepatitis C virus replication in human cell cultures in vitro}; Ataullakhanov RI et al.; Antiviral activity of immunomodulator "Hepon" was evaluated in human cells culture infected with hepatitis C virus . "Hepon" presence protected human cells SW-13 from cytopathogenic effect of hepatitis C virus . Maximum antiviral effect was demonstrated by "Hepon" at concentration 1 mcg/mL . Control antiviral agent reaferon (interferon alfa-2a) was more potent as vitality protecting agent in the case of SW-13 human cells culture . "Hepon" activity is based on changes of cytokins and interferons spectrum so this immunomodulator is expected to be effective against different viruses including herpes virus and encephalocarditis virus.

Altern Lab Anim, 2002 Dec, 30 Suppl 2, 115 - 8
Metabolism-mediated neurotoxicity: the significance of genetically engineered cell lines and new three-dimensional cell cultures; Coecke S et al.; Until now, no in vitro methods for determining neurotoxic effects, on Phase I and Phase II biotransformation-driven metabolite formation or for the evaluation of the metabolism-mediated hazard of a chemical, have been validated . The current test guidelines are based on studies in vivo, involving animals exposed to the test substance . Novel in vitro testing instead of animal testing is required by Directive 86/609/EEC . In the EU White Paper on a Strategy for a Future Chemicals Policy, which may result in up to 20,000 chemicals being screened for toxicity, the use of non-animal test methods is seen as essential and is encouraged . The aim of the present work was to demonstrate the significance of novel technologies, including the use of genetically engineered cell lines and three-dimensional cell culture techniques for direct application in the regulatory hazard-assessment process . Furthermore, attempts were made to make in vitro toxicity tests for specific applications more-readily available for inclusion in the chemical hazard-assessment process, by exploiting advances made in the life sciences.

In Vitro Cell Dev Biol Anim, 2002 Jun, 38(6), 314 - 9
Biphasic culture strategy based on hyperosmotic pressure for improved humanized antibody production in Chinese hamster ovary cell culture; Kim MS et al.; Hyperosmotic pressure increased specific antibody productivity (q(Ab)) of recombinant Chinese hamster ovary (rCHO) cells (SH2-0.32) and it depressed cell growth . Thus, the use of hyperosmolar medium did not increase the maximum antibody concentration substantially . To overcome this drawback, the feasibility of biphasic culture strategy was investigated . In the biphasic culture, cells were first cultivated in the standard medium with physiological osmolality (294 mOsm/kg) for cell growth . When cells reached the late exponential growth phase, the spent standard medium was replaced with the fresh hyperosmolar medium (522 mOsm/kg) for antibody production . The q(Ab) in growth phase with the standard medium was 2.1 microg per 10(6) cells/d, whereas the q(Ab) in antibody production phase with the hyperosmolar medium was 11.1 microg per 10(6) cells/d . Northern blot analysis showed a positive relationship between the relative contents of intracellular immunoglobulin messenger ribonucleic acid and q(Ab) . Because of the enhanced q(Ab) and the increased cell concentration in biphasic culture, the maximum antibody concentration obtained in biphasic culture with 522 mOsm/kg medium exchange was 161% higher than that obtained in batch culture with the standard medium . Taken together, the simple biphasic culture strategy based on hyperosmotic culture is effective in improving antibody production of rCHO cells.

Neurochem Res, 2002 Nov, 27(11), 1401 - 19
Apoptosis in Schwann cell cultures is closely interrelated with the activity of the ubiquitin-proteasome proteolytic pathway; Pasquini LA et al.; Although the participation of the ubiquitin-dependent pathway and of the proteasome in apoptosis has been proposed, its role in this process is not yet clearly defined . In previous studies, we have shown that in the central nervous system of the rat, programmed cell death and the ubiquitin-dependent proteolytic pathway are closely related to each other and that different types of neurons and of glial cells, shown different types of correlation between the two phenomena . In this work, we have used lactacystin, a highly specific inhibitor of the proteasome, to explore in Schwann cell cultures the relationship between the activity of the Ub-dependent pathway and apoptosis . Apoptosis was explored analyzing changes in nuclear morphology, using the Annexin V assay and by flow cytometry . Activity of caspase-3 was also measured . Changes in the levels of ubiquitin-protein conjugates and of the ubiquitin activating enzymes, E1, as well as expression of proteins that instruct the cells to apoptosis (p53, NFkappaB-IkappaB, Bcl2), or that participate in the control and regulation of the cell cycle, were also examined . Our results indicate that the decrease in the activity of the proteasome induced by lactacystin in Schwann cells, induces apoptotic cell death through changes in the concentration of certain key proteins that are involved in the apoptosis-signaling pathways.

Vopr Virusol, 2002 Nov-Dec, 47(6), 17 - 21
{Effect of interferon inductors on infection induced by hepatitis C virus and activity of mRNA cytokines in cell cultures SW-13 and MT-4}; Narovlianskii AN et al.; An experimental model of hepatitis C virus (HCV) infection in cell culture in vitro was used to study the influence of interferon (IFN) inducers on HCV infection activity . In combination with the RT-PCR method, this model was also used to study the dynamics of cytokine mRNA activity for IFN-alpha, IFN-gamma, IL-I beta, IL-2, IL-4, IL-6, IL-8, BL-10, IL-12, IL-18, and TNF-alpha . The research was carried out using long-term cell cultures SW-13 (human paradrenal adenocarcinoma cells) and MT-4 (human cells of lymphoblastoid origin) inoculated with HCV under conditions of acute infection . The obtained data showed that cell cultures SW-13 and MT-4 were sensitive to replication of HCV (cytopathogenic variant) . The addition of IFN inducers Savratz, Kagocel, and Cycloferon to infected cell cultures usually resulted in suppression of HCV reproduction in these cultures . Cycloferon had the greatest antiviral activity (virus titer level decreased by a factor of 2.51 g and 5.51 g TCD50 in cell cultures SW-13 and MT-4, respectively) . It was suggested that induction of IFN-gamma, IL-4 and IL-8 plays a certain role in HCV reproduction suppression . The results of this work provide an opportunity for more efficacious use of IFN inducers in therapy of HCV infection.

Invest Ophthalmol Vis Sci, 2003 Jan, 44(1), 365 - 9
Expression of IGFBP-3 by human retinal endothelial cell cultures: IGFBP-3 involvement in growth inhibition and apoptosis; Spoerri PE et al.; PURPOSE: Growth hormone (GH), insulin-like growth factor (IGF), and somatostatin (SST) modulate each other's actions . SST analogues have been successfully used to treat proliferative diabetic retinopathy (PDR) that is unresponsive to laser therapy and to retard the progression of severe nonproliferative retinopathy to PDR . In this study, the endogenous expression of IGF-binding protein (IGFBP)-3 was examined in human retinal endothelial cells (HRECs), the direct effects of IGFBP-3 on HRECs were evaluated, and the possible involvement of IGFBP-3 in mediating the growth inhibitory effects of SST receptor (SSTR) agonists in HRECs was assessed . METHODS: The cellular localization of IGFBP-3 was examined with anti-IGFBP-3 and fluorescein-conjugated goat anti-rabbit IgG . HRECs were exposed to varying concentrations of human recombinant IGFBP-3, and growth inhibition was evaluated by thiazolyl blue (MTT) conversion . Apoptosis was examined using fluorochrome-annexin V staining . Conditioned media (CM) from SSTR2 agonist (L779976)-treated or SSTR3 agonist (L796778)-treated HRECs were analyzed by ELISA for changes in expression of IGFBP-3 . RESULTS: HREC immunostaining showed cell surface and cytoplasmic IGFBP-3 . Exogenous IGFBP-3 induced a dose-dependent inhibition of HREC proliferation and staining with fluorochrome-annexin V showed numerous apoptotic HRECs . HRECs exposed to the SSTR2 or SSTR3 agonists expressed IGFBP-3 in a concentration-dependent manner . CONCLUSIONS: Cultured HRECs expressed endogenous IGFBP-3 . Exogenous administration of IGFBP-3-induced growth inhibition and apoptosis, supporting a regulatory role for IGFBP-3 in endothelial cells . SSTR agonists mediate their growth-inhibitory effect, in part, by increasing expression of IGFBP-3.

Virology, 2002 Dec 20, 304(2), 302 - 10
Cell-culture propagation of porcine enteric calicivirus mediated by intestinal contents is dependent on the cyclic AMP signaling pathway; Chang KO et al.; Enteric caliciviruses are emerging pathogens in humans and animals, but they do not replicate in cell culture except for the porcine enteric calicivirus (PEC) Cowden strain . The PEC Cowden strain grows in pig kidney (LLC-PK) cells, but only in the presence of intestinal contents (IC) from uninfected gnotobiotic pigs in the medium . In this study, we investigated the relationship between IC and growth of Cowden PEC . Pretreatment of cells or the virus with IC or transfection of viral RNA into cells did not induce virus growth unless the medium was supplemented with IC . Among modulators of cell signal transduction, the G protein uncoupler, suramin, adenylate cyclase (AC) inhibitor, MDL-12,330A, and the cAMP-dependent protein kinase (PKA) inhibitor, N-(2-{bromocinnamulamino}ethyl)-5-isoquinolinesulfonamide (NBEI) inhibited the effect of IC on virus growth for up to 72 h . These data indicate that PEC virus replication may be dependent on an initial cAMP signaling pathway induced by IC .

Biochimie, 2002 Oct, 84(10), 1003 - 11
Pyruvate reverses metabolic effects produced by hypoxia in glioma and hepatoma cell cultures; Perrin A et al.; The intervention of pyruvate in glucose metabolism was investigated during hypoxic stress in tumour cell cultures having respiratory capacities under normoxic conditions . Results obtained with nuclear magnetic resonance (NMR) spectroscopy showed that, under normoxic conditions, rat glioma C6 and human hepatoma Hep G2 cell cultures metabolised {(13)C(1)}glucose into lactate, alanine, glutamate and other less abundant metabolites, as already known from the literature . In the absence of pyruvate, during hypoxia or cyanide poisoning, both cell types dramatically decreased the label into glutamate and accumulated {(13)C(3)}glycerol-3-phosphate . The compound was further identified by 31P NMR spectroscopy . The accumulation of the label in glycerol-3-phosphate, however, did not occur when the cells were incubated in the presence of pyruvate . The fate of the latter, followed under normoxic conditions by incubating cells with {(13)C(3)}pyruvate and natural glucose, showed that the label was mainly found in alanine, lactate and glutamate . Anoxic conditions increased the label in lactate and reduced that of glutamate . The data show a metabolic effect of pyruvate during mitochondrial blockade due to severe lack of oxygen in tumour cell lines.

J Chromatogr B Analyt Technol Biomed Life Sci, 2003 Jan 25, 784(1), 183 - 7
Expanded bed adsorption processing of mammalian cell culture fluid: comparison with packed bed affinity chromatography; Gonzalez Y et al.; A comparison between expanded bed adsorption and conventional packed bed Protein A Fast Flow to purify the anti-rHBsAg mAbs from feedstock is presented in this work . Direct capture by STREAMLINE expanded bed adsorption chromatography resulted in 92% product recovery and sevenfold more concentrated product with similar purity levels compared to that obtained by the standard packed method . The process time and buffer consumption were reduced in the expanded bed adsorption method not only with the binding-elution conditions but also with the use of NaOH during the cleaning-in-place step . The latter is the most widely accepted agent in downstream processing, being a cost effective technique that provides not only efficient cleaning but also sanitizes complete column systems and destroys pirogens.

Biol Pharm Bull, 2002 Dec, 25(12), 1600 - 3
Evaluation of the cytotoxicity effect of dimethyl sulfoxide (DMSO) on Caco2/TC7 colon tumor cell cultures; Da Violante G et al.; Dimethyl sulfoxide (DMSO) is usually used to solubilize poorly soluble drugs in permeation assays such as that using Caco2 enterocyte-like cells . The objective of this study was to evaluate the toxicity of DMSO on Caco2/TC7 cells and determinate the maximal concentration usable in permeation experiments . Caco2/TC7 cells were cultured for 21 d on 96-well plates for evaluation of toxicity . The determination of lactate dehydrogenase (LDH) release in cell supernatant and the measurement of Neutral Red (NR) uptake are used for cytotoxicity assays . DMSO solutions (0-100%) in Hank's balanced salt solution containing HEPES (25 mM), pH 7.4, were incubated with Caco-2/TC7 cells on 96 well plates . Caco2/TC7 cells were cultured on Transwell-Clear inserts to evaluate the influence of DMSO on the apparent permeability of the paracellular marker mannitol . DMSO 10% did not induce any significant increase in LDH release whereas a significant increase in LDH activity (ANOVA, p<0.05) occurred at a DMSO concentration of 20 to 50% . NR incorporation in viable cells was statistically reduced by 27 to 36% at DMSO concentration of 20% up to 100% (ANOVA, p>0.05) . No statistical difference (p<0.05) in apparent mannitol permeability was observed between the control and 10% DMSO groups . In conclusion, at concentrations of up to 10%, DMSO did not produce any significant alteration in apical membrane permeability or on cell-to-cell tight junctional complexes.

J Ethnopharmacol, 2003 Jan, 84(1), 91 - 4
The effects of aqueous extract of Lavandula angustifolia flowers in glutamate-induced neurotoxicity of cerebellar granular cell culture of rat pups; Buyukokuroglu ME et al.; In the present study, neuroprotective effect of Lavandula angustifolia flower aqueous extract in glutamate-induced neurotoxicity in rat pups cerebellar granular cell culture was investigated . The extract at doses of 10 microg ml(-1), 100 microg ml(-1), 1 mg ml(-1) and 10 mg ml(-1) was applied to culture flasks . The extract at doses of 100 microg ml(-1) and 1 mg ml(-1) significantly blocked glutamate-induced neurotoxicity, with the most effective dose being 1 mg ml(-1) . On the other hand, 10 mg ml(-1) dose of extract increased the dead cell with respect to glutamate group, despite being found insignificant statistically . As a result, L . angustifolia protected the neurons against glutamate toxicity .

Prostate Cancer Prostatic Dis, 2000 Dec, 3(4), 229 - 235
In vitro human cell culture models for the study of prostate cancer; Rhim JS; Prostate cancer is the most commonly diagnosed malignancy in the American men and the second leading cause of male cancer death in the United States . Despite its high incidence, the molecular and genetic events involved in prostate cancer progression remain poorly understood . A hurdle in understanding the molecular genetic changes in prostate cancer has been the difficulty in establishing premalignant lesions and primary prostate tumors as in vitro cell cultures . Primary epithelial cells grow for a finite life span and then senesce . Immortalization is defined by continuous growth of otherwise senescing cells and is believed to represent an early stage in tumor progression . In order to examine these early stages, we and others have developed in vitro models of prostate epithelial cell immortalization . Because prostate cancer is a multistep, progressive disease with a typical onset later in life and with an unusually high number of latent cases that do not develop into clinically manifest cancer, the steps in the progression to malignancy are of particular interest . To understand the many factors that are suspected to contribute to the development of this malignancy, there is a need for an in vitro multistep human prostate epithelial (HPE) culture system . These models have been extremely important in identifying genetic and molecular changes involved in prostate cancer progression . Prostate Cancer and Prostatic Diseases (2000) 3, 229-235

J Food Prot, 2002 Dec, 65(12), 1962 - 9
Modified concentration method for the detection of enteric viruses on fruits and vegetables by reverse transcriptase-polymerase chain reaction or cell culture; Dubois E et al.; Fruits and vegetables may act as a vehicle of human enteric virus if they are irrigated with sewage-contaminated water or prepared by infected food handlers . An elution-concentration method was modified to efficiently detect, by reverse transcriptase-polymerase chain reaction (RT-PCR) or by cell culture, contamination by poliovirus, hepatitis A virus (HAV), and Norwalk-like virus (NLV) of various fresh and frozen berries and fresh vegetables . The protocol included washing the fruit or vegetable surface with 100 mM Tris-HCl, 50 mM glycine, and 3% beef extract, pH 9.5 buffer, which favors viral elution from acid-releasing berries, supplemented with 50 mM MgCl2 to reduce the decrease in viral infectivity during the process . The viral concentration method was based on polyethylene glycol precipitation . Copurified RT-PCR inhibitors and cytotoxic compounds were removed from viral concentrates by chloroform-butanol extraction . Viruses from 100 g of vegetal products could be recovered in volumes of 3 to 5 ml . Viral RNAs were isolated by a spin column method before molecular detection or concentrates were filtered (0.22-microm porosity) and inoculated on cell culture for infectious virus detection . About 15% of infectious poliovirus and 20% of infectious HAV were recovered from frozen raspberry surfaces . The percentage of viral RNA recovery was estimated by RT-PCR to be about 13% for NLV, 17% for HAV, and 45 to 100% for poliovirus . By this method, poliovirus and HAV RNA were detected on products inoculated with a titer of about 5 x 10(1) 50% tissue culture infectious dose per 100 g . NLV RNA was detected at an initial inoculum of 1.2 x 10(3) RT-PCR amplifiable units . This method would be useful for the viral analysis of fruits or vegetables during an epidemiological investigation of foodborne diseases.

Invert Neurosci, 1999-2000, 4(1), 25 - 31
The A-like potassium current of a leech neuron increases with age in cell culture; Tribut F et al.; Single leech neurons isolated and maintained in culture sprout and form electrical and chemical synapses, as they do in vivo, retaining most of the electrical properties of the intact membrane . However, some cells, such as Retzius, Anterior Pagoda (AP) cells and motoneurons, exhibit consistent changes of biophysical characteristics, which mimic those induced by axotomy in vivo and are reversed after reconnection . To improve our understanding of the mechanisms involved in these alterations and of their physiological significance, we investigated the early changes in outward currents developed by cultured AP neurons, using the patch-clamp technique in the whole-cell recording configuration . Different currents were isolated and a differential sensitivity to the time spent in culture and to internal calcium was observed . Three potassium currents were dissected: an A-like current, a delayed rectifier and a third unidentified component . The A-like potassium current was significantly increased with neuronal age in cell culture and was a function of the internal Ca(2+) concentration, whereas the two other potassium currents remained unchanged . Intracellular recordings performed from axotomized neurons of cultured ganglia revealed clear-cut alterations in spike adaptation, which might be due to changes of the A-like current.

Endocrinology, 2003 Jan, 144(1), 388 - 99
Stimulation of combinatorial expression of prolactin and glycoprotein hormone alpha-subunit genes by gonadotropin-releasing hormone and estradiol-17beta in single rat pituitary cells during aggregate cell culture; Hauspie A et al.; Previously we showed the existence of rat and mouse anterior pituitary cells coexpressing mRNA from two or more hormone genes in which production and/or storage of the corresponding hormones were not detectable . To substantiate a putative function for these cells, we investigated whether these phenotypes were retained during long-term reaggregate cell culture and whether protagonist regulatory factors could expand cell populations expressing particular hormone mRNA combinations . After 4-wk culture and treatments, aggregates were trypsinized and single cells collected by means of a fluo-rescence-activated cell sorter . Hormone mRNAs were detected by single-cell RT-PCR . Combinatorial hormone mRNA expression was retained in culture . Both estradiol (E2) and GnRH (1 nM) markedly augmented the proportion of cells expressing prolactin (PRL) mRNA together with other hormone mRNAs and cells expressing glycoprotein subunit (GSU)-alpha mRNA together with other hormone mRNAs . GnRH strongly increased the proportion of cells containing alphaGSU mRNA alone, but E2 did not . GnRH and (E2) affected the expansion of a population (approximately 20% of all cells) coexpressing PRL and alphaGSU mRNA without betaGSUs . Immunostaining of stored hormone on tissue sections revealed colocalization of PRL and alphaGSU in the E2- but not in the GnRH-treated cells . The present findings suggest that cells coexpressing different pituitary hormone mRNAs form a distinct population that survives without extrapituitary factors . Their occurrence can be markedly modified by regulatory factors . Certain hormone regimens favor unique coexpressions distinctly at mRNA and protein level . These peculiar characteristics support the notion that combinatorial expression of hormone genes in the pituitary serves a biological role.

J Biomater Sci Polym Ed, 2002, 13(10), 1119 - 34
Synthesis and characterization of glycolide, L-lactide, and PDMS-based terpolymers as a support for cell cultures; Porjazoska A et al.; Poly(lactic acid)/poly(glycolic acid)/poly(dimethylsiloxane) (PLGA/TEGOMER) terpolymers have been synthesized by the ring-opening polymerization of L-lactide and glycolide with alpha,omega-amine-terminated poly(dimethylsiloxane) prepolymer, using stannous octoate as a catalyst . The resulting terpolymers were characterized by various analytical techniques including size exclusion chromatography, 1H-nuclear magnetic resonance (1H-NMR), Fourier transform infrared spectroscopy, and differential scanning calorimetry . The data showed that the terpolymers presented an amorphous structure . The glass transition temperature decreased with increasing TEGOMER unit content . For in vitro degradation studies, porous films were fabricated using a solvent-casting, particulate leaching technique . Degradation of the PLGA/TEGOMER terpolymer was studied in phosphate-buffered saline at pH 7.4 and 37 degrees C . The degradation was followed by intrinsic viscosity, mass loss, and molecular weight measurements, and 1H-NMR spectroscopy . The mass loss after 55 days was 76% for the PLGA/TEGOMER (71/24/5) sample . Cell growth experiments using Swiss 3T3 fibroblasts demonstrated that PLGA/TEGOMER terpolymer matrices allow the attachment and growth of cells.

Cell Tissue Res, 2003 Jan, 311(1), 117 - 30 Epub 2002 Nov 28.
Cell culture of mechanoreceptor neurons innervating proleg sensory hairs in Manduca sexta larvae, and co-culture with target motoneurons; Melville JM et al.; The tip of each proleg in Manduca sexta larvae bears a dense array of mechanosensory hairs termed planta hairs (PHs), each innervated by a single sensory neuron (termed a PH-SN) located in the underlying epidermis . In the CNS, axon terminals of PH-SNs make direct, excitatory, nicotinic cholinergic synapses with proleg retractor motoneurons including the accessory planta retractor (APR) . These synapses mediate a proleg withdrawal reflex, exhibit multiple forms of activity-dependent plasticity and weaken during the prepupal peak of ecdysteroids . In the present study we developed methods to dissociate PH-SNs from the epidermis and culture them alone or with APRs . The PH-SNs were fluorescently labeled in situ by introducing dye through the cut hair shaft or by retrograde axonal staining . Alternatively, unlabeled PH-SNs were utilized . The epidermis beneath the planta hair array was separated from the cuticle, enzymatically treated and mechanically dissociated into single cells . PH-SNs were cultured on glass coverslips coated with concanavalin A and laminin, in modified Leibovitz's IL-15 medium . Supplementation with medium conditioned by an insect cell line produced the best results . Dissociated PH-SNs had somatic diameters of ~10 micro m and typically bore a stout dendrite consisting of the inner and, occasionally, the outer dendritic segment . An axonal stump was sometimes retained . Viable PH-SNs typically extended new processes and often survived for 2-4 weeks . When co-cultured, PH-SNs and APRs exhibited robust growth and made close anatomical appositions . This culture system provides convenient experimental access to PH-SNs and may potentially permit sensorimotor synapses to be investigated in vitro.

FEBS Lett, 2002 Dec 18, 532(3), 441 - 4
Nitric oxide induces neutral ceramidase degradation by the ubiquitin/proteasome complex in renal mesangial cell cultures; Franzen R et al.; The neutral ceramidase is a key enzyme in the regulation of cellular ceramide levels . Previously we have reported that stimulation of rat renal mesangial cells with nitric oxide (NO) donors leads to an inhibition of neutral ceramidase activity which is due to increased degradation of the enzyme . This and the concomitant activation of the sphingomyelinase results in an amplification of ceramide levels . Here, we show that the NO-triggered degradation of neutral ceramidase involves activation of the ubiquitin/proteasome complex . The specific proteasome inhibitor lactacystin completely reverses the NO-induced degradation of ceramidase protein and neutral ceramidase activity . As a consequence, the cellular amount of ceramide, which drastically increases by NO stimulation, is reduced in the presence of lactacystin . Furthermore, ubiquitinated neutral ceramidase accumulates after NO stimulation . In summary, our data clearly show that the ubiquitin/proteasome complex is an important determinant of neutral ceramidase activity and thereby regulates the availability of ceramide.

J Neurosci Res, 2003 Jan 1, 71(1), 38 - 45
Early programmed cell death in human NT2 cell cultures during differentiation induced by all-trans-retinoic acid; Guillemain I et al.; Previous studies have demonstrated that programmed cell death takes place at different stages during the development of the CNS in vivo . Our purpose in this study was to detect early programmed cell death associated with the induction of differentiation by retinoic acid (RA) in the NT2 cell line . By using the annexin V labeling as a marker of apoptosis, a significant apoptotic cell death was quantified during the third and the fourth days of the RA treatment . Double-labeling studies using the staining of the genomic DNA strand breaks with the terminal deoxyribosyl-transferase-mediated dUTP nick end-labeling (TUNEL) assay and either nestin or microtubule-associated protein 2 (MAP2) showed that 1) the early apoptotic cell death affected mostly nestin-positive cells and 2) after 8 days of differentiation, although cells with neuronal phenotypes are present, no colabeled TUNEL/MAP2 cells were detected . With regard to the neuronal protein MAP2, we observed discrete immunolabeling of a few NT2 cells as early as day 3 of the differentiation and a significant emergence of MAP2-immunopositive cells at days 6-8 . Thus, our results show that, when as a whole the differentiating NT2 cell population is considered, 1) the apoptotic cell death observed during the third day of differentiation occurs mostly in undifferentiated cells, 2) this process coincides with the first detection of the neuronal phenotype in NT2 cell cultures, and 3) the end of the cell death period in NT2 cell cultures is marked by both the accumulation of MAP2-positive cells and the beginning of expression of the Bcl-2 protein in the cultures .

Dev Biol (Basel), 2002, 110, 125 - 34
Influenza vaccine technologies and the use of the cell-culture process (cell-culture influenza vaccine); Mabrouk T et al.; Influenza vaccines have been produced for decades in eggs by a classical technology . A range of new vaccine technologies and approaches has been applied to improving these vaccines . Foremost among these are the use of mammalian cells to produce influenza viruses, the use of new adjuvants, and a live attenuated vaccine . Mammalian cell-derived vaccines, produced in nontumorigenic cell lines, have been developed and shown to be effective production systems as alternatives to eggs . This manuscript discusses the advantages and challenges of mammalian cell production, as well as alternative technologies for influenza vaccines.

J Biol Chem, 2003 Feb 21, 278(8), 6371 - 83 Epub 2002 Dec 09.
Analysis of the cytosolic proteome in a cell culture model of familial amyotrophic lateral sclerosis reveals alterations to the proteasome, antioxidant defenses, and nitric oxide synthetic pathways; Allen S et al.; Injury to motor neurons associated with mutant Cu,Zn-superoxide dismutase (SOD1)-related familial amyotrophic lateral sclerosis (FALS) results from a toxic gain-of-function of the enzyme . The mechanisms by which alterations to SOD1 elicit neuronal death remain uncertain despite intensive research effort . Analysis of the cellular proteins that are differentially expressed in the presence of mutant SOD1 represents a novel approach to investigate further this toxic gain-of-function . By using the motor neuron-like cell line NSC34 stably transfected with wild-type, G93A, or G37R mutant human SOD1, we investigated the effects of mutant human SOD1 on protein expression using proteomic approaches . Seven up-regulated proteins were identified as argininosuccinate synthase, argininosuccinate lyase, neuronal nitric-oxide synthase, RNA-binding motif protein 3, peroxiredoxin I, proteasome subunit beta 5 (X), and glutathione S-transferase (GST) Alpha 2 . Seven down-regulated proteins were identified as GST Mu 1, GST Mu 2, GST Mu 5, a hypothetical GST Mu, GST Pi B, leukotriene B(4) 12-hydroxydehydrogenase, and proteasome subunit beta5i (LMP7) . GST assays demonstrated a significant reduction in the total GST activity of cells expressing mutant human SOD1 . Proteasome assays demonstrated significant reductions in chymotrypsin-like, trypsin-like, and post-glutamylhydrolase proteasome activities . Laser capture microdissection of spinal cord motor neurons from human FALS cases, in conjunction with reverse transcriptase-PCR, demonstrated decreased levels of mRNA encoding GST Mu 1, leukotriene B(4) 12-hydroxydehydrogenase, and LMP7 . These combined approaches provide further evidence for involvement of alterations in antioxidant defenses, proteasome function, and nitric oxide metabolism in the pathophysiology of FALS.

Mar Pollut Bull, 2002 Oct, 44(10), 983 - 91
Application of the luciferase cell culture bioassay for the detection of refined petroleum products; Ziccardi MH et al.; A luciferase cell culture-based bioassay, developed to detect 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-like activity of halo-genated and polycyclic aromatic hydrocarbons, was optimized to detect refined petroleum products and to determine their relative inducing potency . Quality control standards from 32 refined products (gasolines and diesels, jet fuels, lubricating oils, fuel oils and weathered products) and three commercial products were evaluated . Induction equivalents (I-EQs) were determined by direct comparison of the EC50 and EC20 values (based on the median and 20% TCDD maximal response, respectively) from dose-response curves for each product to those obtained with TCDD . Most petroleum products were active in the luciferase bioassay, with those products composed of fractions produced later in the distillation process (i.e . fuel oils) inducing higher levels . Additionally, weathering of products reduced their induction potency . Based on the high I-EQ estimates of many products, biological effects associated with exposure may have been previously underestimated using other diagnostic methods.

Biotechnol Bioeng, 2003 Feb 5, 81(3), 329 - 40
Study of caspase inhibitors for limiting death in mammalian cell culture; Sauerwald TM et al.; Apoptosis in mammalian cell culture is associated with decreased bioproduct yields and can be inhibited through altering the intracellular signaling pathways mediating programmed cell death . In this study, we evaluated the capacity to inhibit caspases to maintain high viable cell numbers in CHO and 293 cultures . Two genetic caspase inhibitors, XIAP and CrmA, were examined along with a mutant of each, XIAP-BIR123NC, which contains three BIR domains but lacks the RING finger, and CrmA-DQMD, which has CrmA's pseudosubstrate site replaced with that of another caspase inhibitor, p35 . Stable CHO pooled and 293 clonal cell lines expressing each protein were exposed to apoptotic insults, including spent medium, Sindbis virus, and etoposide . For each insult the mutated protein resulted in higher viabilities than its wild-type counterpart . However, the mutants provided different levels of protection, depending on the insult considered . CrmA-DQMD was the preferred inhibitor for spent medium-induced apoptosis, whereas XIAP-BIR123NC conferred better protection for etoposide-induced death . Addition of Z-VAD.fmk to the genetically engineered cells enhanced viabilities in the presence of spent medium or etoposide; however, the largest increases in viability were experienced by the control cells, indicating an overlap in caspase inhibition between the genetic and chemical inhibitors . Finally, parental 293 cells were treated with caspase-8 and -9 inhibitors, Z-IETD.fmk and Z-LEHD.fmk, in concert with spent medium or etoposide exposure . Spent medium-induced death was delayed more readily with the caspase-8 inhibitors, CrmA-DQMD and Z-IETD.fmk, and etoposide-induced death was stalled more so with XIAP-BIR123NC and Z-LEHD.fmk . These results suggest that the apoptosis pathways induced and the level of protection afforded by a particular caspase inhibitor may vary with the insult considered .

J Orthop Res, 2002 Nov, 20(6), 1305 - 10
Effect of irrigation solutions for arthroscopic surgery on intraarticular tissue: comparison in human meniscus-derived primary cell culture between lactate Ringer's solution and saline solution; Shinjo H et al.; In order to determine whether there is a difference in effect on cell morphology and function between two common arthroscopic irrigation solutions, primary cultures of cells derived from the surgically excised human menisci were incubated for 3 or 6 h in lactated Ringer's solution, isotonic sodium chloride solution, or serum-free cell culture medium (negative-control condition) . Cell integrity was blindly evaluated by three independent examiners scoring photomicrographs of the cell cultures on a battery of five-point scales for abnormality of cell shape, irregularity of cell membrane, change of cell size and cell density . Cell cultures were also quantitatively assayed by semi-quantitative reverse-transcription-polymerase-chain-reaction for mRNA of alpha1 (I) procollagen, alpha1 (II) procollagen, aggrecan and heat-shock protein 70 to assess functional consequences of exposure to the solutions . There was a statistically significant difference in cell integrity scores between either lactated Ringer's solution or serum-free cell-culture medium and isotonic sodium chloride solution with greater damage to cells displayed . Scores for lactated Ringer's solution did not differ from those for serum-free cell-culture medium . There were no significant differences in mRNA expression level among the treatment conditions . It was concluded that the lactated Ringer's solution better maintained human meniscus cell integrity than the isotonic saline.

Anal Biochem, 2002 Dec 15, 311(2), 127 - 32
A streamlined method for the isolation and quantitation of nanomole levels of exported polyamines in cell culture media; Hawel L 3rd et al.; A number of years ago, our laboratory published a method for the isolation of small amounts of polyamines from cell culture media using the ion-exchange resin Bio-Rex 70 . We have used this technique extensively to study the export of putrescine and cadaverine from cultured mammalian cells . Unfortunately, this method was highly inefficient in isolating the polyamines spermidine and spermine and was incapable of recovering the acetylated polyamine N(1)-acetylspermidine . In response to these shortcomings, we modified our previous protocol to quantitatively isolate the polyamines N(1)-acetylspermidine, putrescine, cadaverine, N(1)-acetylspermine, spermidine, and spermine . The new method, which is much faster to perform and more efficient than the one previously described, employs the use of disposable minicolumns and a single resin washing step using a weak solution of sodium carbonate at pH 9.3 . This new protocol also eliminates the column elution step in favor of directly derivatizing the polyamines with dansyl chloride on the ion-exchange resin . High-performance liquid chromatography analysis of the dansylated polyamines isolated by this procedure showed that 75% of N(1)-acetylspermidine and nearly 100% of the other polyamines present in nanomolar levels were recovered from small amounts of cell culture medium . This new protocol is a valuable new tool for the study of the intracellular/extracellular dynamics of polyamine pools in cultured cells . {A detailed laboratory protocol for this procedure (containing all of the information in this paper but in a condensed form) can be requested by e-mailing the authors.}

Biotechnol Prog, 2002 Nov-Dec, 18(6), 1149 - 56
Alterations in Taxol production in plant cell culture via manipulation of the phenylalanine ammonia lyase pathway; Brincat MC et al.; One approach to increasing secondary metabolite production in plant cell culture is to manipulate metabolic pathways to utilize more resources toward production of one desired compound or class of compounds, such as diverting carbon flux from competing secondary pathways . Since phenylalanine provides both the phenylisoserine side chain and the benzoyl moiety at C-2 of Taxol, we speculated that blockage of the phenylpropanoid pathway might divert phenylalanine into Taxol biosynthesis . We used specific enzyme inhibitors to target the first enzyme in the phenylpropanoid pathway, phenylalanine ammonia lyase (PAL), the critical control point for conversion of L-phenylalanine to trans-cinnamic acid . Cinnamic acid acted quickly in reducing PAL activity by 40-50%, without affecting total protein levels, but it generally inhibited the taxane pathway, reducing Taxol by 90% of control levels . Of the taxanes produced, 13-acetyl-9-dihydro-baccatin III and 9-dihydrobaccatin III doubled as a percentage of total taxanes in C93AD and CO93P cells treated with 0.20 and 0.25 mM cinnamic acid, when all other taxanes were lowered . The PAL inhibitor alpha-aminooxyacetic acid (AOA) almost entirely shut down Taxol production at both 0.5 and 1.5 mM, whereas L-alpha-aminooxy-beta-phenylpropionic acid (AOPP) had the opposite effect, slightly enhancing Taxol production at 1 microM but having no effect at 10 microM . The discrepancy in the effectiveness of AOA and AOPP and the lack of effect with addition of phenylalanine or benzoic acid derivatives further indicates that the impact of cinnamic acid on Taxol is related not to its effect on PAL but rather to a specific effect on the taxane pathway . On the basis of these results, a less direct route for inhibiting the phenylpropanoid pathway may be required to avoid unwanted side effects and potentially enhance Taxol production.

Appl Microbiol Biotechnol, 2002 Dec, 60(4), 396 - 402 Epub 2002 Nov 05.
Effects of inoculum size and age on biomass growth and paclitaxel production of elicitor-treated Taxus yunnanensis cell cultures; Zhang CH et al.; Suspension cultures of Taxus yunnanensis cells were inoculated with cells of different culture ages (12-24 days) at various densities {50-250 g fresh weight (fw)/l}, and treated (on day 7) with a mixture of elicitors, including Ag(+), chitosan and methyl jasmonate . The biomass productivity (during the production stage) increased dramatically with inoculum size, but decreased with inoculum age over 16 days . The volumetric yield and productivity of taxol (paclitaxel) also increased with inoculum size, while the specific taxol yield (per cell) was mainly dependent on inoculum age, with an optimum of 20 days, during the early stationary phase . The highest taxol yield and productivity, 39.8 mg/l and 1.9 mg/l per day, respectively, were obtained with a 20-day-old inoculum at 200 g fw/l . Taxol excretion by the cells increased with inoculum age but decreased with inoculum size . The elicitor-induced activities of catalase (CAT) and phenylalanine ammonia-lyase (PAL) also depended mainly on inoculum age; higher PAL activity and lower CAT activity were obtained with an older inoculum, corresponding to a higher taxol yield . The results show that both inoculum size and age are important variables for taxol production, though the latter more profoundly influences elicitor-induced taxol biosynthesis of the cells . Inoculum size and age are also interrelated and should be optimized together in a two-stage culture process.

Biomed Tech (Berl), 2002, 47 Suppl 1 Pt 2, 866 - 7
{Development of a CAN (controller area network) bus for modules of a perfused cell culture system}; Rehn K et al.; For computer-aided data acquisition and automated running of experiments in cell cultivation reactors networking of all sensoric and actoric devices is required . A low cost and high performance solution to this demand can be found by using the widely established CAN (Controller Area Network) bus . If standard PCs running MS Windows are used to control this network special measures have to be taken to prevent loss of data caused by differences in the computing power of the PC on one hand and microcontroller-based devices on the other.

Anesthesiology, 2002 Dec, 97(6), 1466 - 76
Effect of local anesthetic on neuronal cytoplasmic calcium and plasma membrane lysis (necrosis) in a cell culture model; Johnson ME et al.; BACKGROUND: To investigate the mechanism by which rare cases of spinal local anesthetic (LA) neurotoxicity occur, we have tested the hypotheses that LAs elevate cytoplasmic calcium (Ca2+(cyt)), that this is associated with a neurotoxic effect, and that lidocaine and bupivacaine differ in their neurotoxicity . METHODS: Neurons of the ND7 cell culture line, derived from dorsal root ganglion, were loaded with fura-2 and analyzed by digitized video fluorescence microscopy during 60 min LA exposure, allowing determination of Ca2+(cyt) and time of necrotic cell death (plasma membrane lysis) at the single neuron level . RESULTS: Lidocaine 0.1% and bupivacaine 0.025% caused minimal changes in Ca . Lidocaine 0.5-5% and bupivacaine 0.125-0.625% caused an early, small (less than threefold), concentration-dependent increase in Ca2+(cyt) that was transient and returned to near baseline within 10 min . Lidocaine 2.5% and 5% then caused a sustained, greater than ten-fold increase in Ca2+(cyt) and death in some neurons during the 60 min exposure period . Pretreatment with thapsigargin eliminated the initial transient increase in Ca2+(cyt), consistent with endoplasmic reticulum (ER) as its source, and increased neuronal death with 5% lidocaine, suggesting that lidocaine neurotoxicity can be increased by failure of ER to take up elevated Ca2+(cyt) . The later sustained increase in Ca2+(cyt) seen with 2.5 and 5% lidocaine was prevented in Ca2+ -free medium, and restored when Ca2+ was added back to the buffer in the presence of lidocaine, suggesting that higher concentrations of lidocaine increase influx of Ca2+ through the plasma membrane . CONCLUSIONS: In this model, lidocaine greater than 2.5% elevates Ca2+(cyt) to toxic levels . Bupivacaine and lower concentrations of lidocaine transiently alter Ca2+(cyt) homeostasis for several minutes, but without an immediate neurotoxic effect within 60 min.

J Nephrol, 2002 Sep-Oct, 15(5), 539 - 46
The effect of polyamines and dialysate fluid on extracellular matrix synthesis in VERO cell cultures; Stabellini G et al.; BACKGROUND: Polyamines are involved in normal and pathological cell proliferation and differentiation . Like acid radicals, they play an important role in remodelling the extracellular matrix and are considered "uremic toxins" . Proteins and glycosaminoglycans are essential components of the extracellular matrix, and contribute to normal mature organ functions . The aim of this study was to analyse the effects of free polyamines, dialysate fluid components and dialysis fluid on protein and extracellular glycosaminoglycan synthesis in VERO cell cultures . METHODS: The dialysate fluid components were separated with a Sephadex G15 column and the cultures were analysed after incorporation of 3H-leucine and 3H-glucosamine . Cultures were run at pH 7.0 and pH 7.4 . The glycosaminoglycan classes were separated with a DEAE column, and polyamines were determined by high-performance liquid chromatography . Proteins and single glycosaminoglycan classes were quantified by a scintillator . DNA gel electrophoresis was done to detect chromatin fragmentation . RESULTS: Dialysate contained putrescine, spermidine and spermine, chromatography showing four peaks; only peaks I and II indicated polyamines at respectively Da 5000 and 1500 . Polyamines are therefore linked to different carriers . There was an increase of protein and glycosaminoglycan synthesis with dialysis fluid and polyamines, but inhibition with peak II or dialysate . DNA gel electrophoresis showed no chromatin fragmentation . Findings at pH 7.0 and 7.4 were similar . CONCLUSIONS: It would appear that in uremic patients polyamines are conjugated to protein carriers of different molecular weights with different biological actions . As polyamines and dialysis fluid affect changes in extracellular matrix, they could be related to physiological organ functions . However, these in vitro data must be considered with the appropriate limitations when we try to extrapolate them to the in vivo situation.

Int J Impot Res, 2002 Oct, 14(5), 397 - 405
Peyronie's disease cell culture models: phenotypic, genotypic and functional analyses; Mulhall JP et al.; Peyronie's disease is a fibromatosis of the tunica albuginea . While trauma is believed to be the inciting event, the exact pathophysiology of this condition is unknown . In vitro analysis of cell biology can shed light on the pathogenesis of medical conditions and has been used for many decades as a research tool . We have established a cell culture model, which we have used to study the pathobiology of cells derived from Peyronie's disease plaque tissue . In 10 separate cell cultures derived from different individuals, these cells have demonstrated consistent phenotypic, genotypic and functional alterations . In neither of the control cell cultures, neonatal foreskin fibroblasts and normal tunica-derived fibroblasts have any of the above aberrations been demonstrated . The cells studied have been shown to be fibroblasts in nature with a sub-population of myofibroblasts present in culture . The Peyronie's disease plaque tissue-derived fibroblasts have demonstrated (i) consistent morphologic transformation (ii) increased S-phase on flow cytometry (iii) decreased dependence on culture medium (iv) cytogenic instability (v) excess production of fibrogenic cytokines and (vi) stabilization and dysfunctionalization of p53 . Further refinement of this model and future analyses may permit an increased understanding of the pathogenesis of this condition and allow the development of therapeutic strategies.

Biomed Tech (Berl), 2002, 47 Suppl 1 Pt 1, 401 - 3
Patterned polymer surfaces for cell culture applications; Welle A et al.; We studied the physico/chemical effects of deep UV irradiation of polystyrene, PMMA and polycarbonate with respect to cell adhesion and protein immobilization . Photochemical modifications of the polymer surfaces yielded unstable peroxides and carboxylic acid groups . Patterned enzyme and antibody adsorbates were realized by coupling via carbodiimid activation of the COOH-moities . Hepatoma cells (HepG2) and fibroblasts (L929) adhered in the presence of serum proteins in the culture medium on the irradiated regions of the substrate without any further treatment.

Biomed Tech (Berl), 2002, 47 Suppl 1 Pt 1, 377 - 8
{Recording and control of culture parameters in perfused cell culture systems}; Hanke G et al.; For the cultivation of animal cells perfused cell culture systems are advantageous . The measurement of the process parameters for the check of the culture is difficult through the small volumes and dimensions . A developed measuring system allows the measuring of the parameters temperature, pH-value and dissolved oxygen.

Space Med Med Eng (Beijing), 2002 Oct, 15(5), 383 - 6
{Research progress {correction of progresses} of cell culture apparatus}; Tan YJ et al.; Cell culture apparatus (CCA) is necessary to space medicine research and space life-science research . With development of technology, CCA has been fully developed . Nowadays, CCA has been used in scientific research and production widely . In addition, space cell culture apparatus has become an indispensable tool in correlative space research . In this paper, a variety of apparatus used on ground and in space are summarized.

J Neuroimmunol, 2002 Dec, 133(1-2), 20 - 9
IL-4 increases GABAergic phenotype in rat retinal cell cultures: involvement of muscarinic receptors and protein kinase C; Sholl-Franco A et al.; Interleukin-4 (IL-4) is an anti-inflammatory cytokine . During injuries, infections and neurodegenerative diseases, high levels of this molecule are expressed in the brain . In the present work, we investigated the effect of IL-4 on GABAergic differentiation of retinal cells kept in vitro . We analyzed either the uptake of {3H}-gamma-aminobutyric acid (GABA) or the expression of glutamic acid decarboxylase (GAD-67) following IL-4 treatment . We have also investigated the pharmacological modulation of the {3H}-GABA uptake by cholinergic activation . Our results demonstrate that IL-4 increases the uptake of {3H}-GABA after 48 h in culture in a dose-dependent manner (0.5-100 U/ml) . The maximal effect was obtained with 5 U/ml (75% increase) . This effect was blocked by 1 mM of nipecotic acid, demonstrating the involvement of the GAT-1 subtype of GABA transporter . The IL-4 effect depends on M1 muscarinic activity, an increase in intracellular calcium levels, tyrosine kinase activity and protein kinase C (PKC) activity . Treatment with IL-4 for 48 h induced an increase of 90% in the number of GAD- and GABA-immunoreactive cells when compared with control cultures . Our results indicate that IL-4 modulates the GABAergic phenotype of retinal cells in culture . This result can suggest an important role for this cytokine either during the normal development of retinal circuitry or during neuroprotection after injuries.

J Physiol Paris, 2002 Apr-Jun, 96(3-4), 313 - 6
The presence of astrocytes enhances beta amyloid-induced neurotoxicity in hippocampal cell cultures; Domenici MR et al.; A characteristic feature of neuritic plaques in Alzheimer's disease is represented by the presence of activated astrocytes, surrounding dystrophic neurons and beta-amyloid deposition . To explore the role of astrocytes in in vitro beta-amyloid neurotoxicity, we studied the effect of beta-amyloid treatment in hippocampal neurons in two different cell models: pure cultures, where neurons were grown in absence of astrocytes and mixed cultures, where neurons were seeded on a confluent layer of astrocytes . We evaluated two characteristic aspects of in vitro beta-amyloid neurotoxicity: reduction of cell viability and degeneration of the neuritic tree . We demonstrated that neurons growing on astrocytes were more prone to the detrimental effect of the amyloid peptide, with respect to neurons grown in absence of the glial component . Our results support the hypothesis that beta-amyloid-astrocyte interaction can adversely condition neurons and contribute to neuronal damage in Alzheimer's disease .

Nat Prod Lett, 2002 Oct, 16(5), 359 - 63
Pinoresinol from Ipomoea cairica cell cultures; Paska C et al.; Ipomoea cairica cell cultures produced a tetrahydrofuran lignan, (+)-pinoresinol, identified by UV, IR, MS and NMR methods, not yet found in the intact plant, and new in the Convolvulaceae family . Pinoresinol was found to have antioxidant and Ca2+ antagonist properties . As it could be requested for its biological activity, we examined the possibility to raise the pinoresinol yield of I . cairica cultures, as well as we continued investigations on lignans' response to optimization.

Avian Pathol, 2002 Oct, 31(5), 485 - 92
Characterization of the antigenic, immunogenic, and pathogenic variation of infectious bursal disease virus due to propagation in different host systems (bursa, embryo, and cell culture) . III . Pathogenicity; Rodriguez-Chavez IR et al.; Differences in the relative pathogenicity of variant (1084 E and GLS) and standard (Edgar and STC) infectious bursal disease virus (IBDV) strains were observed after propagation in the bursa of Fabricius, embryos, or cell cultures . Bursa-derived IBDV induced the most severe lesions in the bursa of Fabricius when compared with strains propagated in embryos or cell cultures . Embryo-derived IBDV induced moderate gross bursal lesions, whereas cell culture-derived IBDV did not damage the bursa grossly . A high frequency of virus re-isolations was obtained from bursal, spleen, and thymic samples collected from birds inoculated with bursa-derived or embryo-derived IBDV . Virus re-isolation occurred much less frequently from birds inoculated with cell culture-adapted IBDV . Serological evaluations demonstrated that bursa-derived IBDV strains induced a higher neutralizing antibody response than did embryo-derived or cell culture-derived strains . These results document that the relative pathogenicity and immunogenicity of IBDV is reduced following propagation in embryos or cell cultures.

Avian Pathol, 2002 Oct, 31(5), 473 - 83
Characterization of the antigenic, immunogenic, and pathogenic variation of infectious bursal disease virus due to propagation in different host systems (bursa, embryo, and cell culture) . II . Antigenicity at the epitope level; Rodriguez-Chavez IR et al.; Antigenic variation of infectious bursal disease virus (IBDV) due to propagation in different host systems (bursa of Fabricius, embryos, or cell cultures) was determined by enzyme-linked immunosorbent assay (indirect and antigen capture) and western blot analysis . To conduct this study, we used 27 non-neutralizing anti-VP(2) monoclonal antibodies, a reference panel of nine neutralizing monoclonal antibodies, and 13 neutralizing anti-IBDV chicken polyclonal antibodies . Changes occurred in neutralizing, cross-reactive, conformation-dependent epitopes on the VP(2) protein of IBDV . Interestingly, non-neutralizing, cross-reactive, conformation-dependent and confirmation-independent epitopes also changed on VP(2) . These epitope changes were directly associated with the method used to propagate IBDV . These results demonstrate that different host systems may play an important role in the antigenicity of IBDV.

Avian Pathol, 2002 Oct, 31(5), 463 - 71
Characterization of the antigenic, immunogenic, and pathogenic variation of infectious bursal disease virus due to propagation in different host systems (bursa, embryo, and cell culture) . I . Antigenicity and immunogenicity; Rodriguez-Chavez IR et al.; In vitro and in ovo virus neutralization assays were conducted to assess the role of different host systems in infectious bursal disease virus (IBDV) antigenic and immunogenic variation . Four different strains, two variant (1084 E and GLS) and two standard (Edgar and STC), were propagated separately in the bursa of Fabricius and embryos, and were compared with cell culture-adapted preparations of the homologous strains . Chicken polyclonal antisera were prepared against each IBDV and neutralizing antibody titres were determined . Normalized IBDV antibody concentrations were used in neutralization assays against homologous and heterologous IBDVs in 10-day-old specific pathogen free embryos . Both antigenic and immunogenic changes occurred in IBDVs evaluated, as evidenced by differences in the ability of normalized antibody to neutralize IBDV propagated in different host systems . Antibody induced by bursal-derived IBDV neutralized all isolates equally well, whereas antibody induced by cell culture-derived virus neutralized bursal-derived IBDV much less effectively.

Strahlenther Onkol, 2002 Sep, 178(9), 491 - 6
Radiosensitivity and TP 53, EGFR amplification and LOH10 analysis of primary glioma cell cultures; Gerlach B et al.; AIM: Determination of in-vitro radiosensitivity and genetic alterations of cell cultures derived from human glioma biopsy tissue and established glioma cell lines . MATERIAL AND METHODS: Fresh brain tumor specimens of six patients were processed to early passage cell cultures . In addition the cell lines D 384 and Gli 6 were used . Cell cultures were irradiated with doses from 2 to 10 Gy . Following irradiation, cell survival was determined by clonogenic assay and survival curves were generated . The surviving fractions after 2 Gy (SF2) and 4 Gy (SF4) were used as radiosensitivity parameters . Genetic analysis included determination of the mutational and loss of heterozygosity (LOH) status of TP 53 (exons 5-8), the LOH 10- and epidermal growth factor receptor gene (EGFR) amplification status . RESULTS: The SF2 and SF4 values ranged from 0.54 to 0.88 (mean: 0.70) and from 0.13 to 0.52 (mean: 0.32), respectively . Genetic alterations were found in the Gli 6 cell line and in two primary cell cultures . The genetic profile of Gli 6 showed LOH but no TP 53 mutation, complete LOH 10 and no EGFR amplification . The VU 15 cell culture showed TP 53 mutation but no LOH 10 or EGFR amplification, while VU 24 showed incomplete LOH 10, EGFR amplification and no TP 53 mutation . In the other four cell cultures and D 384 cell line no genetic alterations were diagnosed . Histopathological classification of glioblastoma multiforme and/or genetic alterations resulted in lower radiosensitivity . CONCLUSION: In this small series of early passage glioma cell cultures low radiosensitivity and alterations in cell regulatory genes were seen . Further testing of biological behavior in larger series of patient-derived material is ongoing.

J Biotechnol, 2003 Jan 23, 100(2), 177 - 80
Sponge (2',5')oligoadenylate synthetase activity in the whole sponge organism and in a primary cell culture; Kelve M et al.; A high (2',5')oligoadenylate (2-5A) synthetase activity was found in the marine sponge Geodia cydonium . Here we demonstrate that the 2-5A synthetase activity is present also in other sponge species although the level of the 2-5A synthetase activity varies in several magnitudes in different sponges . The 2-5A synthesizing activity was maintained in the primary culture produced from a sponge.

AAPS PharmSci . 2002;4(3):E12.
Transfection efficiency and toxicity of polyethylenimine in differentiated Calu-3 and nondifferentiated COS-1 cell cultures; Florea BI et al.; In the present study, we evaluated polyethylenimine (PEI) of different molecular weights (MWs) as a DNA complexing agent for its efficiency in transfecting nondifferentiated COS-1 (green monkey fibroblasts) and well-differentiated human submucosal airway epithelial cells (Calu-3) . Studying the effect of particle size, zeta potential, presence of serum proteins or chloroquine, it appeared that transfection efficiency depends on the experimental conditions and not on the MW of the PEI used . Comparing transfection efficiencies in both cell lines, we found that PEI was 3 orders of magnitude more effective in COS-1 than in Calu-3 cells, because Calu-3 cells are differentiated and secrete mucins, which impose an additional barrier to gene delivery . Transfection efficiency was strongly correlated to PEI cytotoxicity . Also, some evidence for PEI-induced apoptosis in both cell lines was found . In conclusion, our results indicate that PEI is a useful vector for nonviral transfection in undifferentiated cell lines . However, results from studies in differentiated bronchial epithelial cells suggest that PEI has yet to be optimized for successful gene therapy of cystic fibrosis (CF).

Pharmacol Res, 2002 Nov, 46(5), 409 - 14
The activation of serotonin receptors prevents glutamate-induced neurotoxicity and NMDA-stimulated cGMP accumulation in primary cortical cell cultures; Gandolfi O et al.; In the present study, we performed experiments on primary cell cultures from rat neocortex to assess the effects of the selective serotonergic 5HT(1A), 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) or 5HT(2), (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) agonists on neuronal death induced by 15min exposure to (-)glutamate (300 microM) as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) . The results show that both drugs attenuated (-)glutamate-induced neurotoxicity and this effect was fully antagonized by the selective antagonists of 5HT(1) (NAN-190) or 5HT(2) (ketanserin) receptors.The effects of the selective serotonergic agonists on the production of cyclic GMP (cGMP) accumulation induced by N-methyl-D-aspartate (NMDA) in the same neuronal preparation were also evaluated . Only the 5HT(2) agent, but not 8-OH-DPAT, per se, decreased basal cGMP levels . In contrast, both drugs attenuated the NMDA-induced cGMP accumulation in this cell preparation . The unexpected similar behavior of 5HT(1) and 5HT(2) agonists towards glutamate-induced neurotoxicity and NMDA-induced cGMP accumulation in primary cell cultures is discussed . It is concluded that primary cell cultures from rat cerebral cortex could represent a suitable experimental model to search novel neuroleptics which exert their effects via 5HT receptors.

Anal Biochem, 2002 Sep 15, 308(2), 300 - 6
Quantification of histamine in blood plasma and cell culture supernatants: a validated one-step gas chromatography-mass spectrometry method; Pittertschatscher K et al.; A novel one-step ethylchloroformate (ECF) derivatization of histamine in biological liquid matrices that allows the sensitive quantification by gas chromatography and mass spectroscopic detection (GC-MS) from small volumes of blood plasma or cell culture supernatants within 15 min is described . After addition of ECF/chloroform directly to the crude sample, histamine has been found to be quantitatively derivatized within seconds . Following centrifugation, the organic phase is transferred to a fresh vial, dried by addition of anhydrous sodium sulfate, and subjected to GC-MS analysis . The reliability of the results is verified by use of two different ion pairs for detection . The method is validated according to DIN 38402 . Linearity is given from 0.0054 to 13 microg/ml and the limit of detection is 2 ng/ml (10 pg absolute, at a signal to noise ratio of 3:1) . The limit of quantification, as calculated at a confidence level of 95%, is 15.6 ng/ml . Practical application is exemplified by the determination of the histamine content in blood plasma of birch pollen-sensitized mice and in the culture supernatant of rat basophil leukemia cells after Ca(2+) ionophore-mediated degranulation.

J Ocul Pharmacol Ther, 2002 Oct, 18(5), 455 - 68
Pilocarpine permeability across ocular tissues and cell cultures: influence of formulation parameters; Scholz M et al.; In vitro permeation studies of drugs across biological barriers are promising tools for estimating the quality and quantity of drug transport in vivo . The objective of this work was to compare the permeability of the hydrophilic model drug pilocarpine-HCl (P-HCl) through different ocular tissues and cell cultures: isolated pig cornea (PCr) and sclera (PSc), rabbit conjunctiva (RCo), and rabbit conjunctival (RCoEC) or corneal epithelial cell culture (RCrEC) . Furthermore, the study included investigations about the influence of the excipients benzalkonium chloride (BAC) and ethylene diamine tetra acetic acid disodium salt (EDTA) on the permeability of the small drug . In general, BAC caused a facilitated drug transport, while EDTA hardly influenced the P-HCl concentration on the acceptor side, except for RCoEC . Additionally, the impact of variation in buffer solution pH and tonicity on drug transport in both cell cultures was tested . The higher the tonicity of the buffer solution (80, 300, and 600 mOsm/kg) the lower the permeability coefficient (P(eff)) . At different pH values (6.4, 7.4, and 8.4) the P(eff) showed a directly proportional demeanor . In summary, a good correlation between the isolated tissues and cell cultures with regard to P-HCl transport could be observed.

BMC Med Genet . 2002 Nov 05;3(1):12.
Gene expression patterns vary in clonal cell cultures from Rett syndrome females with eight different MECP2 mutations; Traynor J et al.; BACKGROUND: Females with the neurological disorder Rett syndrome are heterozygous for mutations in X-linked MECP2 that encodes methyl-CpG binding protein 2 (MeCP2) thought to act as a transcriptional repressor . To identify target genes for MeCP2 modulation, we studied global gene expression in single cell-derived wild-type and mutant MECP2 expressing fibroblast clones with four common mutations (R106W, R306C, 705delG, 1155del32) and in lymphoblastoid cell lines (LCLs) that included four mutant MeCP2 (T158M, 803delG, R168X and 1159del28) expressing, and five (1159del28, R106W, R255X, 803delG, 803delG) wild-type MeCP2 expressing lines . METHODS: Clonality and mutation status were verified by androgen receptor methylation assays for X-inactivation and by sequencing MECP2 transcripts . Expression studies were done with oligonucleotide microarrays (Affymetrix U95) and verified with real-time quantitative RT-PCR using Sybr Green . RESULTS: Expression of 49 transcripts was increased, and expression of 21 transcripts was decreased, in at least 3 of 4 mutant/wild-type fibroblast comparisons . Transcript levels of 11 genes, determined by quantitative RT-PCR, were highly correlated with the microarray data . Therefore, multiple additional clones from two Rett individuals were tested by RT-PCR only . Striking expression differences were found in both mutant and wildtype MeCP2 expressing clones . Comparing expression profiles of lymphoblastoid cell lines yielded 16 differentially expressed genes . CONCLUSIONS: MeCP2 deficiency does not lead to global deregulation of gene expression . Either MeCP2's in vivo function does not involve widespread transcriptional repression, or its function is redundant in cell types that also express other methyl-CpG binding proteins . Our data suggest that clonal fibroblast strains may show substantial inter-strain variation, making them a difficult and unstable resource for genome-wide expression profiling studies.

Nutr Cancer, 2002, 42(2), 233 - 40
The cruciferous nitrile crambene has bioactivity similar to sulforaphane when administered to Fischer 344 rats but is far less potent in cell culture; Keck AS et al.; The anticarcinogenic properties of broccoli are believed to be due to modification of detoxification enzymes by a group of isothiocyanates, hydrolysis products of glucosinolates, particularly sulforaphane . We previously showed that the nitrile crambene (1-cyano-2-hydroxy-3-butene), present in most Brassica vegetables, induces hepatic quinone reductase activity when administered to rats . In this study, we compared the effects of seven daily oral doses of crambene (50 mg/kg rat/day) and sulforaphane (50 mg/kg rat/day) on induction of hepatic quinone reductase activity in Fischer 344 rats . The two treatments produced similar effects, with crambene and sulforaphane producing 1.5- and 1.7-fold induction in hepatic quinone reductase activity, respectively . Additionally, we evaluated the effect of crambene on quinone reductase activity in Hepa 1c1c7 cells, because this system had been shown to possess high sensitivity to sulforaphane and is commonly used for screening anticarcinogenic compounds . Crambene (5 mM) induced quinone reductase activity and caused cell cycle arrest in the G2/M phase in mouse Hepa 1c1c7 cells, rat H4IIEC3 cells, and human Hep G2 cells (> 95% viability) . Doses of crambene needed for induction of quinone reductase in cell culture were approximately 100-fold greater than effective doses of sulforaphane . These findings indicate that hepatoma cell lines may not accurately reflect relative potency of anticarcinogens in Fischer 344 rats.

Electrophoresis, 2002 Oct, 23(20), 3623 - 9
Determination of monoclonal antibody production in cell culture using novel microfluidic and traditional assays; Ohashi R et al.; This study compares microfluidic technology (Protein 200 LabChip Assay kit, Agilent 2100 Bioanalyzer, referred to here as Protein 200) to the traditional approach for protein analysis, one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), for the sizing and quantification of immunoglobulin G (IgG) in hybridoma cell cultures . Internal references differ between each method: purified IgG was used alone in SDS-PAGE while myosin (the upper marker) was added to each sample in Protein 200 . The IgG used here were produced in cultures propagated in either a serum-free or a serum-containing medium . With serum-containing samples, there was a significant difference in the IgG concentrations (p < 0.05) between SDS-PAGE and Protein 200 . The concentration determined by SDS-PAGE was significantly higher (> 30%) than by Protein 200 or by high-pressure liquid chromatography (HPLC) because the large amounts of serum albumin in the samples affect the accuracy of SDS-PAGE . Protein 200 can determine size similarly to SDS-PAGE in serum-free samples (standard error of the mean, SEM, < 1%, 95% confidence < +/-1%), unlike in serum-containing samples . The Protein 200 assay was more effective than the traditional one-dimensional SDS-PAGE in determining concentration and size of IgG in cell culture samples and it provided a miniaturized and convenient platform for rapid analysis.

Anal Chem, 2002 Oct 15, 74(20), 5227 - 36
The influence of correlated calibration samples on the prediction performance of multivariate models based on mid-infrared spectra of animal cell cultures; Rhiel MH et al.; The effect of the presence of metabolism-induced concentration correlations in the calibration samples on the prediction performance of partial least-squares regression (PLSR) models and mid-infrared spectra from Chinese hamster ovary cell cultures was investigated . Samples collected from batch cultures contained highly correlated metabolite concentrations as a result of metabolic relations . Calibrations based on such samples could only be used to predict concentrations in new samples if a similar correlation structure was present and failed when the new samples were randomly spiked with the analytes . On the other hand, such models were able to predict glucose correctly even if they were based on a spectral range in which glucose does not absorb, provided that the correlations in the calibration and in the new samples were similar . If however, samples from a calibration culture were randomly spiked with the main analytes, much more robust PLSR models resulted . It was possible to predict analyte concentrations in new samples irrespective of whether the correlation structure was maintained or not . Validity of all established models for any given use could be predicted a priori by computing the space inclusion and observer conditions . Predictions from these computations agreed in all cases with the experimental test of model validity.

Photochem Photobiol, 2002 Sep, 76(3), 301 - 9
UVB irradiation of normal human skin favors the development of type-2 T-cells in vivo and in primary dermal cell cultures; Di Nuzzo S et al.; To determine the effect of UVB exposure on the balance of type-1 or type-2 T-cells in skin, we examined the expression of key markers interferon (IFN)-gamma and interleukin (IL)-4 in cryostat sections . IFN-gamma mRNA was clearly detectable in nonirradiated control skin, and IFN-gamma protein was found in 2% of the dermal CD3pos T-cells, whereas IL-4 mRNA was hardly detectable, and no IL-4 protein was found . In contrast, IL-4 mRNA expression increased upon irradiation, and IL-4 was found in 2% of the T-cells at day 2 after UVB-exposure . Concomitantly, IFN-gamma mRNA expression decreased, and IFN-gamma protein became absent . We also analyzed T-cells present in primary dermal cell cultures, which were used as an in vitro equivalent of the in vivo situation . As compared with T-cells from control skin, T-cells in dermal cell cultures from UVB-exposed skin displayed an increased IL-4 and decreased IFN-gamma expression . No such skewing occurred when the T-cells from irradiated skin were cloned in the absence of a dermal microenvironment . Except for an occasional positive T-cell, type-1-associated cell-surface markers (CCR5, CXCR3) or type-2 markers (CCR3, CD30, CRTH2) were undetectable in situ . But these markers were expressed on cultured dermal T-cells from UVB-exposed and control skin at a comparable level, but did not correlate with the IFN-gamma and IL-4 production . Altogether, UVB-induced changes of the dermal microenvironment favor the development of type-2 T-cells.

Cloning Stem Cells, 2002, 4(3), 223 - 9
Mitochondrial activity in response to serum starvation in bovine (Bos taurus) cell culture; Takeda K et al.; In nuclear transfer procedures, in addition to nuclei, donor cell mitochondria are routinely transferred into recipient oocytes, and mitochondrial heteroplasmy has been reported . However, various protocols have resulted in either homoplasmy for recipient oocyte mitochondria or varying heteroplasmic levels in cloned animals . In nuclear transfer protocols, donor cells are subjected to serum-starvation prior to electroporation . Therefore, the relationship between culture conditions and mitochondrial activity was explored . Fibroblast cell lines were propagated from bovine ear epithelium, skin, skeletal muscle, or cumulus cells . In vitro mitochondrial viability was assessed in proliferative and confluent cells, cultured under serum-starvation or supplemented conditions . Cells were stained with MitoTracker Red CMXRos and comparative fluorescence intensities were assessed . The mitochondrial activity per cell was highest under proliferation, significantly lower at confluency (p < 0.001), and remained depressed after serum starvation for within a week (p < 0.001) . Serum starvation induced an increase in mitochondrial viability in confluent cells . These results demonstrate that mitochondrial viability is dramatically affected by cell culture conditions . Consequently, specific cell culture parameters provide one explanation for the varying incidence of heteroplasmy identified in cloned animals . Future research should reveal whether specific cell culture parameters represent one of the factors for the varying incidence of heteroplasmy identified in cloned animals.

Am J Respir Cell Mol Biol, 2002 Nov, 27(5), 536 - 41
Interleukin-13 induces goblet cell differentiation in primary cell culture from Guinea pig tracheal epithelium; Kondo M et al.; The Th2 cytokines, interleukin (IL)-4 and IL-13, bind to IL-4Ralpha, and cause goblet cell metaplasia/hyperplasia with increased mucin expression in vivo . However, there is not enough evidence that these cytokines directly induce mucin production in vitro . In this study, primary epithelial cells from guinea pig trachea were cultured at an air-liquid interface, and immediately after achieving confluence at Day 7 they were treated with human recombinant IL-4 or IL-13 for 14 d . IL-13-treated cells consisted of a large number of fully mature goblet cells with a smaller number of ciliated cells . Secretory granules of the goblet cells were positive for both periodic acid-Schiff and toluidine blue, and showed exocytosis . By contrast, IL-4 failed to induce goblet cell differentiation . The electric resistances of IL-13-treated cells were lower than those of IL-4-treated cells and nontreated cells, suggesting leaky epithelia . MUC5AC protein level in cell lysates measured by ELISA was several-fold higher in IL-13-treated cells than in nontreated cells, whereas the level in IL-4-treated cells was not changed . These data suggest that human recombinant IL-13, but not IL-4, can induce differentiation into mature goblet cells that produce MUC5AC protein in guinea pig tracheal epithelial cells in vitro.

Bioelectromagnetics, 2002 Dec, 23(8), 592 - 8
Cylindrical waveguide applicator for in vitro exposure of cell culture samples to 1.9-GHz radiofrequency fields; Gajda GB et al.; An applicator for in vitro cell culture exposure was developed based on a circularly polarized, cylindrical waveguide for the 1.9-GHz frequency band used by Personal Communications Services (PCS) in Canada . The applicator consists of two coaxial Petri dishes that sit on the open end of the cylindrical waveguide . The inner 60-mm Petri dish contains the cell culture while the outer 150-mm dish contains coolant water, which is circulated from a pump . A dosimetric evaluation was made using thermometric and E-field probe techniques . The latter allowed the entire inner dish to be scanned to determine the range of specific absorption rates (SARs) pertinent to the expected position of the cells . A representative SAR rate (SAR per unit of input power) of 8.6 +/- 2.1 W/kg/W (95th percentile) was determined 1 mm from the bottom, for a 10 ml sample volume of standard medium . Evaluation of the cooling system demonstrated that following an initial 0.3 degrees C temperature increase, a constant temperature was maintained for 24 h when the waveguide was energized to achieve an average sample SAR of 10 W/kg . These properties enable both acute and sub-acute in vitro bio-effect studies to be performed on a variety of cell culture samples .

Mol Genet Genomics, 2002 Oct, 268(2), 160 - 8 Epub 2002 Sep 17.
Renilla luciferase- Aequorea GFP (Ruc-GFP) fusion protein, a novel dual reporter for real-time imaging of gene expression in cell cultures and in live animals; Wang Y et al.; Light-emitting reporter proteins play an increasing role in the study of gene expression in vitro and in vivo . Here we present a ruc-gfp fusion gene construct generated by fusing a cDNA for Renilla luciferase (ruc) in-frame with a cDNA encoding the "humanized" GFP (gfp) from Aequorea . A plasmid containing the fusion gene construct was successfully transformed into, and expressed in, mammalian cells . The transformed cells exhibited both Renilla luciferase activity in the presence of coelenterazine and GFP fluorescence upon excitation with UV light . Spectrofluorometry of cells containing the Ruc-GFP fusion protein, in the absence of wavelengths capable of exciting GFP fluorescence but in the presence of the luciferase substrate, coelenterazine, showed an emission spectrum with two peaks at 475 nm and 508 nm . These two peaks correspond to the emission maximum of Renilla luciferase at 475 nm and that of GFP at 508 nm . The peak at 508 nm generated in the presence of coelenterazine alone (without UV excitation) is the result of intramolecular energy transfer from Renilla luciferase to Aequorea GFP . Southern analysis of genomic DNA purified from transformed Chinese hamster ovary (CHO) cells and fluorescence in situ hybridization (FISH) to metaphase chromosomes confirmed the integration of the ruc-gfp fusion gene on a single chromosome . The bifunctional Ruc-GFP fusion protein allows the detection of gene expression at the single-cell level based on green fluorescence, and in a group of cells based on luminescence emission . Furthermore, animal experiments revealed that light emission from the Ruc-GFP fusion protein can be detected externally in the organs or tissues of live animals bearing the gene construct.

Neuroreport, 2002 Oct 28, 13(15), 1945 - 50
Caffeine-induced neuronal death in neonatal rat brain and cortical cell cultures; Kang SH et al.; We examined the potential neurotoxicity of caffeine and . Intraperitoneal administration of caffeine (50 mg/kg, 3 times a day) produced neuronal death in various brain areas of neonatal rats 24 h later . Caffeine at doses > 300 microM was also neurotoxic in murine cortical cell cultures . Caffeine-induced neuronal death was accompanied by cell body shrinkage and attenuated by anti-apoptotic drugs including cycloheximide, high potassium, and growth factors . Two necrotic pathways, excitotoxicity and oxidative stress, did not mediate caffeine neurotoxicity . The pro-apoptotic protease caspase-3 was activated to mediate neuronal death following exposure to caffeine . The present findings suggest that caffeine may cause caspase-3-dependent neuronal cell apoptosis in neonatal rat as well as.

J Virol Methods, 2002 Dec, 106(2), 175 - 84
Comparison of cell culture systems for duck hepatitis B virus using SyBr green quantitative PCR; Wang CY et al.; The Hepadnaviridae family contains DNA viruses such as human hepatitis B virus (HBV), woodchuck hepatitis B virus (WHV), and duck hepatitis B virus (DHBV) . DHBV is distributed in both wild and domestic ducks . HBV is a worldwide health problem with carriers at risk of developing cirrhosis and liver cancer . All medical staff and scientists working with HBV must be vaccinated, because of its highly contagious nature . DHBV is a safe surrogate for HBV because of their similarities . Several cell culture systems have been developed to study anti-DHBV drugs and disinfectants . However, differences in their capabilities to support DHBV propagation have not been reported . Therefore, a sensitive and reproducible quantitative PCR based on SyBr green dye was developed . This system does not need electrophoresis for analysis of PCR products, thus reducing processing time and potential for cross-contamination . It allowed precise quantification of DHBV over 8-logarithm dynamic range with a good correlation (R(2) = 0.9689) and showed minimal run-to-run deviation . Sensitivity was 820 copies of DHBV genome and specificity was confirmed by melting curve analysis . It demonstrated good repeatability in quantification of DHBV loads from serum of infected ducks . This assay compared DHBV yields from different cultured cells . All cells had similar kinetic curves for DHBV replication and replication peaks appeared 4 days post-infection . Duck embryonic hepatocytes showed the highest (P > 0.05) replication peak for DHBV . Therefore, duck embryonic hepatocytes and quantitative PCR based on SyBr green dye are a good choice for anti-DHBV drug and disinfectant testing .

Eur J Pharmacol, 2002 Oct 18, 453(1), 27 - 32
Diacetylmorphine degradation to 6-monoacetylmorphine and morphine in cell culture: implications for in vitro studies; Hutchinson MR et al.; Diacetylmorphine deacetylates rapidly to 6-monoacetylmorphine and then to morphine . The immunomodulatory effects of diacetylmorphine are under investigation by several groups utilising various methods including in vitro cell culture; however, diacetylmorphine stability under these conditions is unknown . The aim of this study was to quantify diacetylmorphine degradation under cell culture conditions and to determine the mechanism by which this occurs . Diacetylmorphine degradation in a mouse splenocyte mitogenesis assay was investigated . Morphine and 6-monoacetylmorphine were quantified using HPLC with UV detection . After 6 h, approximately 73% of diacetylmorphine had been hydrolysed in the presence of cells . The half-life of diacetylmorphine was 1.4 h in cell media alone and 1.2-2.2 h in incubations containing cells, while the half-life of 6-monoacetylmorphine was 3.1 h in cell media alone and 0.99-1.2 h in incubations containing cells . 6-Monoacetylmorphine and morphine formation were found to be dependent on incubation time and diacetylmorphine concentration, and were not dependent on esterase activity, mitogen concentration, presence of erythrocytes and cell media evaporation . Only morphine formation was dependent on lymphocyte concentration . 6-Monoacetylmorphine formation was independent of cells and appeared to be due to the conditions of the cell culture (pH and temperature), while morphine formation was dependent to a greater extent on cells, but independent of esterase activity . The study highlights the limitations of conclusions made in previous studies which have not recognised diacetylmorphine instability .

J Appl Microbiol, 2002, 93(5), 745 - 50
Evaluation of integrated cell culture-PCR (C-PCR) for virological analysis of environmental samples; Greening GE et al.; AIMS: The aims of this study were to establish an integrated culture-polymerase chain reaction (C-PCR) method for detection of enteric viruses in environmental samples, and to evaluate it for sensitivity, speed and provision of virus infectivity data . METHODS AND RESULTS: C-PCR, direct reverse transcription (RT)-PCR, PCR and plaque assay methods were used to detect enteroviruses and adenoviruses in seeded and naturally contaminated environmental samples . Using C-PCR, infectious enterovirus presence was confirmed in 3 d and adenovirus presence in 5 d, compared with up to 10 d required by conventional cell culture methods . CONCLUSIONS: C-PCR was the preferred method for detection of enteric viruses in environmental samples containing high viral concentrations . It was less successful for samples with low viral concentrations or containing toxic materials or inhibitors . SIGNIFICANCE AND IMPACT OF THE STUDY: C-PCR provides sensitive, specific results within 2-5 d and is useful as a rapid screen for environmental samples of low toxicity.

Neurol Res, 2002 Oct, 24(7), 725 - 9
MK 801 attenuates c-Fos and c-Jun expression after in vitro ischemia in rat neuronal cell cultures but not in PC 12 cells; Gerlach R et al.; Cellular homeostatic adaptation to cerebral ischemia is complex and contains changes in receptor mediated gene expression and signaling pathways . The proteins of the immediate early genes c-Fos and c-Jun are thought to be involved in coupling neuronal excitation to target gene expression, due to formation of heterodimers and binding to the AP1 promotor region . We used an in vitro model to compare ischemia induced c-Fos and c-Jun expression in rat neuronal cell cultures and nerve growth factor (NGF) differentiated PC 12 cells . Since activation of glutamate receptors is known to mediate ischemic injury we determined the effect of the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK 801 on c-Fos and c-Jun expression in both cell culture systems during ischemia . Neuron rich cultures and NGF differentiated PC 12 cells were exposed to sublethal in vitro ischemia using an hypoxic chamber flushed with argon/CO2 (95 %/5%) . C-Fos and c-Jun mRNA expression was analyzed by competitive reverse transcription-polymerase chain reaction using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal standard . One hour of in vitro ischemia significantly increased c-Fos and c-Jun mRNA levels in both cell culture systems . In neuron rich cultures a 10-fold (c-Fos) and 7-fold (c-Jun) mRNA increase was observed . The mRNA rise was less pronounced in PC 12 cells (5.5-fold and 2-fold) for c-Fos and c-Jun, respectively . The addition of MK 801 significantly reduced the expression of c-Fos and c-Jun mRNA in neuronal cultures, whereas no effect was detectable in PC 12 cells . Since MK 801 failed to reduce the c-Fos and c-Jun expression in NGF differentiated PC 12 cells different signaling pathways may initiate c-Fos and c-Jun expression in both cell culture systems.

Biol Pharm Bull, 2002 Oct, 25(10), 1295 - 7
Nitric oxide donor sodium nitroprusside induces neurotoxicity in cerebellar granular cell culture in rats by an independent mechanism from L-type or dantrolene-sensitive calcium channels; Gepdiremen A et al.; The effects of sodium nitroprusside (SNP) in rat cerebellar granular cell culture were investigated in the present study . All doses of the SNP (10, 25, 50, 100, 250, 500 microM) were able to induce cell death compared with control values (p < 0.001 for all groups tested) . Interestingly enough, a nonlinear dose-response curve was obtained for SNP-induced neurotoxicity . We also investigated the possible neuroprotective effects of nimodipine and dantrolene, alone or in combination . Both drugs failed to prevent neuronal cell death at the doses tested, either alone or in combination . Despite the fact that the most effective dose was a dantrolene concentration of 10 microM with SNP 500 microM and a concentration of 1 microM with SNP 50 microM, the differences were insignificant statistically . According to our results, SNP-induced cerebellar toxicity appears to be an independent reaction from L-type or endoplasmic reticulum calcium currents.

Microsc Res Tech, 2002 Nov 1, 59(3), 249 - 55
Carotid body chemoreceptors in dissociated cell culture; Nurse CA et al.; Carotid body (CB) glomus or type 1 cells act as peripheral chemoreceptors which detect changes in arterial PO(2), PCO(2), and pH and help maintain homeostasis via the reflex control of ventilation . Over the last approximately 12 years significant progress has been made towards understanding chemotransduction mechanisms using freshly isolated or cultured type 1 cells . The latter preparation allows several powerful experimental manipulations (e.g., co-culture with sensory neurons) resulting in significant advances in our understanding of CB chemoreception . Here, we review several properties of type 1 cells after several days to weeks in culture . Typically, cultured type 1 cells grow in monolayer clusters enveloped by glial-like, type II, or sustentacular cells, which are immunopositive for the glial marker, glial fibrillary acid protein (GFAP) . These cells can undergo DNA synthesis, evidenced by uptake of bromodeoxyuridine (BrdU), and show a limited capacity for cell division . Mitosis and survival of type 1 cells can be regulated by oxygen tension and/or growth factors (e.g., bFGF, insulin) . In the rat, type 1 cells are immunopositive for several monoaminergic markers, including tyrosine hydroxylase (TH), dopamine transporter (DAT), and 5-HT . They also express cholinergic markers (e.g., vesicular acetylcholine transporter; VAChT), the highly conserved synaptic vesicle protein (SV2), and gap junctional proteins including Connexin 32 (Cx32) . Moreover, in long-term culture ( approximately 2 weeks) they retain expression of O(2)-sensitive, TASK-1-like, and Ca(2+)-dependent (BK), K(+) channels as revealed by immunocytochemistry or RT-PCR analysis of mRNA extracted from type 1 clusters after removal from the culture surface .

Brain Res, 2002 Oct 25, 953(1-2), 157 - 69
Bradykinin increases permeability by calcium and 5-lipoxygenase in the ECV304/C6 cell culture model of the blood-brain barrier; Easton AS et al.; The blood-brain barrier (BBB) was modelled in this study using ECV304 cells in co-culture with rat C6 glioma cells, which resulted in elevated transendothelial electrical resistance (TEER) . The inflammatory mediator bradykinin (1 microM) was studied and found to induce a fall in TEER; the link between this change and intracellular free calcium concentration ({Ca(2+)}(i)) was then examined . 1 microM bradykinin produced a peak-plateau increase in {Ca(2+)}(i) . The peak showed desensitization and was dose dependent (over 0.1 nM to 1 microM) . The {Ca(2+)}(i) increase was blocked by the B(2) antagonist HOE 140 (1 microM) without effect from a B(1) agonist and antagonist . The plateau response was abolished in Ca(2+)-free solution containing 2 mM EDTA, and also by the Ca(2+) channel blockers lanthanum, La(3+) (10 microM), and SKF 96365 (100 microM) . The store Ca(2+)ATPase inhibitor thapsigargin (1 microM) abolished the peak response . The putative phospholipase C inhibitors, U73122 (20 microM) and ETH-18-OCH(3) (100 microM), unexpectedly increased {Ca(2+)}(i); after their application, bradykinin was ineffective . Agents without effect on Ca(2+) responses to bradykinin included the phospholipase A(2) (PLA(2)) inhibitor aristolochic acid (0.5 mM), cyclooxygenase inhibitor indomethacin (100 microM), 5-lipoxygenase inhibitor nordihydroguaiaretic acid, NDGA (100 microM), calphostin C (0.5 microM), L-NAME (1 mM) and nifedipine (10 microM) . The fall in TEER from bradykinin was blocked by HOE 140, U73122 and thapsigargin combined with La(3+), and also by aristolochic acid and NDGA, but not indomethacin, calphostin C or L-NAME . U73122 increased TEER while ETH-18-OCH(3) reduced it . Thus bradykinin reduced TEER through B(2) receptor-linked release of Ca(2+) from thapsigargin-sensitive stores, leading to activation of PLA(2) and metabolism of arachidonic acid by 5-lipoxygenase.

Vet Microbiol, 2002 Nov 6, 89(4), 291 - 302
Evaluation of different media and a BGM cell culture assay for isolation of Borrelia burgdorferi sensu lato from ticks and dogs; Speck S et al.; A co-culture assay for isolation of Borrelia burgdorferi sensu lato (sl.) from naturally infected ticks and dogs suspected of Lyme borreliosis (LB) was evaluated using buffalo-green-monkey (BGM) cells as the mammalian component . Four different media were tested for their ability to provide sufficient growth conditions for spirochetes and BGM cells . A total of 176 Ixodes ricinus ticks and 268 specimens from 98 dogs were used to compare cell-free culture with the BGM co-culture . A 1:1 mixture of Barbour-Stoenner-Kelly medium (BSK) and Eagle's minimum essential medium (EMEM) supported the growth of the two test strains, B . burgdorferi sensu stricto B31 and B . valaisiana VS116 to the same extent as BSK medium and the growth as well as the viability of BGM cells in this medium were the same as in EMEM . Using the 1:1 mixture of BSK and EMEM, borrelial growth measured in co-culture with BGM cells did not differ significantly from corresponding values obtained in cell-free cultures . In cell-free culture the isolation rate of B . burgdorferi sl . from ticks was significantly higher in BSK/EMEM 1:1 than in BSK medium (P < 0.01) . Co-culture with BGM cells had no significant influence on the isolation rate of borreliae from ticks . However, a significant amount of isolates were obtained by one of the procedures only . Analysing canine specimens accordingly, spirochetes were grown from the blood of one dog after four weeks in BGM cell co-culture . The isolate was classified as B . afzelii by PCR-coupled restriction fragment length polymorphism analysis.

J Chromatogr B Analyt Technol Biomed Life Sci, 2002 Nov 15, 780(1), 137 - 44
Liquid chromatography/electrospray ionisation-mass spectrometry method for the quantification of sphingosine and sphinganine in cell cultures exposed to fumonisins; Seefelder W et al.; Fumonisins, mycotoxins produced by Fusarium verticillioides, are potent inhibitors of the de novo sphingolipid biosynthesis via inhibition of the key enzyme ceramide synthase . The cellular response to a fumonisin exposure is obvious as an alteration of the ratio of the sphingoid bases sphingosine (SO) and sphinganine (SA) . We developed a new column liquid chromatography/electrospray ionisation-mass spectrometry (LC-ESI-MS) method for the rapid, simultaneous and quantitative determination of these bases in cell cultures of immortalised human kidney epithelial cells (IHKE cells) . For sample preparation, cell lysates were only diluted, centrifuged and directly used for LC-MS measurements . Quantification was carried out using phytosphingosine (PSO) as an internal standard . Detecting the protonated molecule {M+H}(+) signals of SO (m/z 300) and SA (m/z 302) in the selected ion monitoring (SIM) mode, detection limits of 10 pg for SO (signal-to-noise ratio S/N=3:1) and 25 pg for SA (S/N=3:1) were established . The average recovery for SO and SA was higher than 90% for control IHKE-cells, respectively . The developed LC-ESI-MS method allows the sensitive, selective and rapid monitoring of sphingosine and sphinganine in cell matrices with a drastically reduced time for sample preparation.

Ann N Y Acad Sci, 2002 Oct, 969, 141 - 6
Amino acid content of cell cultures infected with Cowdria ruminantium propagated in a protein-free medium; Josemans AI et al.; The in vitro culture of Cowdria ruminantium, the causative agent of heartwater in domestic ruminants, was first achieved in 1985 . Culture media were usually supplemented with serum and tryptose phosphate broth, both undefined components, contributing to great variability . Recently, we reported about the propagation of stocks of C . ruminantium in a protein-free culture medium referred to as SFMC-23, which is chemically fully defined . To clarify whether the amino acid composition in SFMC-23 is adequate for the in vitro propagation of Cowdria, the Welgevonden stock was propagated in SFMC-23 medium . After a 3-day culture period, samples were taken from uninfected and infected bovine endothelial cell cultures . They were analyzed for free amino acids by the Pico Taq reversed-phase HPLC precolumn derivatization method . Eighteen different amino acids were examined . A considerable decrease in concentration was observed with proline (29%) and glutamine (62%) . Further dramatic changes were observed with amino acids which accumulated in the culture medium: aspartic acid, serine, asparagine, tryptophane, glycine, and alanine . The concentration of alanine increased by approximately 660% . The concentrations of all other amino acids analyzed remained within a 25% range, either increasing or decreasing . These results suggest that only glutamine may run short during in vitro cultivation . It seems more likely that accumulation of various amino acids may impact negatively on long-term Cowdria propagation.

World J Gastroenterol, 2002 Oct, 8(5), 872 - 8
Mutational characteristics in consecutive passage of rapidly replicating variants of hepatitis A virus strain H2 during cell culture adaptation; Hu NZ et al.; AIM: To investigate the molecular mechanism of cell adaptation and rapid replication of hepatitis A virus strain H2 in KBM17 cells . METHODS: Virus of strain H2 at passage 7 was consecutively passaged in KBM17 cells for 22 passages, every passage was incubated for 14 days . Antigenic and infectious titers of every passage and one-step growth dynamics of passage 22 were determined with ELISA . Genomes of passage 6, passage 12, passage 18 and passage 22 were sequenced and compared with H2K7 . RESULTS: During continuous passage of vaccine strain H2 at passage K7 in KMB17 cells, infectious and antigenic titers increased with the increase of passages, infectious titers at day 14 reached 6.77LgCCID(50)ml(-1) for passage 6 (P6), 7.0 LgCCID(50)ml(-1) for passage 12 (P12), 7.33 LgCCID(50)ml(-1) for passage 18 (P18) and 7.83 LgCCID(50)ml(-1) for passage 22 (P22), respectively . The one-step growth dynamics showed that replicating peak of P22 appeared at day 14 with infectious titers of 7.83 LgCCID(50)ml(-1) and antigenic titer of 1:1024 . After passage 22 a new cell-adapted variant (P22) of H2K7 with rapid and shortened replication cycle from 28 days to 14 days was obtained . Sequencing and comparisons of genomes of P6, P12, P18 and P22 showed that mutational numbers in genomes of different passages increased with adaptive passages, and mutations scattered over the genome . In comparison with that of K7, P6 had only 6 nucleotides (nt) mutations, P12 had 7 mutational changes, in addition to 6 same mutations with P6, there appeared a new mutation in 5'NTR at nucleotide position 591 resulting in a nucleotide exchange from A to G . P18 had 10 nt mutations, among the 10 mutations, 7 mutational changes were same as with P12, three new mutational changes appeared in the genome, one in 5'NTR, one in 3C coding region, one in 3D coding region, at P22 there appeared 18 nucleotide changes in the genome, on the basis of P18,there occurred additional 8 nucleotide mutations, two in 5'NTR, three in 2C, one in 3A, one in 3C and one in 3D . The results suggested that although H2K7 was already an attenuated strain, the mutations of genome is not sufficient to completely adapt the KMB17, further mutations caused rapid replication adaptation . CONCLUSION: 18-nt changes scattering over the genome are cooperatively responsible for further adaptation characterized by rapid and shortened replication cycle from 28 days to 14 days in KMB17 cells . The mutations in 2C coding region play more important role in increase of infectious titer than other mutations, the mutations in 2B coding region show less important role than it usually does in cell adaptation, nucleotide changes in 5' NTR seem to be not relevant to cell adaptation during initial stages (before P6), but do in late stages.

Di Yi Jun Yi Da Xue Xue Bao, 2002 Jul, 22(7), 588 - 91
Effect of conditioned media from decidual cell culture on the expression of genes regulating the invasion of trophoblastic cells; Zhang XQ et al.; OBJECTIVE: To investigate the effect of the conditioned media from decidual cell cultures (DCM) on the expression of genes regulating the invasion of trophoblastic cells . METHODS: In vitro culture of decidual cells obtained from healthy women of both early (within the first trimester) and full-term pregnancy respectively was performed to prepare conditioned media of decidual cells (DCM) . Trophoblastic cells were also obtained in these subjects and treated with DCM for 24 h in in vitro culture, to observe the effect of DCM, with the help of semi-quantitative reverse transcriptase-PCR, on the expression of genes in these cells that regulate their invasion . RESULTS: Expression of matrix metallsoproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinases (TIMP)-1 and urokinase-type plasminogen activator (u-PA), other than that of TIMP-2 and plasminogen activator inhibitor type (PAI)-1, was observed in normal trophoblastic cells in in vitro culture . DCM derived from wemon of early and full-term pregnancy down-regulated the expression of MMP-2, MMP-9, u-PA while up-regulated the expression of TIMP-1, PAI-1 . CONCLUSION: DCM may exhibit its anti-invasive activity by regulating the expression of genes involved in the regulation of trophoblastic cell invasion, such as MMP-2, MMP-9, TIMP-1, u-PA and PAI-1.

Amino Acids, 2002, 23(1-3), 19 - 25
Inhibition of phosphatidylcholine synthesis is associated with excitotoxic cell death in cerebellar granule cell cultures; Gasull T et al.; Glucose deprivation (GD) enhances the sensitivity of cerebellar granule cells to die by excitotoxicity . Neither 70 min of GD, a treatment that depletes cell energy resources, nor exposure to 20 microM glutamate (GLU) for 30 min, induce significant cell death in cultures of cerebellar granule cells . However, the combined treatment with GLU and GD induces choline (Cho) release before excitotoxic cell death . We investigated whether the neurotoxic effect of this treatment is related with inhibition of phosphatidylcholine (PC) synthesis . We found that exposure to GLU for 30 min, to GD for 70 min, and to the combination of both, inhibited PC synthesis at the end of treatment by 71%, 92% and 91%, respectively . The inhibition of PC synthesis was accompanied by a decrease in the incorporation of {(3)H}Cho into phosphocholine and by an increase of the intracellular content of free {(3)H}Cho, indicating that these treatments inhibit the synthesis of PC by inhibiting choline kinase activity . However, only the combined treatment with GLU and GD induced a prolonged inhibition of PC synthesis that extended after the end of treatment . These results show that excitotoxic death is associated with sustained inhibition of PC synthesis and suggest that this effect of the combined treatment with GLU and GD on PC synthesis is produced by an action on an enzymatic step downstream of choline kinase activity.

Environ Toxicol Chem, 2002 Oct, 21(10), 2027 - 33
Application of the luciferase recombinant cell culture bioassay system for the analysis of polycyclic aromatic hydrocarbons; Ziccardi MH et al.; An aryl hydrocarbon (Ah) receptor-based luciferase cell culture bioassay developed to detect 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other halogenated aromatics was modified and optimized to detect and quantitate polycyclic aromatics (PAHs) . Twenty-four PAHs were analyzed, and subsequent EC50 and EC20 concentrations (based on the median and 20% TCDD maximal response, respectively) and appropriate induction equivalency factors (calculated by comparison to the response obtained with TCDD) were determined from dose-response experiments . Six compounds were shown to be active in the system, with benzo{k}fluoranthene > benz{a,h}anthracene indeno{1,2,3-cd}pyrene > benzo{a}pyrene > benzo{b}fluoranthene > chrysene . A complex mixture of 16 PAHs was also analyzed using this system, and overall induction equivalency (or 1-EQ) of the mixture was shown to be very similar to that predicted from the sum of the activity estimated for each individual PAH . Overall, our results strongly support the use of this system for the detection and relative quantitation of Ah receptor-active PAHs.

Curr Drug Metab, 2002 Oct, 3(5), 551 - 7
Permeability characteristics of endocrine-disrupting chemicals using an in vitro cell culture model, Caco-2 cells; Yoshikawa Y et al.; The purpose of this study was to evaluate the permeability characteristics of endocrine disrupting chemicals utilizing epithelial monolayers of Caco-2 cells . The drugs tested in this study were bisphenol A (BPA), tert-octylphenol (tOP), tert-butylphenol (tBP), di(2-ethylhexyl)phthalate (DOP), dibutylphthalate (DBP), and butylbenzylphthalate (BBP), all of which are used in plastic materials . The Caco-2 cell line was grown on cell culture inserts with polyethylene terephthalate membranes, and Hank's balanced salt solution (HBSS, pH 7.4) was used for the transport experiments . The barrier properties were assessed by measuring transepithelial electrical resistance (TEER) using a volt ohmmeter, and transport of these endocrine disrupting chemicals was examined in both directions . The permeated amounts of these chemicals within 180 min in the apical to basolateral (A-to-B) and the basolateral to apical (B-to-A) directions without verapamil, a P-glycoprotein (P-gp) inhibitor, were in the rank order of tBP > tOP > BPA > DOP > DBP > BBP and BPA >> tBP > tOP > DOP > DBP > BBP, respectively . In the presence of 100 microM verapamil, the permeated amounts of BPA, tOP and tBP within 180 min in the B-to-A direction decreased by 12-, 2.6- and 3.1-fold, respectively . In the case of phthalate esters, the permeated amount of DOP within 180 min in the B-to-A direction decreased by 1.6-fold, while that of DBP and BBP showed no significant changes . The ratios of apparent permeability coefficient of B-to-A against A-to-B, P(app) ratios, for BPA, tOP and tBP were markedly decreased in the presence of 100 microM verapamil . These findings indicated that both BPA and alkyl phenols are substrates of the P-gp located in the apical side of Caco-2 cells, and suggested that the P-gp in the small intestine may act as an organic barrier against BPA and alkyl phenols.

J Virol Methods, 2002 Oct, 106(1), 39 - 50
Development of viral disinfectant assays for duck hepatitis B virus using cell culture/PCR; Wang CY et al.; Human hepatitis B virus (HBV) is a worldwide public health problem with chronic carriers at risk for developing cirrhosis and hepatocellular carcinoma . Accidental nosocomial infections from inadequately disinfected equipment or exposure to blood and body fluids from patients are major routes . To solve such problems, disinfectants to inactivate HBV must be validated . Duck hepatitis B virus (DHBV) is accepted as a surrogate for HBV, due to their similar sensitivities to disinfectants and its safety . Ducklings are used for disinfectant efficacy assays; however, the same virus titer is obtained using duck embryonic hepatocytes . Viral titration in disinfectant efficacy assay is conducted using Southern hybridization of infected duck serum . However, this test requires radioisotopes . Therefore, disinfectant assessment protocols were developed using duck embryonic hepatocytes with polymerase chain reaction (PCR) or nested PCR . The ease of handling, lowered cost and enhanced sensitivity make PCR desirable . Chicken embryonic hepatocytes were applied to DHBV disinfectant efficacy assay . Results were consistent and could be used under certain conditions . The virucidal activities of two quaternary ammonium chloride disinfectants, n-alkyl dimethyl benzyl ammonium chloride and alkyl dimethyl benzyl ammonium chloride (10C-12C) were compared and effective concentrations were 1200 and 1800 ppm, respectively . Efficacies of these disinfectants were validated using real-time quantitative PCR . Results confirmed that the efficacy of n-alkyl dimethyl benzyl ammonium chloride was higher than alkyl dimethyl benzyl ammonium chloride (10C-12C) . This assay was useful for rapid discrimination of killing potentials of disinfectants . In conclusion, these assays can be applied to other viruses that are unable to cause CPE in cell cultures and broadened the utility of DHBV as animal model for HBV.

Histochem J, 2002 Jan-Feb, 34(1-2), 27 - 33
Cyclosporine A-induced toxicity in two renal cell culture models (LLC-PK1 and MDCK); Rezzani R et al.; Renal damage caused by therapeutic treatment with cyclosporine A has been well documented . Clinical experiences have shown that cyclosporine A nephrotoxicity is determined by interstitial fibrosis with tubular atrophy . However, the exact mechanism by which this drug causes nephrotoxicity has not yet been clarified . This study used an in vitro model in an attempt to identify the cellular mechanisms underlying kidney cyclosporine A damage . We used two cell lines with the characteristics of proximal and distal tubule cells (pig kidney proximal tubular epithelial cell line {LLC-PK1} and Madin-Darby canine kidney cell line {MDCK} . The cell lines were treated with cyclosporine A for 24 h . After the treatment, the cells were stained with Trypan Blue to estimate cell viability and processed by histochemical reactions to evaluate their cellular metabolism . Four enzymes (acid phosphatase, alkaline phosphatase, lactate dehydrogenase and succinate dehydrogenase) were considered . The cell viability assay showed that the LLC-PK1 cell line was more sensitive to cyclosporine A than MDCK . Remarkably, the LLC-PK1 cells disappeared with cyclosporine A treatment . As for the hydrolytic enzymes, only acid phosphatases showed an increased positivity in the treated LLC-PK1 cells . Similarly, lactate dehydrogenase showed a different activity histochemically . No statistically significant alterations were observed in the succinate dehydrogenase reaction . The cyclosporine A-treated MDCK cell lines did not show any difference in either their hydrolytic or succinate dehydrogenase enzyme positivity with respect to the control line . In contrast, there was a significant increase in lactate dehydrogenase activity . This study allowed the possible mechanism of cyclosporine A-induced damage in renal tubular cells to be evaluated . The enzymatic changes happened rapidly (during the 24 h of treatment), suggesting that this alteration was one of the steps by which cyclosporine A induced toxicity . Moreover, since acid phosphatase is a marker of protein catabolism, the variation in the activity of this enzyme, in the LLC-PK1 line only, showed that cyclosporine can induce alterations leading to cellular toxicity . The modifications in lactate dehydrogenase activity, in both lines, suggested that this drug caused cell stress, inducing the production of lactic acid from glucose in the presence of oxygen . In conclusion, cyclosporine A treatment may force LLC-PK1 and MDCK cells to use anaerobic glycolysis preferentially . Further, these enzyme alterations may represent an epiphenomenon or a consequence of cyclosporine A toxicity.

J Med Chem, 2002 Oct 10, 45(21), 4748 - 54
The effect of exchanging various substituents at the 2-position of 2-methoxyestradiol on cytotoxicity in human cancer cell cultures and inhibition of tubulin polymerization; Cushman M et al.; A new set of estradiol derivatives bearing various substituents at the 2-position were synthesized in order to further elucidate the structural parameters associated with the antitubulin activity and cytotoxicity of 2-substituted estradiols . The potencies of the new compounds as inhibitors of tubulin polymerization were determined, and the cytotoxicities of the analogues in human cancer cell cultures were investigated . The substituents introduced into the 2-position of estradiol included E-3'-hydroxy-1'-propenyl, 2'-hydroxyethoxy, 3-N,N-dimethylaminoethylideneamino, 2'-hydroxyethylineneamino, (beta-3,4,5-trimethoxyphenyl)ethenyl, phenylethynyl, ethynly, 1'-propynyl, and cyano . The substituents conferring the ability to inhibit tubulin polymerization included E-3'-hydroxy-1'-propenyl, 2'-hydroxyethoxy, ethynyl, and 1'-propynyl . The remaining compounds were all inactive as inhibitors of tubulin polymerization when tested at concentrations of up to 40 microM . All of the compounds were cytotoxic in a panel of 55 human cancer cell cultures, and in general, the most cytotoxic compounds were also the most potent as inhibitors of tubulin polymerization . 2-(1'-Propynyl)estradiol displayed significant anticancer activity in the in vivo hollow fiber animal model.

Berl Munch Tierarztl Wochenschr, 2002 Sep-Oct, 115(9-10), 381 - 4
{Definitive ability of Stamp-staining, antigen-ELISA, PCR and cell culture for the detection of Coxiella burnetii}; Henning K et al.; Many assays are used for the detection of the aetiological agent of Q fever, Coxiella burnetii, i.e . staining according to the method of Stamp, capture ELISA, PCR or isolation by cell culture . In this study the results of these four assays are compared for their sensitivity and specificity . Staining smears according to the method of Stamp gave many false positive or false negative results . The capture ELISA seems to be a very sensitive assay for the detection of Coxiella burnetii but it has a lack in specificity . It is a useful test system for the screening of large scales of samples . Positive ELISA results should be confirmed by PCR, a very sensitive and specific method for the detection of Coxiella burnetii but more time consumptive than the ELISA . Isolation of the agent using cell cultures was not completely satisfactory because of its lack in sensitivity . Therefore it should only be used in special cases.

Planta, 2002 Oct, 215(6), 1031 - 9 Epub 2002 Jul 25.
Biosynthesis of podophyllotoxin in Linum album cell cultures; Seidel V et al.; Cell cultures of Linum album Kotschy ex Boiss . (Linaceae) showing high accumulation of the lignan podophyllotoxin (PTOX) were established . Enzymological studies revealed highest activities of phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, 4-hydroxycinnamate:CoA ligase and cinnamoyl-CoA:NADP oxidoreductase immediately prior to PTOX accumulation . To investigate PTOX biosynthesis, feeding experiments were performed with {2-(13)C}3',4'-dimethoxycinnamic acid, {2-(13)C}3',4'-methylenedioxycinnamic acid (MDCA), {2-(13)C}3',4',5'-trimethoxycinnamic acid, {2-(13)C}sinapic acid, {2-(13)C}- and {2,3-(13)C(2)}ferulic acid . Analysis of the metabolites by HPLC coupled to tandem mass spectrometry revealed incorporation of label from ferulic acid into PTOX and deoxypodophyllotoxin (DOP) . In addition, MDCA was also unambiguously incorporated intact into PTOX . These observations suggest that in L . album both ferulic acid and methylenedioxy-substituted cinnamic acid can be incorporated into lignans . Furthermore, it appears that, in this species, the hydroxylation of DOP is a rate-limiting point in the pathway leading to PTOX . Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/wo.1007/s00425-002-0834-1.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2002 Sep, 94(3), 361 - 5
Cytotoxicity of resin-based restorative materials on human pulp cell cultures; Huang FM et al.; OBJECTIVE: The objective of this study was to determine the cytocompatibility of 5 different extracts of resin-based restorative materials (2 resin-modified glass ionomer cements, 1 compomer, and 2 composite resins) on human pulp cells . STUDY DESIGN: Set specimens from 2 resin-modified glass-ionomer cements (Fuji II LC and Fuji IX), 1 compomer (Dyract), and 2 composite resins (Tetric and Superfil) were eluted with culture medium for 2 and 5 days . The effects of resin-based restorative materials on human pulp cells were evaluated with cytotoxicity and mitochondrial activity assays . RESULTS: The results showed that the eluates from resin-modified glass-ionomer, compomer, and composite resins were cytotoxic to primary human pulp cells . In addition, Superfil, Fuji IX, and Tetric demonstrated an inhibitory effect on mitochondrial activity of human pulp cells . It was found that composite resin Superfil was the most toxic restorative material among the chemicals tested . CONCLUSION: The influence of the cytotoxicity depended on the materials tested . Compomer or light-curing resin-modified glass ionomer may initially react more favorably to pulp cells.

Biomaterials, 2002 Dec, 23(23), 4615 - 9
Effect of mammalian cell culture medium on the gelation properties of Pluronic F127; Matthew JE et al.; We investigate the gelation of a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) copolymer, Pluronic F127, in mammalian cell culture medium for applications in tissue engineering and cell encapsulation . In both minimum essential medium (MEM) and MEM with added fetal bovine serum (MEM-FBS), the gel-phase boundary shifts to lower temperatures and concentrations as compared to pure water . The thermodynamics of gel formation are similar in MEM, MEM-FBS, and pure water, suggesting that the mechanism of gelation is similar in all three solvents . The shift of the sol-gel boundary to lower concentrations is particularly significant for development of cell encapsulation protocols using Pluronics and applications where copolymer concentration must be minimized due to toxicity concerns.

Biomaterials, 2002 Dec, 23(23), 4549 - 55
Easy assessment of the biocompatibility of Ni-Ti alloys by in vitro cell culture experiments on a functionally graded Ni-NiTi-Ti material; Bogdanski D et al.; The biocompatibility of nickel-titanium alloys was investigated by single-culture experiments on functionally graded samples with a stepwise change in composition from pure nickel to pure titanium, including an Ni-Ti shape memory alloy for a 50:50 mixture . This approach permitted a considerable decrease of experimental resources by simultaneously studying a full variation of composition . The results indicate a good biocompatibility for a nickel content up to about 50% . The cells used in the biocompatibility studies comprised osteoblast-like osteosarcoma cells (SAOS-2, MG-63), primary human osteoblasts (HOB), and murine fibroblasts (3T3).

Toxicology, 2002 Oct 15, 179(3), 233 - 45
Cadmium induces direct morphological changes in mesangial cell culture; L'Azou B et al.; The cadmium produced by industrial and agricultural practice represents a major environmental pollutant which may induce severe damage, especially in the kidney where cadmium accumulates . While cadmium is known to severely impair renal tubular functions, glomerular structures are also potential targets . The present study investigated the effects of cadmium on glomerular mesangial cell cultures after short- and long-term exposures, requiring for each endpoint specific culture conditions . After 30 min exposure to 1 microM CdCl(2), used as non-lethal concentration, 0.14 ng/microg proteins of cadmium was internalized by the cells as evaluated by atomic emision spectrometry and induced a significant, cell surface reduction (8.9+/-1.9%) . These morphological changes could be correlated to smooth muscle alpha-actin disorganization, without quantitative change in its protein expression level as evaluated by Western-blot and Northern-blot analysis (SMAmRNA/28sRNA, 1.78 CdCl(2) vs . 1.42 control) . For longer exposure times, in complex medium, cadmium uptake was efficient (0.36 ng/microg proteins) and induced changes in the actin cytoskeleton with no loss of cell membrane integrity . This study suggests that cultured mesangial cells provide an alternative model to study the effect of cadmium, and underlines the importance of using well-defined conditions to study further intracellular mechanisms.

Photochem Photobiol, 1997 Dec, 66(6), 837 - 41
Nonlinear dynamics of intracellular methylene blue during light activation of cell cultures; Ruck A et al.; Methylene blue (MB+) is a well-known dye in medicine and has been discussed as an easily applicable drug for topical treatment in photodynamic therapy (PDT) . Methylene blue can potentially be used as a redox indicator to detect the important redox reactions that are induced during PDT . The kinetics of this process was analyzed on a subcellular level with confocal laser scanning microscopy . BKEz-7 endothelial cells were incubated 4 h with 1 microM MB+ . The fluorescence dynamics of MB+ during irradiation with 633 nm light was observed with subcellular resolution . Images were acquired at 0.5 s intervals (frame rate 1 image/0.5 s) . Fluorescence was observed in the red channel of the laser scanning microscope . Synchronously, the phase-contrast image was visualized with the green channel . Morphological changes could therefore be correlated with the dynamics of MB+ . In addition, the light-dose-dependent phototoxicity at 633 nm irradiation was determined by viable cell counting . After an induction period (phase I), fast fluorescent spikes could be observed in the whole cytoplasm, which decayed with a time constant of about 20 s (phase II), followed by a period of nearly constant fluorescence intensity (phase III) and exponential photobleaching (phase IV) . Phase II exhibits highly nonlinear kinetics, which is hypothesized to correlate probably with a nonlinear quantal production of reactive oxygen species (ROS) . Morphological cell changes were not observed during phase II . During phase III, a pycnotic cell nucleus developed . From the determination of viable cells we can conclude that a light dose applied within phase II was only sublethal in correlation with morphological observations . Overproduction of ROS leading finally to cell killing during phases III and IV is discussed.

Biosens Bioelectron, 2002 Oct, 17(10), 883 - 91
Analysis of glycoproteins in cell culture supernatants using a lectin immunosensor technique; Liljeblad M et al.; A method based on a surface plasmon resonance technique for detection of changes in concentration and glycosylation of proteins in cell culture supernatant is described . The method was used to analyze alpha(1)-acid glycoprotein (AGP) produced by a human hepatoma cell line (HepG2) . Cell culture supernatant was injected to a BIACORE 2000 instrument and AGP was captured on the sensor chip by immobilized antibodies . The captured glycoprotein was then analyzed for content of carbohydrate epitopes using three different lectins, Aleuria aurantia lectin (AAL), Sambucus nigra agglutinin (SNA), and Triticum vulgaris agglutinin (wheat germ agglutinin, WGA) . The method was used to analyze changes in concentration and glycosylation of AGP produced by HepG2 cells grown with or without three different cytokines, interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and transforming growth factor beta-1 (TGF beta(1)) . Using the described method it was shown that when HepG2 cells were grown in the presence of IL-6 both AGP concentration and fucosylation increased . When HepG2 cells instead were grown in the presence of TGF beta(1) AGP fucosylation increased whereas AGP concentration decreased.

Vet Microbiol, 2002 Oct 22, 89(2-3), 239 - 251
Vaccination of cattle with Anaplasma marginale derived from tick cell culture and bovine erythrocytes followed by challenge-exposure with infected ticks; de la Fuente J et al.; Anaplasmosis, a hemolytic disease of cattle caused by the tick-borne pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) has been controlled using killed vaccines made with antigen harvested from infected bovine erythrocytes . We recently developed a cell culture system for propagation of A . marginale in a continuous tick cell line . In this study, we performed a cattle trial to compare the bovine response to vaccination with A . marginale harvested from tick cell culture or bovine erythrocytes . All immunized and control cattle were then challenge-exposed by allowing male Dermacentor variabilis infected with A . marginale to feed and transmit the pathogen . Nine yearling cattle (three per group) were used for this study and were immunized with cell culture-derived A . marginale, erythrocyte-derived A . marginale or received adjuvant only to serve as controls . Each vaccine dose contained approximately 2 x 10(10) A . marginale and three immunizations were administered at weeks 1, 4 and 6 . At week 8, cattle were challenge-exposed by allowing 60 D . variabilis male that were infected with A . marginale as adults to feed on the cattle . Antibody responses of cattle against major surface proteins (MSP) 1a, 1b and 5, as determined by ELISAs, peaked 2 weeks after the last immunization . Cattle immunized with infected IDE8 cell-derived antigens had a preferential recognition for MSP1b while cattle immunized with erythrocyte-derived antigens had a preferential recognition for MSP1a . Protection efficacy was evaluated using the percent infected erythrocytes (PPE), the packed cell volume (PCV), and the prepatent period . A . marginale-immunized cattle showed lower PPE and higher PCV values when compared to control animals and did not display clinical anaplasmosis . The cell culture-derived A . marginale shows promise for use as antigen in development of a new killed vaccine for anaplasmosis.

Cell Tissue Res, 2002 Oct, 310(1), 19 - 29 Epub 2002 Aug 24.
Expression and adhesive properties of basement membrane proteins in cerebral capillary endothelial cell cultures; Tilling T et al.; Previous studies have indicated the importance of basement membrane components both for cellular differentiation in general and for the barrier properties of cerebral microvascular endothelial cells in particular . Therefore, we have examined the expression of basement membrane proteins in primary capillary endothelial cell cultures from adult porcine brain . By indirect immunofluorescence, we could detect type IV collagen, fibronectin, and laminin both in vivo (basal lamina of cerebral capillaries) and in vitro (primary culture of cerebral capillary endothelial cells) . In culture, these proteins were secreted at the subcellular matrix . Moreover, the interaction between basement membrane constituents and cerebral capillary endothelial cells was studied in adhesion assays . Type IV collagen, fibronectin, and laminin proved to be good adhesive substrata for these cells . Although the number of adherent cells did not differ significantly between the individual proteins, spreading on fibronectin was more pronounced than on type IV collagen or laminin . Our results suggest that type IV collagen, fibronectin, and laminin are not only major components of the cerebral microvascular basal lamina, but also assemble into a protein network, which resembles basement membrane, in cerebral capillary endothelial cell cultures.

J Neurosci Res, 2002 Oct 1, 70(1), 57 - 64
Low-density lipoprotein receptor-related protein mediates in PC12 cell cultures the inhibition of nerve growth factor-promoted neurite outgrowth by pregnancy zone protein and alpha2-macroglobulin; Chiabrando GA et al.; Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein closely related to human alpha(2)-macroglobulin (alpha(2)M) . It has been demonstrated that monoamine-activated forms of human and rat alpha(2)M and rat alpha(1)M can bind to TrkA and, respectively, inhibit and stimulate NGF-promoted neurite outgrowth, Trk phosphorylation, and intracellular signal transduction in PC12 cells . However, the effect of PZP on neurons is unknown, and the molecular mechanism of neuroinhibition by monoamine-activated alpha(2)M is still unclear . In this report, we show that methylamine-activated PZP (MA-PZP), like MA-alpha(2)M, inhibits in a dose-dependent way the NGF-promoted neurite extension and TrkA phosphorylation in PC12 cells . On the other hand, normal PZP (N-PZP) had little or no effect . In addition, the inhibitory effect of activated alpha-macroglobulins (alphaMs) was reversible upon its removal from the cell culture . In addition, PZP, as well as alpha(2)M, is neuroinhibitory without being directly cytotoxic . It is known that the activated alphaMs bind to the multiligand receptor termed low-density lipoprotein receptor-related protein (LRP) and that the receptor-associated protein (RAP) specifically blocks uptake of all known LRP ligands . To investigate the potential role of LRP in neuromodulation by activated PZP/alpha(2)M, the effect of RAP on the neuroinhibitory activities of these alphaMs was also studied . Data presented here show that RAP blocked the neurite- and Trk-inhibitory activities of both MA-PZP and MA-alpha(2)M, whereas RAP itself had no neuromodulatory effect . Hence, we conclude that these data suggest that the LRP receptor and its alphaM ligands may play a role in regulating Trk receptors .

Eur J Cell Biol, 2002 Aug, 81(8), 419 - 35
Tight junctions and compositionally related junctional structures in mammalian stratified epithelia and cell cultures derived therefrom; Langbein L et al.; The occurrence of extended tight junction (TJ) structures, including zonulae occludentes (ZO), and the spatial arrangement of TJ proteins in stratified mammalian epithelia has long been controversially discussed . Therefore, we have systematically examined the localization of TJ proteins in diverse stratified epithelial tissues (e.g., epidermis, heel pad, snout, gingiva, tongue, esophagus, exocervix, vagina, urothelium, cornea) of various species (human, bovine, rodents) as well as in human cell culture lines derived from stratified epithelia, by electron microscopy as well as by immunocytochemistry at both the light and the electron microscopic level, using antibodies to TJ proteins such as occludin, claudins 1 and 4, protein ZO-1, cingulin and symplekin . We have found an unexpected diversity of TJ-related structures of which only those showing colocalization with the most restricted transmembrane TJ marker protein, occludin, are presented here . While in epidermis and urothelium occludin is restricted to the uppermost living cell layer, TJ-related junctions are abundant in the upper third or even in the majority of the suprabasal cell layers in other stratified epithelia . Interfollicular epidermis contains, in the stratum granulosum, extended, probably continuous ZO-like structures which can also be traced at least through the Henle cell layer of hair follicles . Similar apical ZO-like structures have been seen in the upper living cell layers of all other stratified epithelia and cell cultures examined, but in most of them we have noticed, in addition, junctional regions showing relatively broad, ribbon-like membrane contacts which in cross-section often appear pentalaminar, with an electron-dense middle lamella ("lamellated TJs", coniunctiones laminosae) . In suprabasal layers of several stratified epithelia we have further observed TJ protein-containing junctions of variable sizes which are characterized by a 10-30-nm dense lamina interposed between the two membranes ("sandwich junctions"; iuncturae structae) . Moreover, we have often observed variously sized regions in which the intermembrane distance is rather regularly bridged by short rod-like elements ("cross-bridged cell walls"; parietes transtillati), often in close vicinity of TJ-related structures or desmosomes . The significance of these structures and their possible biological importance are discussed.

Plant Physiol, 1994 Sep, 106(1), 271 - 279
Biosynthesis of p-Hydroxybenzoate from p-Coumarate and p-Coumaroyl-Coenzyme A in Cell-Free Extracts of Lithospermum erythrorhizon Cell Cultures; Loscher R et al.; The enzymatic formation of p-hydroxybenzoate from p-coumarate in cell-free extracts of cell cultures of Lithospermum erythrorhizon Sieb . et Zucc . was investigated . p-Coumaroyl-coenzyme A (p-coumaroyl-CoA) is the activated intermediate in this biosynthetic reaction . It is formed by an ATP-, Mg2+ -, and CoA-dependent 4-hydroxycinnamate:CoA ligase reaction . p-Coumaroyl-CoA is oxidized and cleaved to p-hydroxybenzoyl-CoA and acetyl-CoA in a thioclastic reaction in which NAD is an essential cofactor . These CoA esters are rapidly hydrolyzed to acetate and p-hydroxybenzoate, probably by thioesterases . The enzymes involved in the formation of p-hydroxybenzoate are soluble . p-Hydroxybenzalde-hyde is not an intermediate in this conversion, and S-denosylmethionine and uridine-5{prime}-diphosphoglucose do not enhance formation of p-hydroxybenzoate in our system.

Plant Physiol, 1993 May, 102(1), 205 - 211
Purification and Characterization of Geranyl Diphosphate Synthase from Vitis vinifera L . cv Muscat de Frontignan Cell Cultures; Clastre M et al.; A geranyl diphosphate synthase (EC 2.5.1.1), which catalyzes the formation of geranyl diphosphate from dimethylallyl diphosphate and isopentenyl diphosphate, was isolated from Vitis vinifera L . cv Muscat de Frontignan cell cultures . Purification of the enzyme was achieved successively by ammonium sulfate precipitation and chromatography on DEAE-Sephacel, hydroxylapatite, Mono Q, Phenyl Superose, Superose 12, and preparative nondenaturing polyacrylamide gels . The enzyme formed only geranyl diphosphate as a product . In all cases, neither neryl diphosphate, the cis isomer, nor farnesyl diphosphate was detected . The enzyme showed a native molecular mass of 68 {plus or minus} 5 kD as determined by gel permeation . On sodium dodecyl sulfate polyacrylamide gels, geranyl diphosphate synthase purified to electrophoretic homogeneity migrated with a molecular mass of 66 {plus or minus} 2 kD . Michaelis constants for isopentenyl diphosphate and dimethylallyl diphosphate were 8.5 and 56.8 {mu}M, respectively . The enzyme required Mn2+ and Mg2+ as cofactors and its activity was enhanced by Triton X-100 . Inorganic pyrophosphate, aminophenylethyl diphosphate, and geranyl diphosphate had inhibitory effects on the enzyme.

Biotechnol Bioeng, 2002 Nov 5, 80(3), 257 - 67
Impact of cell culture process changes on endogenous retrovirus expression; Brorson K et al.; Cell culture process changes (e.g., changes in scale, medium formulation, operational conditions) and cell line changes are common during the development life cycle of a therapeutic protein . To ensure that the impact of such process changes on product quality and safety is minimal, it is standard practice to compare critical product quality and safety attributes before and after the changes . One potential concern introduced by cell culture process improvements is the possibility of increased endogenous retrovirus expression to a level above the clearance capability of the subsequent purification process . To address this, retrovirus expression was measured in scaled down and full production scaled Chinese hamster ovary (CHO) cell cultures of four monoclonal antibodies and one recombinant protein before and after process changes . Two highly sensitive, quantitative (Q)-PCR-based assays were used to measure endogenous retroviruses . It is shown that cell culture process changes that primarily alter media components, nutrient feed volume, seed density, cell bank source (i.e., master cell bank vs . working cell bank), and vial size, or culture scale, singly or in combination, do not impact the rate of retrovirus expression to an extent greater than the variability of the Q-PCR assays (0.2-0.5 log(10)) . Cell culture changes that significantly alter the metabolic state of the cells and/or rates of protein expression (e.g., pH and temperature shifts, NaButyrate addition) measurably impact the rate of retrovirus synthesis (up to 2 log(10)) . The greatest degree of variation in endogenous retrovirus expression was observed between individual cell lines (up to 3 log(10)) . These data support the practice of measuring endogenous retrovirus output for each new cell line introduced into manufacturing or after process changes that significantly increase product-specific productivity or alter the metabolic state, but suggest that reassessment of retrovirus expression after other process changes may be unnecessary .

Plant Physiol, 1996 Oct, 112(2), 659 - 667
Subcellular Location of O-Acetylserine Sulfhydrylase Isoenzymes in Cell Cultures and Plant Tissues of Datura innoxia Mill; Kuske CR et al.; O-Acetylserine sulfhydrylase (OASS; EC 4.2.99.8) catalyzes the formation of L-cysteine from O-acetylserine and inorganic sulfide . Three OASS isoenzymes that differ in molecular mass and subunit structure are present in shoot and root tissues and in cadmium-resistant and cadmium-susceptible cell cultures of Datura innoxia Mill . Different OASS forms predominate in leaves, roots, and suspension-cell cultures . To determine the subcellular location of the OASS isoenzymes, purified mitochondria, chloroplasts, and cytosolic fractions from protoplasts were obtained . The isoenzymes are compartmentalized in D . innoxia cells, with a different isoenzyme predominant in the chloroplast, cytosol, and mitochondria, suggesting that they serve different functions in the plant cell . The chloroplast form is most abundant in green leaves and leaf protoplasts . The cytosolic form is most abundant in roots and cell cultures . A mitochondrial form is abundant in cell cultures, but is a minor form in leaves or roots . Cadmium-tolerant cell cultures contain 1.8 times as much constitutive OASS activity as the wild-type cell line, and 2.9 times more than the cadmium-hypersensitive cell line . This may facilitate rapid production of glutathione and metal-binding phytochelatins when these cultures are exposed to cadmium.

Plant Physiol, 1996 Jun, 111(2), 605 - 612
Cell-Wall Changes and Cell Tension in Response to Cold Acclimation and Exogenous Abscisic Acid in Leaves and Cell Cultures; Rajashekar CB et al.; Freeze-induced cell tensions were determined by cell water relations in leaves of broadleaf evergreen species and cell cultures of grapes (Vitis spp.) and apple (Malus domestica) . Cell tensions increased in response to cold acclimation in leaves of broadleaf evergreen species during extracellular freezing, indicating a higher resistance to cell volume changes during freezing in cold-hardened leaves than in unhardened leaves . Unhardened leaves, typically, did not develop tension greater than 3.67 MPa, whereas cold-hardened leaves attained tensions up to 12 MPa . With further freezing there was a rapid decline and a loss of tension in unhardened leaves of all the broadleaf evergreen species studied . Also, similar results were observed in cold-hardened leaves of all of the species except in those of inkberry (Ilex glabra) and Euonymus fortunei, in which negative pressures persisted below -40{deg}C . Abscisic acid treatment of inkberry and Euonymus kiautschovica resulted in increases in freeze-induced tensions in leaves, suggesting that both cold acclimation and abscisic acid have similar effects on freezing behavior{mdash} specifically on the ability of cell walls to undergo deformation . Decreases in peak tensions were generally associated with lethal freezing injury and may suggest cavitation of cellular water . However, in suspension-cultured cells of grapes and apple, no cell tension was observed during freezing . Cold acclimation of these cells resulted in an increase in the cell-wall strength and a decrease in the limiting cell-wall pore size from 35 to 22 A in grape cells and from 29 to 22 A in apple cells.

Brain Res Mol Brain Res, 2002 Aug 15, 104(2), 227 - 39
Genes associated with pro-apoptotic and protective mechanisms are affected differently on exposure of neuronal cell cultures to arsenite . No indication for endoplasmic reticulum stress despite activation of grp78 and gadd153 expression; Mengesdorf T et al.; The effect of arsenite exposure on cell viability, protein synthesis, energy metabolism and the expression of genes coding for cytoplasmic (hsp70) and endoplasmic reticulum (ER; gadd153, grp78, grp94) stress proteins was investigated in primary neuronal cell cultures . Furthermore, signs of ER stress were evaluated by investigating xbp1 mRNA processing . Arsenite levels of 30 and 100 microM induced severe cell injury . Protein synthesis was reduced to below 20% of control in cultures exposed to 30 and 100 microM arsenite for 1 h, and it remained markedly suppressed until 24 h of exposure . Arsenite induced a transient inhibition of energy metabolism after 1 h of exposure, but energy state recovered completely after 3 h . Arsenite exposure affected the expression and translation of genes coding for HSP70 and GRP78, GRP94, GADD153 to different extents . While hsp70 mRNA levels rose drastically, approximally 550-fold after 6 h exposure, HSP70 protein levels did not change over the first 6 h . On the other hand, gadd153 mRNA levels rose only approximately 14-fold after 6 h exposure, while GADD153 protein levels were markedly increased after 3 and 6 h exposure . HSP70 protein levels were markedly increased and GADD153 protein levels decreased to almost control levels in cultures left in arsenite solution for 24 h, i.e . when only a small fraction of cells had escaped arsenite toxicity . Arsenite exposure of neurons thus induced an imbalance between pro-apoptotic and survival-activating pathways . Despite the marked increase in gadd153 mRNA levels, we did not observe signs of xbp1 processing in arsenite exposed cultures, indicating that arsenite did not produce ER stress.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2002 Jun, 19(2), 268 - 72
{Human peripheral blood monocyte derived dendritic cell culture and mature regulation}; Li S et al.; Mature dendritic cells are potent antigen-presenting cells that initiate primary immune responses, while immature dendritic cells have quite different properties from mature dendritic cells and are tolerance inducer actually . Here we describe the method of using monocyte condition medium to generate dendritic cells of different maturation phases from nonproliferating progenitors in human peripheral blood . The procedure involves two steps . The first step(or priming phase) is to work on a 6-7-day culture of plastic-adherent blood monocyte in medium supplement with GM-CSF and IL-4 . The second step (or differentiation phase) requires the exposure to monocyte conditioned medium . Only the dendritic cells generated by the first step are actually immature, with strong immature dendritic cell features such as active endocytosis, the same expression of monocyte marker CD14, and much of the MHC class II still lies within intracellular compartments (MIIC) . The second stage dendritic cells have all the features of mature dendritic cell, including a stellate shape, nonadherence to plastic, the expression of dendritic cells restricted marker CD83, and very strong T cell stimulatory function . All of these dendritic cell properties are retained for at least 3 days when the cytokines are removed, suggesting that these populations are stable and terminally differentiated . Since progression from immature to mature dendritic cell is entirely dependent on exogenously added growth factor such as monocyte condition medium, the peripheral blood monocyte may help to harness synchronized population of mature and immature dendritic cells for studies or therapies.

FASEB J, 2002 Nov, 16(13), 1847 - 9 Epub 2002 Sep 05.
Cell culture on thin tissue sections commonly prepared for histopathology; Takezawa T et al.; Thin tissue sections commonly prepared on a glass slide for histopathology retain many in vivo biochemical attributes related not only to structure but also function . We hypothesized that such tissue sections might serve as novel cell culture substrata that would reflect tissue conditions in vivo . Here we report the applicability of tissue section substrata to tissue reconstruction and serum-free culture . Four different cell types were cultured on section and acellularized section substrata prepared from a bovine placenta . The labyrinth region of the substratum induced cell differentiation to elicit the formation of multicellular spheroids of BeWo cells (human choriocarcinoma cells), a capillary network-like structure for CPAE cells (bovine pulmonary artery endothelial cells), and a neuronal network-like structure for PC-12 cells (rat pheochromocytoma cells) . The substratum provided a microenvironment that maintained the viability of PC-12 cells in a serum-free culture . We also succeeded in preparing a multicellular mass of normal human dermal fibroblasts (NHDFs) involving acellularized section-derived components . This technology offers the novel investigation of cell behaviors induced by culturing different cell types on various tissue sections and will be a useful tool for identifying cell characteristics and clarifying the molecular mechanisms that regulate the behavior of each cell type.

Brain Res Dev Brain Res, 2002 Aug 30, 137(2), 139 - 48
Phosphorylation of glial fibrillary acidic protein is stimulated by glutamate via NMDA receptors in cortical microslices and in mixed neuronal/glial cell cultures prepared from the cerebellum; Kommers T et al.; In previous work we showed that phosphorylation of glial fibrillary acidic protein (GFAP), an astrocyte marker, is increased by glutamate in hippocampal slices from immature rats via a type II metabotropic receptor . In the present work we show that glutamate also stimulates GFAP phosphorylation in microslices prepared from immature cerebellar cortex, but by a different receptor mechanism from that observed in the hippocampus . Thus, in cerebellar microslices, NMDA consistently stimulated GFAP phosphorylation, whereas no effect of metabotropic or non-NMDA ionotropic agonists was observed . Glutamate and NMDA also stimulated GFAP phosphorylation in mixed neuronal/glial cell cultures from the cerebellum, although no effect of these agonists was observed in primary cultures of cerebellar astrocytes . In both models, the effects of glutamate and NMDA were dependent on external Ca(2+), were reversed by the NMDA receptor antagonist AP5 and were not blocked by tetrodotoxin . In the slice study the effect of NMDA was confined to a period starting with the first detectable expression of GFAP at 10 days and finishing at 16 days postnatal, as previously observed with metabotropic agonists in hippocampal slices . This period in the rat corresponds to the start of synaptogenesis when astrocyte hypertrophy is occurring . The results are discussed in the light of information in the literature on the occurrence of functional NMDA receptor subunits in glia.

Clin Exp Allergy, 2002 Sep, 32(9), 1285 - 92
Effect of ozone and nitrogen dioxide on the permeability of bronchial epithelial cell cultures of non-asthmatic and asthmatic subjects; Bayram H et al.; BACKGROUND: Although epidemiological as well as in vivo exposure studies suggest that ozone (O3) and nitrogen dioxide (NO2) may play a role in airway diseases such as asthma, the underlying mechanisms are not clear . OBJECTIVE: Our aim was to investigate the effect of O3 and NO2 on the permeability of human bronchial epithelial cell (HBEC) cultures obtained from non-atopic non-asthmatic (non-asthmatics) and atopic mild asthmatic (asthmatics) individuals . METHODS: We cultured HBECs from bronchial biopsies of non-asthmatics and asthmatics, and exposed these for 6 h to air, 10 to 100 parts per billion (p.p.b.) O3, or to 100 to 400 p.p.b . NO2, and assessed changes in electrical resistance (ER) and movement of 14C-BSA across the cell cultures . RESULTS: Although exposure to either O3 or NO2 did not alter the permeability of HBEC cultures of non-asthmatics, 10 to 100 p.p.b . O3 and 400 p.p.b . NO2 significantly decreased the ER of HBEC cultures of asthmatics, when compared with exposure to air . Additionally, 10, 50 and 100 p.p.b . O3 led to a significant increase in the movement of 14C-BSA across asthmatic HBEC cultures, after 6 h of exposure (medians = 1.73%; P < 0.01, 1.50%; P < 0.05 and 1.53%, P < 0.05, respectively), compared with air exposed cultures (median = 0.89%) . Similarly, exposure for 6 h to both 200 and 400 p.p.b . NO2 significantly increased the movement of 14C-BSA across asthmatic HBEC cultures, when compared with air exposure . A comparison of data obtained from the two study groups demonstrated that 10 to 100 p.p.b . O3- and 200 to 400 p.p.b . NO2-induced epithelial permeability was greater in cultures of asthmatics compared with non-asthmatics . CONCLUSION: These results suggest that HBECs of asthmatics may be more susceptible to the deleterious effects of these pollutants . Whether in patients with asthma the greater susceptibility of bronchial epithelial cells to O3 and NO2 contributes to the development of the disease, or is a secondary characteristic of this condition, remains to be determined.

Hua Xi Yi Ke Da Xue Xue Bao, 1999 Mar, 30(1), 23 - 5
{The behaviour of rat mandibular condylar cartilage in cell culture}; Yang H et al.; This study aimed to identify the behaviour of mandibular condylar chondrocytes in vitro . Cells were harvested from the mandibular condyles of 3 week-old S . D . rats . Cell structure, morphology and characteristics were assessed by phase-contrast microscopy, scanning electron microscopy, enzymohistochemistry and immunohistochemistry . The cultured condylar chondrocytes, stellated or spindle-shaped, could grow in several layers and form many cell colonies . They could secret proteoglycans, alkaline phosphatase, type II collagen, et al . The methods of isolation, culture and identification of condylar chondrocytes were presented and discussed, which can be adopted in probing further into the cellular mechanism of functional orthopedics.

J Pept Sci, 2002 Aug, 8(8), 438 - 52
Antiproliferative action of valorphin in cell cultures; Blishchenko E et al.; The antiproliferative effects of the haemoglobin beta-chain fragment (33-39) (valorphin or VV-haemorphin-5) were studied in a panel of tumour cell lines and normal cells of different origin, using various methods of activity determination (trypan blue inclusion test, sulphorhodamine B staining, MTT staining, flow cytometry and clonogenic test) . Valorphin suppressed the proliferation of tumour cells by 25%-95%, depending on the cell line . The maximal valorphin activity was detected in transformed cells of fibroblastic (L929) and epithelial (MCF-7) origin, transformed haematopoietic cells (K562, HL-60) being less sensitive . In normal cells, valorphin activity was several fold lower (10%-15%) . A study of the dynamics of cell proliferation in L929 cells using a visual cell count and flow cytometry showed that valorphin induced reversible and relatively short (24 h) S-phase arrest of cell proliferation, accompanied by a reversible increase of cell size . The proliferation delay was followed by a comparatively long period of reversible resistance of the cells to the peptide (96 h) when the cells are dividing at normal rate . The same dynamics were demonstrated for A549, MCF-7 and primary murine breast carcinoma cells . On the basis of the data obtained, a pattern of regulation of cell growth by valorphin is suggested.

Growth Dev Aging, 2002 Summer, 66(1), 11 - 26
Secretion of insulin-like growth factor (IGF)-I and -II and IGF binding proteins (IGFBPs) in fetal stromal-vascular (S-V) cell cultures obtained before and after the onset of adipogenesis in vivo; Hausman GJ et al.; The present study examined the influence of dexamethasone (DEX) treatment on preadipocyte differentiation and insulin-like growth factor binding protein (IGFBP) secretion in stromal-vascular (S-V) cell cultures established from subcutaneous adipose tissue obtained from nine 75 day and four 50 day pig fetuses . Cultures of S-V cells from four young pigs (5-7 days old) were also studied . Each fetal S-V cell culture represented 1 pool of S-V cells/dam . Cultures were seeded and plated in 10% FBS from day 0-3 and treated with insulin (ITS) + 10 nM DEX from day 3-6 (late DEX treatment) . Alternatively, cultures were seeded and plated in 10% FBS + 80 nM DEX from day 0-3 and treated with insulin alone from day 3-6 (early DEX treatment) . Conditioned media was collected on day 6 of culture after 3 days of conditioning, and prepared for subsequent 125I-IGF-I ligand blot analysis for IGFBPs and RIA for IGF-I and IGF-II . Early and late DEX increased (P<0.05) preadipocyte (AD-3+) recruitment but only early DEX increased preadipocyte differentiation (lipid + and C/EBP alpha+) by day 6 in S-V cultures from 75 day fetuses . Levels of IGFBP-2, IGFBP-4, IGF-I and IGF-II in media conditioned by 75 day fetal S-V cultures were not influenced by late DEX . However, late DEX reduced levels of 29 kDa IGFBPs and markedly increased (P<0.05) IGFBP-3 levels in 75 day S-V media . Late DEX also markedly increased (P<0.05) IGFBP-3 levels in 50 day S-V media but had little influence on other IGFBPs . Early DEX treatment increased (P<0.05) IGFBP-4 levels in 75 day S-V media but had little to no influence on levels of IGF-I, IGF-II and other IGFBPs . These studies indicate that IGFBP-4 may regulate local metabolism during preadipocyte differentiation, whereas IGFBP-3 may antagonize preadipocyte differentiation by targeting IGF-I away from differentiating cells and towards growing cells.

J Neurosci Res, 2002 Sep 1, 69(5), 687 - 91
Human bone marrow stromal cell cultures conditioned by traumatic brain tissue extracts: growth factor production; Chen X et al.; Treatment of traumatic brain injury (TBI) with bone marrow stromal cells (MSCs) improves functional outcome in the rat . However, the specific mechanisms by which introduced MSCs provide benefit remain to be elucidated . Currently, the ability of therapeutically transplanted MSCs to replace injured parenchymal CNS tissue appears limited at best . Tissue replacement, however, is not the only possible compensatory avenue in cell transplantation therapy . Various growth factors have been shown to mediate the repair and replacement of damaged tissue, so trophic support provided by transplanted MSCs may play a role in the treatment of damaged tissue . We therefore investigated the temporal profile of various growth factors, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF), within cultures of human MSCs (hMSCs) conditioned with cerebral tissue extract from TBI . hMSCs were cultured with TBI extracts of rat brain in vitro and quantitative sandwich enzyme-linked immunosorbent assays (ELISAs) were performed . TBI-conditioned hMSCs cultures demonstrated a time-dependent increase of BDNF, NGF, VEGF, and HGF, indicating a responsive production of these growth factors by the hMSCs . The ELISA data suggest that transplanted hMSCs may provide therapeutic benefit via a responsive secretion of an array of growth factors that can foster neuroprotection and angiogenesis .

Electrophoresis, 2002 Jul, 23(14), 2223 - 32
Proteomic evaluation of cell preparation methods in primary hepatocyte cell culture; Witzmann FA et al.; In vitro liver preparations are being used increasingly to study various aspects of chemical hepatotoxicity and thus have become powerful alternatives to in vivo toxicologic models . Primary hepatocyte culture systems are especially useful in screening cytotoxic and genotoxic compounds and assessing biochemical lesions associated with chemical exposure . We have begun to use this approach in combination with proteomic analysis to construct a molecular "toxicoproteomic" test system for a broad range of relevant and potentially toxic chemicals . Using a highly parallel two-dimensional electrophoretic (2-DE) protein separation system to analyze cells from culture systems, we previously observed significant variations in protein expression that were unrelated to chemical exposure . We hypothesized these artifactual protein alterations were the result of the variations in the culture conditions or cell manipulations, or both . Therefore, we conducted a study to assess the expression of hepatocyte proteins cultured on 6-well plates and recovered for analysis either by scraping/pelleting or direct in-well solubilization . Following incubation of 1.2 x 10(6) hepatocytes in six-well plate, recovery and solubilization of the cells and 2-DE of the solubilized lysates of 100 000 cells, we detected 1388 proteins in the in-well solubilized samples compared to 899 proteins in the washed/scraped/pelleted cell samples, a loss of 35% . Based on protein identification by peptide mass fingerprinting, the subcellular location of nearly all of the proteins whose abundance decreased were cytosolic and those few that increased were either microsomal, mitochondrial, or cytoskeletal proteins . These results emphasize the variation introduced by cell-handling during recovery of hepatocytes from culture plates and may explain at least some of the artifactual differences observed in earlier in vitro experiments.

J Biomed Mater Res, 2002 Dec 5, 62(3), 438 - 46
Fabrication and surface modification of macroporous poly(L-lactic acid) and poly(L-lactic-co-glycolic acid) (70/30) cell scaffolds for human skin fibroblast cell culture; Yang J et al.; The fabrication and surface modification of a porous cell scaffold are very important in tissue engineering . Of most concern are high-density cell seeding, nutrient and oxygen supply, and cell affinity . In the present study, poly(L-lactic acid) and poly(L-lactic-co-glycolic acid) (70/30) cell scaffolds with different pore structures were fabricated . An improved method based on Archimedes' Principle for measuring the porosity of scaffolds, using a density bottle, was developed . Anhydrous ammonia plasma treatment was used to modify surface properties to improve the cell affinity of the scaffolds . The results show that hydrophilicity and surface energy were improved . The polar N-containing groups and positive charged groups also were incorporated into the sample surface . A low-temperature treatment was used to maintain the plasma-modified surface properties effectively . It would do help to the further application of plasma treatment technique . Cell culture results showed that pores smaller than 160 microm are suitable for human skin fibroblast cell growth . Cell seeding efficiency was maintained at above 99%, which is better than the efficiency achieved with the common method of prewetting by ethanol . The plasma-treatment method also helped to resolve the problem of cell loss during cell seeding, and the negative effects of the ethanol trace on cell culture were avoided . The results suggest that anhydrous ammonia plasma treatment enhances the cell affinity of porous scaffolds . Mass transport issues also have been considered .

Arch Virol, 2002 Sep, 147(9), 1665 - 83
High fidelity of homologous retroviral recombination in cell culture; Bircher LA et al.; Genetic variation continues to be a major obstacle in the development of therapies and vaccines against retroviral infections and contributes extensively to viral pathogenesis and persistence . Recombination is one mechanism that increases retroviral variation by shuffling mutations from different genomes . Recent studies suggest that recombination not only shuffles the mutations but also generates them at high rates during reverse transcription . In contrast to these recent studies, this investigation shows that recombination does not generate mutations during recombination . A spleen necrosis virus (SNV)-based homologous recombination system was used to test the hypothesis that retroviral recombination is a high-fidelity process during replication of the virus in cell culture . The system consisted of a pair of SNV vectors expressing two drug resistance genes . The vectors were constructed so that cells containing recombinant proviruses could be selected by a double drug-resistant phenotype . Restriction enzyme digestion and agarose gel electrophoresis were used to map the location of recombination within 182 proviruses . Sequencing and single-strand conformation polymorphism techniques were then used to check for mutations within the recombinant proviruses . Since no mutations were detected among the 182 recombinants that were analyzed, homologous recombination is a high-fidelity process for retroviruses in cell culture.

J Neurosci Res, 2002 Sep 15, 69(6), 810 - 25
Selective specification of CNS stem cells into oligodendroglial or neuronal cell lineage: cell culture and transplant studies; Espinosa-Jeffrey A et al.; Neural stem cells (NSCs) were isolated from embryonic day 16 Sprague-Dawley rats and cultured in a novel serum-free stem cell medium that selected for the growth of NSCs and against the growth of GFAP(+) cells (astrocytes) . NSCs maintained in culture for extended periods of time retained immunoreactivity for both nestin and PSA-NCAM, two markers characteristic of the stem cell phenotype . Moreover, using an oligodendrocyte (OL) specification medium, NSCs differentiated into OL as evidenced by their morphology and expression of multiple oligodendrocyte/myelin-specific markers . In addition, NSCs are capable of acquiring a neuronal phenotype as evidenced by expressing neuronal markers, such as neurofilament (NF) and NeuN when cultured in a defined medium for neurons indicating that these cells are also a good source of neuroblasts, which could be used to replace neuronal populations in the brain . We also showed successful propagation and differentiation of NSCs into OL after cryostorage, allowing for the later use of stored NSCs . The long-term goal of culturing NSCs and committed oligodendrocyte progenitors (OLP) is to obtain homogeneous populations for transplantation with the goal of remyelinating the myelin-deficient CNS . Our preliminary experiments carried out on normal and myelin deficient rats demonstrate that these cells survive and migrate extensively in both types of hosts . NSCs grafted as such, as well as cells derived from NSCs exposed to selective specification before grafting, are able to differentiate within the host brain . As expected, NSCs are capable of giving rise to astrocytes in a medium favoring this phenotype .

Toxicology, 2002 Sep 30, 179(1-2), 171 - 80
ESR and cell culture studies on free radical-scavenging and antioxidant activities of isoflavonoids; Guo Q et al.; Isoflavonoids are thought to be the biologically active components in soy that play a role in the prevention of coronary heart disease and breast and prostate cancer . Mechanisms to explain how isoflavonoids mediate beneficial effects have not yet been clearly established . This study was undertaken to investigate the free radical-scavenging and antioxidant activities of various structure-related isoflavonoids including genistein, daidzein, biochanin A, and genistin in a cell-free and an endothelial cell model system . Electron spin resonance spectroscopy and spin trapping techniques were applied to evaluate the ability of isoflavonoids to scavenge hydroxyl, superoxide, nitric oxide, diphenylpicrylhydrazyl, galvinoxyl, and lipid-derived radicals . All isoflavonoids tested had no significant scavenging effects on the aforementioned radicals in concentrations up to 1.0 mM . However, at a physiologically achievable concentration of 5 nM, both genistein and daidzein slightly increased intracellular-reduced glutathione levels approximately by 10 and 30%, respectively, in human endothelial cells, whereas cellular alpha-tocopherol and uric acid remained unchanged by the isoflavonoid treatments . Present data indicate that free radical-scavenging activities of the isoflavonoids tested probably do not substantially contribute to their antioxidant properties . The ability of genistein and daidzein to increase cellular GSH (reduced glutathione) might be important for their action in biological system.

Int J Parasitol, 2002 Sep, 32(10), 1253 - 65
Exogenous nitric oxide triggers Neospora caninum tachyzoite-to-bradyzoite stage conversion in murine epidermal keratinocyte cell cultures; Vonlaufen N et al.; Neospora caninum, like Toxoplasma gondii, undergoes stage conversion in chronically infected animals, and forms tissue cysts which contain the slowly proliferating bradyzoite stage . These tissue cysts are delineated by a cyst wall, protect the parasite from physiological and immunological reactions on part of the host, and bradyzoites remain viable within an infected host for many years . However, unlike T . gondii, N . caninum bradyzoites have been difficult to obtain using in vitro culture techniques, and current protocols, based on those developed for T . gondii, have been shown to be not very efficient in promoting tachyzoite-to-bradyzoite stage conversion . We report here an alternative in vitro culture method to obtain stage conversion of N . caninum from the proliferative to the cystic stage by using the Nc-Liverpool isolate, murine epidermal keratinocytes as host cells, and continuous treatment of infected cultures with 70 microM sodium nitroprusside for up to 8 days . This treatment significantly reduced parasite proliferation as assessed by Neospora-specific quantitative real-time PCR . The expression of bradyzoite markers was analysed by immunofluorescence following 4 and 8 days of in vitro culture using antibodies directed against bradyzoite antigen 1, the mAbCC2, and the lectin Dolichos biflorus agglutinin . Expression of the tachyzoite-specific immunodominant antigen NcSAG1 and the tachyzoite antigen NcMIC1 was also assessed . Transmission electron microscopy revealed that the majority of parasitophorous vacuoles were in the process of forming a distinct cyst wall through accumulation of granular material at the periphery of the vacuole, and parasites exhibited the typical features of bradyzoites . These findings demonstrate the usefulness of this culture technique as a promising way to study tachyzoite-to-bradyzoite stage conversion in N . caninum in vitro.

Calcif Tissue Int, 2002 Jul, 71(1), 36 - 44 Epub 2002 Jun 24.
Maintenance of osteoblastic and adipocytic differentiation potential with age and osteoporosis in human marrow stromal cell cultures; Justesen J et al.; Osteoblasts and adipocytes share a common precursor cell in the bone marrow stroma, termed marrow stromal cell (MSC) . As the volume of bone adipose tissue increases in vivo with age, we hypothesized that decreased bone formation observed during aging and in patients with osteoporosis (OP) is the result of enhanced adipogenesis and decreased osteoblastogenesis from the MSCs . Thus, cultures of MSCs were established from young donors (age 18-42, n = 34), elderly healthy donors (age 66-78, n = 20), and patients with OP (age 58-76, n = 15) . Cells were cultured for 2 weeks in an adipogenic medium (containing 15% horse serum and 100 nM dexamethasone), osteogenic medium (containing 10% fetal calf serum {FCS} and 10 nM calcitriol), or control medium (10% FCS) . The MSCs were identified by their abilities to form colonies . Total number of colonies, osteoblastic colonies stained positive for alkaline phosphatase (AP+), and adipocytic colonies containing adipocytes (Ad+) were quantitated . In addition, steady state mRNA levels of gene markers of adipocytic and osteoblastic phenotypes were determined using reverse-transcriptase polymerase chain reaction (RT-PCR) . The adipogenic and osteogenic media induced cell differentiation and the expression of adipocytic and osteoblastic lineage-specific markers, respectively . We found no age-related changes in the osteoblastic or adipocytic colony formation or the steady state levels of mRNA of the adipogenic or osteogenic gene markers . Cells obtained from patients with OP showed a pattern of differentiation similar to those of age-matched controls . In conclusion, MSCs maintain their differentiation potential during aging and in patients with OP . Other mechanisms responsible for age-related decrease in bone formation need to be determined.

Biochem Biophys Res Commun, 2002 Aug 30, 296(4), 1000 - 4
Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo; Bertrand JR et al.; Efficiencies of a nuclease resistant antisense oligonucleotide and of siRNA both being targeted against the green fluorescent protein stably expressed in HeLa cells are compared in cell cultures and in xenografted mice . Using Cytofectin GSV to deliver both inhibitors, the siRNAs appear to be quantitatively more efficient and its effect is lasting for a longer time in cell culture . In mice, we observed an activity of siRNAs but not of antisense oligonucleotides . The absence of efficiency of antisense oligonucleotides is probably due to their lower resistance to nuclease degradation.

Biomed Pharmacother, 2002 Jul, 56(5), 254 - 7
The effect of high molecular weight dextran sulfate on the production of interleukin-8 in monocyte cell culture; Jagodzinski PP et al.; It has been demonstrated that high molecular weight dextran sulfate (HMDS) is involved in the activation of immune cells . We have shown that HMDS increases the concentration of interleukin (IL)-8 in the medium of monocyte cell culture, in a dose-dependent fashion, whereas under the same conditions, low molecular weight dextran sulfate (LMDS) does not exhibit any effect on IL-8 biosynthesis . The effect of HMDS on IL-8 production is additive to that of IL-1beta and tumor necrosis factor-a (TNFalpha) . Flow cytometric analysis revealed the biosynthesis of IL-8 in monocytes incubated in the presence of the HMDS . We hereby postulate that HMDS induces IL-8 biosynthesis in monocyte cell culture.

J Parasitol, 2002 Aug, 88(4), 794 - 6
Total anaerobiosis during in vitro culture in conventional cell culture media is required for retaining infectivity of Trichinella spiralis L1 larvae; Bolas-Fernandez F; The infectivity of Trichinella spiralis L1 larvae was examined in Swiss CD-1 mice after their maintenance in conventional cell culture media under different atmospheric conditions . Larvae isolated from the infected mouse carcasses were cultured for 24 hr in Roswell Park Memorial Institute (RPMI) medium, minimum essential medium (MEM), 199 medium, and Hank's balanced salt solution (HBSS) medium under anaerobic, microaerobic, and 5% CO2 conditions . Only those larvae maintained under anaerobiosis in all media retained their infectivity in mice . The larvae maintained microaerobically and under 5% CO2 lost more of their infectivity when cultured in RPMI medium and MEM (> 96%) than in 199 and HBSS (> 78%).

J Biol Chem, 2002 Oct 25, 277(43), 40650 - 8 Epub 2002 Aug 19.
Activation of human meprin-alpha in a cell culture model of colorectal cancer is triggered by the plasminogen-activating system; Rosmann S et al.; The activation of latent proenzymes is an important mechanism for the regulation of localized proteolytic activity . Human meprin-alpha, an astacin-like zinc metalloprotease expressed in normal colon epithelial cells, is secreted as a zymogen into the intestinal lumen . Here, meprin is activated after propeptide cleavage by trypsin . In contrast, colorectal cancer cells secrete meprin-alpha in a non-polarized way, leading to accumulation and increased activity of meprin-alpha in the tumor stroma . We have analyzed the activation mechanism of promeprin-alpha in colorectal cancer using a co-culture model of the intestinal mucosa composed of colorectal adenocarcinoma cells (Caco-2) cultivated on filter supports and intestinal fibroblasts grown in the companion dish . We provide evidence that meprin-alpha is activated by plasmin and show that the presence of plasminogen in the basolateral compartment of the co-cultures is sufficient for promeprin-alpha activation . Analysis of the plasminogen-activating system in the co-cultures revealed that plasminogen activators produced and secreted by fibroblasts converted plasminogen to active plasmin, which in turn generated active meprin-alpha . This activation mechanism offers an explanation for the observed meprin-alpha activity in the tumor stroma, a prerequisite for a potential role of this protease in colorectal cancer.

J Agric Food Chem, 2002 Aug 28, 50(18), 5202 - 6
Transport of amino acids from in vitro digested legume proteins or casein in Caco-2 cell cultures; Rubio LA et al.; Purified legume storage proteins (chickpea 11S and 7S globulins, faba bean globulins, and lupin globulins) and casein (casein) were subjected to an in vitro enzyme (pepsin + pancreatin) digestion process . Protein digests were then used in a bicameral Caco-2 cell culture system to determine amino acid transport across the cell monolayer . With digests from legume proteins, absolute amounts of aspartate, glycine, and arginine transported were higher than those found in digested casein, whereas amounts of glutamate, proline, tyrosine, valine, and lysine were lower . However, proportions of amino acids in the basolateral chamber as compared with amounts added in the apical chamber were lower than casein controls for all amino acids except cystine . Results confirm previous in vivo observations that amino acids from legume proteins are probably absorbed at rates different from those in other proteins of animal origin such as casein.

Biotechniques, 2002 Aug, 33(2), 420 - 3
Multichannel plating unit for high-throughput plating of cell cultures; Hamilton CM et al.; High-throughput genomic approaches to gene function or target identification have led to the development and implementation of the 96-well format for many standard molecular biology manipulations . The apparatus described here, a Multichannel Plating Unit, is designed to plate out individual cultures efficientlyfrom standard 96-well culture blocks . Following transformation, aliquots of culture are loaded onto sterile beads that are rolled along individual channels of agar media . After the beads traverse the channel, they drop into the exit alley for disposal via an exit pore . The apparatus presented has 12 individual lanes, and the spacing is compatible with a standard 12-channel pipettor Thus, the unit allows for the rapid plating of 12 individual cultures at a time . For one 96-well block of transformants, this method reduces the labeling and plating effort from 96 culture dishes that are spread individually to eight multichannel plates . The savings in time, materials, and storage space is significant

Klin Lab Diagn, 2002 Jul, (7), 5 - 7
{Gas chromatography analysis of lipids in cell culture in viral infections and in serum of patients with various pathological conditions}; Krotenko AV et al.; The results of gas chromatographic analysis of fatty acid composition of cell cultures infected with Coxsackie B viruses (CBV) and of sera of patients with unstable angina, psoriasis, and after stroke indicate that the presence of CBV in the organism can be one of the causes of lipid dysmetabolism.

J Virol, 2002 Sep, 76(18), 9218 - 24
Cleavage at the furin consensus sequence RAR/KR(109) and presence of the intervening peptide of the respiratory syncytial virus fusion protein are dispensable for virus replication in cell culture; Zimmer G et al.; Proteolytic processing of the respiratory syncytial virus F (fusion) protein results in the generation of the disulfide-linked subunits F1 and F2 and in the release of pep27, a glycopeptide originally located between the two furin cleavage sites FCS-1 (RKRR(136)) and FCS-2 (RAR/KR(109)) . We made use of reverse genetics to study the importance of FCS-2 and of pep27 for BRSV replication in cell culture . Replacement of FCS-2 in the F protein of recombinant viruses by either of the sequences NANR(109), RANN(109) or SANN(109), respectively, abolished proteolytic processing at this position, whereas the cleavage of FCS-1 was not affected . All mutants replicated in calf kidney and Vero cells in the absence of exogenous trypsin, although somewhat higher titers of BRSV containing the NANR(109) or the RANN(109) motif were achieved in the presence of trypsin . The virus mutants showed a reduced cytopathic effect which was lowest in the case of the SANN(109) mutant . These findings demonstrate that cleavage at FCS-2 is dispensable for replication of respiratory syncytial virus in cell culture . A deletion mutant containing FCS-1 but lacking FCS-2 and most of pep27 replicated in cell culture as efficiently as the parental virus, indicating that this domain of the F protein is not essential for virus maturation and infectivity.

J Gen Virol, 2002 Sep, 83(Pt 9), 2135 - 43
Apoptosis induction by the Therien and vaccine RA27/3 strains of rubella virus causes depletion of oligodendrocytes from rat neural cell cultures; Domegan LM et al.; The induction of cell death by the Therien strain of rubella virus (RVT), and the vaccine RA27/3 strain, was investigated in mixed glial cell cultures derived from the rat CNS . Cell death induction in Vero and rat glial cells by RVT and RA27/3 was dependent on virus replication . In both cell types and for both virus strains, cell death induction had the hallmarks of apoptosis, as detected by DNA laddering, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling staining and Annexin V staining . For rat mixed glial cells, the depletion of oligodendrocytes was due to the induction of apoptosis for both virus strains . The induction of apoptosis in H358a cells, which carry a homozygous deletion of the p53 gene, indicated that a p53-independent pathway can be involved . The induction of cell death by RVT and RA27/3 in Vero and rat glial cells was associated with caspase-3 activity . It is concluded that rubella virus (RV) induces apoptosis in oligodendrocytes in rat glial cell cultures by a caspase-dependent pathway and that similar mechanisms occur for both the RVT laboratory strain and the vaccine RA27/3 strain . The tropism of both strains of RV for oligodendrocytes and the induction of apoptosis in such cells may have important implications for the mechanism of virus neuropathogenesis.

J Aerosol Med, 2002 Summer, 15(2), 131 - 9
Drug absorption by the respiratory mucosa: cell culture models and particulate drug carriers; Ehrhardt C et al.; The inhalation route is of increasing interest for both local and systemic drug delivery, including macromolecular biopharmaceuticals, such as peptides, proteins, and gene therapeutics . In addition to appropriate aerosolization for deposition in relevant areas of the respiratory tract, therapeutic molecules may require an advanced carrier system for safe and efficient delivery to their target . Two approaches to obtain novel carrier systems for pulmonary drug delivery are large porous microparticles with a low aerodynamic diameter and lectin-functionalized liposomes . Epithelial cells of alveolar or bronchial origin, obtained either from patient material or from established cell lines, can be grown on permeable filter supports, resulting in polarized monolayers with functional intercellular junctions . With such in vitro models, transport of drugs into pulmonary epithelial cells and/or across the air-blood barrier, as well as the effect and efficacy of novel drug carrier systems can be systematically studied.

J Appl Physiol, 2002 Sep, 93(3), 1047 - 56
Spaceflight affects bone formation in rhesus monkeys: a histological and cell culture study; Zerath E et al.; Using analyses of iliac crest cell and tissue, back-scattered electron imaging, and biochemical techniques, we characterized the effects of a 14-day spaceflight (Bion 11) on bone structure and bone formation in two 3- to 4-yr-old male rhesus monkeys compared with eight age-matched Earth-control monkeys . We found that postflight bone volume was 35% lower than preflight values in flight monkeys . This was associated with reduced osteoid (-40%) and mineralizing (-32%) surfaces and decreased bone formation rate (-53%) . Moreover, flight monkeys exhibited trends to lower values of mineralization profile in iliac bone (back-scattered electron imaging) and to decreased osteocalcin serum levels (P = 0.08) . The initial number of trabecular bone cells yielded in cultures did not differ in flight and control animals before or after the flight . However, osteoblastic cell proliferation was markedly lower in postflight vs . preflight at 9 and 14 days of culture in one flight monkey . This study suggests that a 14-day spaceflight reduces iliac bone formation, osteoblastic activity, and/or recruitment in young rhesus monkeys, resulting in decreased trabecular bone volume.

Antivir Chem Chemother, 2002 Jan, 13(1), 61 - 6
Antiviral activity of brassinosteroids derivatives against measles virus in cell cultures; Wachsman MB et al.; Twenty-seven brassinosteroid derivatives were tested for antiviral activity against measles virus (MV) via a virus-yield reduction assay . Compounds 6b {(22S,235)-3beta-bromo-5alpha,22,23-trihydroxystigmastan-6-one}, 1d {(22R,23R)-2alpha,3alpha,22,23-tetrahydroxy-beta-Homo-7-oxa-stigmastan-6-one}, 8a {(22R,23R)-3beta-fluoro-22,23-dihydroxystigmastan-6-one}, 9b {(22S,23S)-3beta-fluoro-5alpha,22,23-trihydroxystigmastan-6-one} and 10b {(22S,23S)-5alpha-fluor-3beta,22,23-trihydroxystigmastan-6-one}, with selectivity indexes (SI) of 40, 57, 31, 37 and 53, are the derivatives with good antiviral activity against MV . These SI values are higher than those obtained with ribavirin (used as reference drug) . A comparative analysis of 50% cytotoxic concentration (CC50) values, using confluent non-growing cells, gives and indication of structure-activity relationship . According to their degree of cytotoxicity the compounds were divided in three groups: low, intermediate and high cytotoxicity . By observing the chemical structures of compounds belonging to the first group we can see that less cytotoxic activities are related to the presence of a 3beta-hydroxy group on C-3 (ring A) and a double bond between C-22 and C-23 (side chain) . The replacement of a 5alpha-hydroxy group by a 5alpha-fluoro group enhances cytotoxicity . Halogenated brassinosteroid derivatives in C-3 position are more cytotoxic than those with an acetoxy group in the same position . For compounds 1d, 6b, 10b and ribavirin, cytotoxicity measurements were also done with replicating cells; CC50 values were low, but they still competed favourably with ribavirin against MV.

Pharm Res, 2002 Jul, 19(7), 1038 - 45
Differential modulation of P-glycoprotein expression and activity by non-nucleoside HIV-1 reverse transcriptase inhibitors in cell culture; Stormer E et al.; PURPOSE: This study investigated the effects of the non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTI) nevirapine (NVR), efavirenz (EFV), and delavirdine (DLV) on P-glycoprotein (P-gp) activity and expression to anticipate P-gp related drug-drug interactions associated with combination therapy . METHODS: NNRTIs were evaluated as P-gp substrates by measuring differential transport across Caco-2 cell monolayers . Inhibition of P-gp mediated rhodaminel23 (Rh123) transport in Caco-2 cells was used to assess P-gp inhibition by NNRTIs . Induction of P-gp expression and activity in LS180V cells following 3-day exposure to NNRTIs was measured by western blot analysis and cellular Rh123 uptake, respectively . RESULTS: The NNRTIs showed no differential transport between the basolateral to apical and apical to basolateral direction . NNRTI transport in either direction was not affected by the P-gp inhibitor verapamil . DLV inhibited Rh123 transport, causing a reduction to 15% of control at 100 microM (IC50 = 30 microM) . NVR caused a concentration-dependent induction of P-gp expression in LS180V cells resulting in a 3.5-fold increase in immunoreactive P-gp at 100 microM NVR . Induction attributable to EFV and DLV was quantitatively smaller . NVR significantly reduced cellular uptake of Rh123 into LS180V cells, indicating increased drug efflux due to induced P-gp activity; effects of EFV and DLV were smaller . Acute DLV treatment of LS180V cells previously induced with NVR or ritonavir did not reverse the decreased Rh123 cell accumulation . CONCLUSIONS: NNRTIs show differential effects on P-gp activity and expression in vitro . Clinical studies are required to elucidate the clinical importance of potential drug interactions.

Mol Gen Mikrobiol Virusol, 2002, (2), 27 - 30
{Submicroscopic characteristics of Marburg virus and its mini genome analog replication in cell cultures}; Cheusova TB et al.; Marburg virus (Filoviridae) causes severe hemorrhagic fevers in humans and some lower primates with high mortality . The virus genome is formed by a single strand RNA of negative polarity, coding for seven structural proteins . We studied the ultrastructure of Marburg virus replicative cycle and replication of its minigenome RNA (coding for the terminal areas of the genome) in the presence of helper virus in VERO fibroblastoid cell culture and epithelioid MDCK cell culture . Ultrastructural parameters of Marburg virus multiplication in these cell cultures are virtually the same . The virus nucleocapsid assembly is performed on the outer side of EPR membrane and is not associated with preliminary accumulation of the precursor material . Virions form by budding on plasmalemma and are located on the entire surface in Vero cells and only on the basolateral surface of MDCK cells . Replication of minigenome analog of marburg virus is associated with impairment of the helper virus morphogenesis and formation of spherical pseudoviral particles.

Curr Biol, 2002 Jul 23, 12(14), 1169 - 77
New class of HIV integrase inhibitors that block viral replication in cell culture; Pannecouque C et al.; BACKGROUND: To improve the existing combination therapies of infection with the human immunodeficiency virus (HIV) and to cope with virus strains that are resistant to multiple drugs, we initiated a search for effective inhibitors of HIV integrase, the enzyme responsible for inserting the viral cDNA into the host cell chromosome.RESULTS: We have now identified a series of 5H-pyrano{2,3-d:-6,5-d'}dipyrimidines that block the replication of various strains of HIV-1 and HIV-2 . The most potent congener, 5-(4-nitrophenyl)-2,8-dithiol-4,6-dihydroxy-5H-pyrano{2,3-d:-6,5-d'}dipyrimidine (V-165), inhibited the replication of HIV-1(III(B)) in MT-4 cells at a 50% effective concentration (EC(50)) of 8.9 microM, which is 14-fold below its cytotoxic concentration . V-165 was equally active against virus strains that were resistant toward inhibitors of viral entry or reverse transcriptase . In combination regimens in cell culture, V-165 acted subsynergistically with zidovudine or nelfinavir and synergistically with nevirapine . V-165 inhibited both reverse transcriptase and integrase activities in enzymatic assays at micromolar concentrations, but only a close correlation was found between the anti-HIV activity observed in cell culture and the inhibitory activity in the integrase strand transfer assays . Time-of-addition experiments indicated that V-165 interfered with the viral replication cycle at a time point coinciding with integration . Quantitative Alu-PCR corroborated that the anti-HIV activity of V-165 is based upon the inhibition of proviral DNA integration.CONCLUSIONS: Based on their mode of action, which is different from that of clinically approved anti-HIV drugs, PDPs are good candidates for further development into new drugs and to be included in future combination regimens.

Biosci Rep, 2001 Dec, 21(6), 765 - 78
Different molecular capacity in the induction of apoptosis by polychlorinated biphenyl congeners in rat renal tubular cell cultures; Perez-Reyes PL et al.; The effects of a commercial polychlorinated biphenyl (PCB) mixture (Aroclor 1248) and two individual PCB congeners were evaluated on rat renal proximal tubule culture cell viability and internucleosomal DNA fragmentation (DNA ladder) characteristic of apoptosis . Treatment with Aroclor 1248 caused the loss of cell viability and promoted apoptosis in a concentration- and time-dependent manner . The two PCB congeners assessed can also induce apoptosis . However, the extent of apoptosis generated was greater for the non-ortho-substituted planar congener (3,3',4,4-tetrachlorobiphenyl) than for the di-ortho-substituted nonplanar congener (2,2',4,4',5,5'-hexachlorobiphenyl) . This correlated with the loss of cell viability since the planar compound is much more cytotoxic . The results suggest a different molecular mechanism in the induction of apoptosis by planar or nonplanar PCB congeners.

Hum Mol Genet, 2002 Aug 15, 11(17), 2061 - 75
Differential gene expression in a cell culture model of SOD1-related familial motor neurone disease; Kirby J et al.; Motor neurone disease is caused by mutations in Cu/Zn superoxide dismutase (SOD1) in 15-20% of familial cases, due to a toxic gain of function by the mutant enzyme . However, the underlying mechanism of SOD1-mediated neurodegeneration remains uncertain . By investigating alterations in gene expression in the presence of mutant Cu/Zn SOD, we aimed to identify pathways that contribute to motor neurone injury and cell death . Using a cellular model of familial motor neurone disease, the motor neuronal cell line NSC34 was stably transfected with either normal or mutant (G37R, G93A, I113T) SOD1 cDNAs, and the effect of the presence of these proteins on gene expression was analysed . This model allowed gene expression changes to be studied specifically in cells with a motor neurone phenotype, without interference from genes expressed by glia, astrocytes and other cell types located in the central nervous system . Using a commercially available cDNA membrane array, we investigated the expression levels of 588 genes from key biological pathways . Gene expression was studied in the cells under both basal culture conditions and following oxidative stress induced by serum withdrawal . Twenty-nine differentially expressed genes were identified, 7 of which were specifically downregulated in the presence of the mutant Cu/Zn SOD protein, and whose expression was further studied by real-time PCR . Presence of the mutant Cu/Zn SOD was confirmed to lead to a decrease in expression of KIF3B, a kinesin-like protein, which forms part of the KIF3 molecular motor . c-Fes, thought to be involved in intracellular vesicle transport was also decreased, further implicating the involvement of vesicular trafficking as a mode of action for mutant Cu/Zn SOD . In addition, a decrease was confirmed in ICAM1, a response in part due to the increased expression of SOD1, and decreased Bag1 expression was confirmed in two of the three mutant cell lines, providing further support for the involvement of apoptosis in SOD1-associated motor neurone death.

Acta Physiol Hung, 2001, 88(3-4), 173 - 96
Effect of capsaicin on voltage-gated currents of trigeminal neurones in cell culture and slice preparations; Balla Z et al.; Effects of capsaicin on voltage-gated currents were examined in vitro by whole-cell patch-clamp recordings from small neurones of rat trigeminal ganglia either in slice preparations or in different cell cultures . Cells were classified as sensitive to capsaicin if they responded with inward current and/or conductance change to the agent in nanomolar concentration . Capsaicin (150 to 330 nM) in sensitive cells reduced the mixed inward current evoked by depolarizing step or ramp commands in all preparations . In cultured cells, the inward current was depressed to 32.78 +/- 26.42% (n = 27) of the control . Both the tetrodotoxin-sensitive and -resistant inward currents were affected . The data support the concept that capsaicin besides acting on VR-1 receptors inhibits also some voltage gated channels . In 34 cultured cells, capsaicin increased the slope conductance to 170.5 +/- 68% . Percentage of capsaicin sensitive cells observed in nerve growth factor-treated cultured cell populations was higher (77.8%) than in the two other preparations (14.3 or 38.8%) . It is concluded that 1) depression of the voltage-gated currents may play an important role in the functional desensitization of the sensory receptors and in the analgesic effect induced by the agent and 2) cell body of sensory neurones under native condition seems less sensitive to capsaicin then that of cells cultured in the presence of nerve growth factor.

Eur J Neurosci, 2002 Jul, 16(1), 44 - 54
ERK activation and nuclear translocation in amyloid-beta peptide- and iron-stressed neuronal cell cultures; Kuperstein F et al.; Oxidative stress in the human brain has been strongly implicated as the cause of neuronal cell losses in Alzheimer's disease patients, but the exact mechanism still remains unknown . In this report several oxidative stress parameters and an associated signalling transduction cascade predating neuronal cell death in cultures treated with the oxidative stressors Fe(2+) (5 microm) and the amyloid beta (A beta(1-40)) peptide (5 microm) were studied . Production of reactive oxygen species as detected by dichlorofluorescein staining was apparent within 5 min in the presence of both agents . Lipid peroxide content increased by approximately 10-fold after 2 h, while mitochondrial activity was impaired by 40% after 6 h . Caspase-3 activity was elevated 5-6 fold, all indicative of oxidative cell stress . The combined presence of A beta(1-40) and Fe(2+) resulted in a rapid (5 min) ERK activation followed by a decline by 30 min and a second activation that continued up to 24 h when nuclear translocation was noticed . Neither treatment with Fe(2+) nor that with A beta(1-40) alone caused similar changes . Addition of either deferroxamine (DFe, 25 microm), catalase (0.4 mg/mL) or N-acetyl cysteine (0.5 mm) - the last two known as suppressants of oxidative stress - attenuated ERK activation and nuclear translocation . The mitogen-activated protein/ERK kinase (MEK) inhibitor U0126 blocked ERK and caspase 3 activation, suppressed ERK translocation and reduced the number of apoptotic cells, suggesting a central role for the ERK signalling cascade in A beta(1-40) plus Fe(2+) (A beta(1-40)/Fe(2+)) -induced apoptotic death . The full peptide A beta(1-42) was very effective at 0.5 microm while the inverse peptide A beta(40-1) at 5 microm was ineffective . The acetyl-amyloid-beta protein amide fragment 15-20 (V-pep) known to be an A beta aggregation inhibitor, prevented A beta(1-40)/Fe(2+)-induced toxicity . These findings indicate that metal ions chelators and antioxidants suppress the A beta(1-40)/Fe(2+)-induced oxidative stress cascade and may be beneficial in reducing the severity of Alzheimer's disease.

Biotechnol Prog, 2002 Jul-Aug, 18(4), 855 - 61
Insect cell culture medium supplementation with fetal bovine serum and bovine serum albumin: effects on baculovirus adsorption and infection kinetics; Maranga L et al.; The effects of insect cell culture medium supplementation with FBS were investigated . BSA was found to be the factor responsible for the increased baculovirus infection rate of FBS-supplemented cultures in a concentration-dependent form up to 25 g L(-)(1) . Lower rates of baculovirus binding to cells were observed with FBS- and BSA-supplemented cultures compared with infections carried out in serum-free media . Virus attachment constants were found to depend on medium matrix composition . An efficiency factor dependent on the medium matrix composition was introduced to account for these effects, and a mathematical model was developed to describe the virus-cell interactions . It was shown that BSA acts by minimizing the nonspecific virus binding leading to an increased cell infection rate . Cell specific Porcine parvovirusvirus-like particles (PPV-VLPs) expression was unaffected by medium supplementation pointing out that BSA and/or FBS affects mainly the initial phase of the baculovirus infection cycle . Implications for process definition are discussed.

Neuroscience, 2002, 113(3), 641 - 6
Production of beta-amyloid by primary human foetal mixed brain cell cultures and its modulation by exogenous soluble beta-amyloid; Hayes GM et al.; Previous studies on beta-amyloid production have been carried out using transfected cells and cell lines . We measured the 40 and 42 amino acid forms of beta-amyloid released into the culture medium by primary human foetal mixed brain cell aggregate culture over 3 months . In this model, neurones and supporting cells are maintained in serum-free defined medium . The secretion of significant amounts of beta-amyloid 40 and 42 was observed throughout culture for three separate cultures . Levels of beta-amyloid 40 and 42 closely followed the neuronal content of the cultures as estimated by cellular neurone-specific enolase . Addition of synthetic beta-amyloid 1-40 to the cultures for 1 week at 35 days in vitro resulted in a dose-related reduction in cellular neurone-specific enolase levels . Primary human aggregate brain cell cultures produced multimeric beta-amyloid, as determined by immunoassay . beta-Amyloid-treated cultures released diminishing amounts of multimeric beta-amyloid and contained increasing amounts of intracellular multimeric beta-amyloid with increasing exogenous beta-amyloid.These results suggest that release of multimeric beta-amyloid into the extracellular environment by human primary neurones can be affected by the presence of extracellular beta-amyloid . This has implications for Alzheimer's disease in that beta-amyloid released into the extracellular environment by dead/dying neurones could modulate beta-amyloid release by surrounding neurones, potentially causing amplification of toxicity . Moreover, intracellular beta-amyloid oligomer-dependent neurotoxicity may be a component of neurodegeneration in Alzheimer's disease, and other conditions with increased beta-amyloid synthesis, suggesting anti-amyloid therapies for Alzheimer's disease may have to target intracellular beta-amyloid.

Free Radic Res, 2002 May, 36(5), 593 - 9
Hydrogen peroxide generation in caco-2 cell culture medium by addition of phenolic compounds: effect of ascorbic acid; Roques SC et al.; Phenolic compounds have recently attracted special attention due to their beneficial health effects; their intestinal absorption and bioavailability need, therefore, to be investigated and Caco-2 cell culture model appeared as a promising tool . We have shown herein that the addition of a grape seed extract (GSE) to Dulbecco's modified Eagle's medium (DMEM) used for Caco-2 cell culture leads to a substantial loss of catechin, epicatechin and B2 and B3 dimers from GSE in the medium after 24 h and to a production of hydrogen peroxide (H2O2) . When 1420 microM ascorbic acid is added to the DMEM, such H2O2 production was prevented . This hydrogen peroxide generation substantially involves inorganic salts from the DMEM . We recommend that ascorbic acid be added to circumvent such a risk.

Biol Cell, 2002 May, 94(2), 65 - 76
Calpain and myogenesis: development of a convenient cell culture model; Dedieu S et al.; Previous studies have led us to hypothesize that m-calpain plays a pivotal role in myoblast fusion through its involvement in cell membrane and cytoskeleton component reorganization . To support this hypothesis, a convenient and simple myoblast culture model using frozen embryonic myoblasts was developed, which resolved a number of problems inherent to cell primary culture . Biological assays on cultured myoblasts using different media to define the characteristics of the fusion process were first conducted . Proteinase was detectable before the initiation of the fusion process and was closely correlated to the phenomenon of fusion under each culture condition studied . In addition, the study of calpastatin showed that the initiation of fusion does not require a decrease in the level of this endogenous inhibitor of calpains and also confirmed that calpastatin may be implicated in the determination of the end of fusion . On the other hand, analysis of the evolution of myogenic factors revealed that myogenins, MyoD and Myf5, increase very significantly during the formation of multinucleated myotubes . Moreover, the antisense technique against myogenin is capable of preventing the process of fusion by 50%, confirming the pivotal role of this factor in the early stages of differentiation . The possible role of myogenic regulator factors on m-calpain gene expression is discussed.

Invest Ophthalmol Vis Sci, 2002 Aug, 43(8), 2666 - 76
Comparison of the neuroprotective effects of adrenoceptor drugs in retinal cell culture and intact retina; Baptiste DC et al.; PURPOSE: The efficacy of beta1-adrenoceptor (AR)-selective (betaxolol and metoprolol) and nonselective (timolol) antagonists and the alpha2-AR agonist UK14,304 as retinal neuroprotectants was compared and contrasted in an in vitro glutamate excitotoxicity model . The ability of UK14,304, brimonidine, and betaxolol to alter glutamate-receptor-induced changes in intracellular calcium ({Ca2+}i) was also determined in isolated retinal neurons and retinal ganglion cells (RGCs) in an intact retina preparation . METHODS: Neuronal survival was measured in mixed retinal cell cultures treated for 24 hours with media containing 100 microM glutamate, with or without the addition of each of the drugs (1-1000 microM) . Effects of glutamate on glia were also investigated in a C6 glioma cell line . Glutamate-induced changes in {Ca2+}i with and without UK14,304, and its analogue brimonidine were assessed by calcium-imaging techniques in retinal neurons in culture . The effect of betaxolol on {Ca2+}i was investigated in RGCs in intact rabbit retina . RESULTS: In cell cultures, 10-1000 microM glutamate resulted in a dose-dependent loss of neurons, but not of glia . The absence of glutamate toxicity in glia was confirmed in C6 glioma cells . Betaxolol, but not timolol or metoprolol, significantly increased survival (from 52% of control in glutamate-only to 78% with 10 microM betaxolol) after excitotoxic insult . UK14,304 also increased survival (from 62% of control in glutamate only to 109% and 101% of control with 10 and 100 microM UK14,304, respectively) . This effect was blocked by the specific alpha2-antagonist, yohimbine . Both UK14,304 and brimonidine (10-100 microM) reduced glutamate-induced {Ca2+}i increases in retinal neurons in culture . The actions of the alpha2-agonists in reducing glutamate-induced {Ca2+}i increases were reduced by yohimbine (1 microM) . Betaxolol (100 microM) reduced N-methyl-D-aspartate (NMDA)-induced increases of {Ca2+}i in RGCs in intact retina . CONCLUSIONS: Betaxolol reduced glutamate excitotoxicity in retinal neurons in vitro through a mechanism independent of beta-AR interactions . UK14,304, acting through alpha2-ARs, was also neuroprotective in vitro . The neuroprotective actions of betaxolol and the alpha2-agonists on retinal neurons may be due, at least in part, to a direct reduction of glutamate receptor-mediated increases of {Ca2+}i.

Appl Environ Microbiol, 2002 Aug, 68(8), 3809 - 17
Comparison of in vitro cell culture and a mouse assay for measuring infectivity of Cryptosporidium parvum; Rochelle PA et al.; In vitro cell cultures were compared to neonatal mice for measuring the infectivity of five genotype 2 isolates of Cryptosporidium parvum . Oocyst doses were enumerated by flow cytometry and delivered to animals and cell monolayers by using standardized procedures . Each dose of oocysts was inoculated into up to nine replicates of 9 to 12 mice or 6 to 10 cell culture wells . Infections were detected by hematoxylin and eosin staining in CD-1 mice, by reverse transcriptase PCR in HCT-8 and Caco-2 cells, and by immunofluorescence microscopy in Madin-Darby canine kidney (MDCK) cells . Infectivity was expressed as a logistic transformation of the proportion of animals or cell culture wells that developed infection at each dose . In most instances, the slopes of the dose-response curves were not significantly different when we compared the infectivity models for each isolate . The 50% infective doses for the different isolates varied depending on the method of calculation but were in the range from 16 to 347 oocysts for CD-1 mice and in the ranges from 27 to 106, 31 to 629, and 13 to 18 oocysts for HCT-8, Caco-2, and MDCK cells, respectively . The average standard deviations for the percentages of infectivity for all replicates of all isolates were 13.9, 11.5, 13.2, and 10.7% for CD-1 mice, HCT-8 cells, Caco-2 cells, and MDCK cells, respectively, demonstrating that the levels of variability were similar in all assays . There was a good correlation between the average infectivity for HCT-8 cells and the results for CD-1 mice across all isolates for untreated oocysts (r = 0.85, n = 25) and for oocysts exposed to ozone and UV light (r = 0.89, n = 29) . This study demonstrated that in vitro cell culture was equivalent to the "gold standard," mouse infectivity, for measuring the infectivity of C . parvum and should therefore be considered a practical and accurate alternative for assessing oocyst infectivity and inactivation . However, the high levels of variability displayed by all assays indicated that infectivity and disinfection experiments should be limited to discerning relatively large differences.

Biochem Pharmacol, 2002 Aug 1, 64(3), 413 - 24
Cell culture model for acetaminophen-induced hepatocyte death in vivo; Pierce RH et al.; Overdose of the popular, and relatively safe, analgesic acetaminophen (N-acetyl-p-aminophenol, APAP, paracetamol) can produce a fatal centrilobular liver injury . APAP-induced cell death was investigated in a differentiated, transforming growth factor alpha (TGFalpha)-overexpressing, hepatocyte cell line and found to occur at concentrations, and over time frames, relevant to clinical overdose situations . Coordinated multiorganellar collapse was evident during APAP-induced cytotoxicity with widespread, yet selective, protein degradation events in vitro . Cellular proteasomal activity was inhibited with APAP treatment but not with the comparatively nonhepatotoxic APAP regioisomer, N-acetyl-m-aminophenol (AMAP) . Low concentrations of the proteasome-directed inhibitor MG132 (N-carbobenzoxyl-Leu-Leu-Leucinal) increased chromatin condensation and cellular stress responses preferentially in AMAP-treated cultures, suggesting a contribution of the proteasome in APAP- but not AMAP-mediated cell death . APAP-specific alterations to mitochondria were observed morphologically with evidence of mitochondrial proliferation in vitro . Biochemical alterations to cellular proteolytic events were also found in vivo, including APAP- or AMAP-mediated inhibition of caspase-3 processing . These results indicate that, although retaining some attributes of apoptosis, both APAP- and AMAP-mediated cell death have additional distinctive features consistent with longer term necrosis.

ASAIO J, 2002 Jul-Aug, 48(4), 346 - 9
Advances in the mechanisms of cell delivery to cardiovascular scaffolds: comparison of two rotating cell culture systems; Sutherland FW et al.; Having a reliable method of delivering cells to polymer scaffolds in vitro is fundamental to the development of tissue engineered structures . This paper compares the efficacy of two rotating systems for this purpose . Ten conduits, measuring 40 mm by 10 mm, were fabricated from polyglycolic acid mesh and poly-4-hydrobutyrate . Five conduits were placed in a rotating wall vessel (RWV, Synthecon Inc., Houston, TX), developed by the National Aeronautics and Space Administration (NASA); five conduits were also placed in rotating individual sealed tubes (RISTs) . Medium in the RWV was left unchanged for the duration of the experiment; medium in the RISTs required daily change . Samples of the discarded medium and samples from the RWV were analyzed for pH, pCO2, pO2, and lactate concentration . Constructs were assayed for DNA content as a surrogate for cell number . In the RWV, pH, pCO2, and pO2 remained stable, while the lactate concentration gradually increased . The measure of PO2 did not differ significantly between the RWV and the RISTs, but the pH was lower and the pCO2 and the lactate concentration measurements were higher in the RIST system at each time point (p = 0.001) . After 6 days (p = 0.001), the total DNA per conduit was 226+/-7 microg for the conduits seeded in the RISTs and 396+/-18 microg for the conduits in the RWV, suggesting that the RWV is superior to the RIST system for delivering cells to polymer scaffolds.

Biotechniques, 2002 Jul, 33(1), 212 - 4, 216, 218
Calcium-alginate gel bead cross-linked with gelatin as microcarrier for anchorage-dependent cell culture; Kwon YJ et al.; Valuable products obtainedfrom the cultivation of anchorage-dependent mammalian cells require large-scale processes to obtain commercially useful quantities . It is generally accepted that suspension culture is the ideal mode of operation . Because anchorage-dependent cells need surfaces to be able to attach and spread, the incorporation of microcarriers to suspension culture is indispensable . Since the dextran-based microcarrier wasfirst introduced, many different types of microcarriers have been developed and commercialized . In this study, alginate-based microcarriers were made in the following order: (i) calcium-alginate gel beads prepared by dropping a blend of sodium alginate and propylene glycol alginate (PGA) into calcium chloride solution, (ii) the PGA section of gel beads cross-linked with gelatin in alkaline solution (i.e., via the transacylation reaction between the ester group of PGA and amino group of gelatin), and (iii) gelatin membrane around the beads further cross-linked by glutaraldehyde . The glutaraldehyde-treated gelatintransacylated PGA/alginate microcarrier showed superior features in high stability under phosphate-containing solution, density close to that of culture medium, and transparency . Moreover, the Chinese hamster ovary CHO-KI and amphotropic retrovirus producer PA317 cells cultivated on the newly synthesized microcarriers exhibited similar growth kinetics of these two types of cell lines cultured on commercial polystyrene microcarriers . However, cell morphology was easily monitored on the transparent microcarriers made in this study.

Brain Res, 2002 Aug 9, 946(1), 130 - 8
Early biochemical and histological changes during hyperbaric or normobaric reoxygenation after in vitro ischaemia in primary corticoencephalic cell cultures of rats; Gunther A et al.; In a first series of experiments, the morphological changes of corticoencephalic cells by ischaemia were determined by staining with celestine blue-acid fuchsin in order to classify cells as intact, dark basophilic (supposedly reversibly injured) and preacidophilic or acidophilic (profoundly injured) . Hypoxia and glucose-deprivation (in vitro ischaemia) markedly decreased the number of intact cells and correspondingly increased the number of both reversibly and profoundly damaged cells . The morphological characteristics indicated a partial recovery during reoxygenation either in the absence or presence of glucose and irrespective of whether normobaric or hyperbaric oxygen was used . In a second series of experiments, nucleoside triphosphate and diphosphate levels were determined in corticoencephalic cultures by high-performance liquid chromatography . Hypoxia in combination with glucose-deficiency markedly decreased the ATP:ADP, GTP:GDP and UTP:UDP ratios . A still larger fall of these ratios was observed both after normobaric and hyperbaric reoxygenation . In contrast, both normobaric and hyperbaric reoxygenation in the presence of glucose led to an almost complete recovery near the control normoxic values . In conclusion, the histological changes were not adequately reflected by changes in the nucleoside triphosphate:diphosphate ratios and, in addition, hyperbaric oxygen had neither favourable nor unfavourable effects on the early morphological and functional restitution of ischaemically damaged cells under the conditions of the present study.

Brain Res, 2002 Aug 9, 946(1), 87 - 95
Platelet-activating factor increases prostaglandin E(2) release from astrocyte-enriched cortical cell cultures; Teather LA et al.; The phospholipid mediator platelet-activating factor (PAF) increased the release of prostaglandin E(2) (PGE(2)) from astrocyte-enriched cortical cell cultures in a concentration- and time-dependent manner . The nonhydrolyzable PAF analog methylcarbamyl-PAF (mc-PAF), the PAF intermediate lyso-PAF, and arachidonic acid (AA) also produced this effect . In contrast, phosphatidlycholine (PC) and lyso-PC, lipids that are structurally similar to PAF and lyso-PAF, had no effect on PGE(2) production, suggesting that PAF-induced PGE(2) release is not the consequence of nonspecific phospholipid-induced membrane perturbation . Antagonism of intracellular PAF binding sites completely abolished the ability of mc-PAF and lyso-PAF to mobilize PGE(2,) and attenuated the AA effect . Antagonism of the G-protein-coupled PAF receptor in plasma membranes had no significant effect on mc-PAF, lyso-PAF or AA-induced PGE(2) release . Based on the present findings, we hypothesize that intracellular PAF is a physiologic stimulus of PGE(2) production in astrocytes.

Eur J Pharm Sci, 2002 Aug, 16(3), 151 - 7
Nicotine permeability across the buccal TR146 cell culture model and porcine buccal mucosa in vitro: effect of pH and concentration; Nielsen HM et al.; The present study was conducted to investigate and compare the effect of pH and drug concentration on nicotine permeability across the TR146 cell culture model and porcine buccal mucosa in vitro . As a further characterization of the TR146 cell culture model, it was explored whether the results were comparable for bi-directional and uni-directional transport in the presence of a transmembrane pH gradient . Nicotine concentrations between 10(-5) and 10(-2) M were applied to the apical side of the TR146 cell culture model or the mucosal side of porcine buccal mucosa . Buffers with pH values of 5.5, 7.4 and 8.1 were used to obtain different fractions of non- and mono-ionized nicotine . The apparent permeability (P(app)) of nicotine across both models increased significantly with increasing pH, and the P(app) values obtained with the two models could be correlated in a linear manner . With increasing concentrations of nicotine, the P(app) values decreased, which can partly be explained by an effect on the paracellular pathway . Similar results were also obtained when using the models for bi-directional as well as for uni-directional studies . The TR146 cell culture model may be used as model for buccal epithelium in studies with ionized drugs and a transmembrane pH gradient.

Ideggyogy Sz, 2002 May 20, 55(5-6), 173 - 9
Identification of gliomas by morphological and immunocytochemical analysis in cell cultures; Fazekas I et al.; INTRODUCTION: The morphology and immunocytochemical properties of 250 different monolayer cultures derived from various human brain tumor specimens were investigated on purpose to support and complement the neuropathological diagnosis . In this study analyses of 124 glioma cases are presented . METHODS: The tumor samples were mechanically dissociated and seeded on glass coverslips . After the formation of the monolayer cultures were fixed and stained by May-Grunwald-Giemsa method for the morphological examination . Semi-quantitative immunocytochemical labeling included several different types of mono- and polyclonal primary antibodies using avidin-biotin visualization system . In nine cases of the glioblastomas the sufficient proliferation made possible to establish cell lines from the primary cultures . RESULTS: The glial origin of the tumors was identified in 124 cases based upon the presence of glial fibrillary acidic protein . A negative correlation between the intensity of glial fibrillary acidic protein immunostaining and the grade of tumor malignancy was found . During long-term cultivation of the higher grade gliomas the incidence and intensity of glial fibrillary acidic protein labeled cells was decreasing . Both the vimentin and the neuron specific enolase labeling were in general stronger than the glial fibrillary acidic protein and almost all the cells were stained . The incidence of Ki-67 positive cells increased with the grade of malignancy . Concerning the tumor classification our immunocytochemical results correlated with the routine histopathological examination . CONCLUSIONS: On the basis of these results we conclude that monolayer cultures obtained from tumor specimens can support and complement the correct diagnosis of the various human brain tumors.

J Math Biol, 2001 Jul, 43(1), 22 - 36
An alternative stochastic model of generation of oligodendrocytes in cell culture; Boucher K et al.; According to our previous model, oligodendrocyte--type 2 (O-2A) astrocyte progenitor cells become competent for differentiation in vitro after they complete a certain number of critical mitotic cycles . After attaining the competency to differentiate, progenitor cells divide with fixed probability p in subsequent cycles . The number of critical cycles is random; analysis of data suggests that it varies from zero to two . The present paper presents an alternative model in which there are no critical cycles, and the probability that a progenitor cell will divide again decreases gradually to a plateau value as the number of completed mitotic cycles increases . In particular all progenitor cells have the ability to differentiate from the time of plating . The Kiefer-Wolfowitz procedure is used to fit the new model to experimental data on the clonal growth of purified O-2A progenitor cells obtained from the optic nerves of 7 day old rats . The new model is shown to fit the experimental data well, indicating that it is not possible to determine whether critical cycles exist on the basis of these experimental data . In contrast to the fit of the previous model, which suggested that the addition of thyroid hormone increased the limiting probability of differentiation as the number of mitotic cycles increases, the fit of the new model suggests that the addition of thyroid hormone has almost no effect on the limiting probability of differentiation.

Am J Vet Res, 2002 Jul, 63(7), 963 - 8
Validation of a cell culture bioassay for detection of petroleum exposure in mink (Mustela vison) as a model for detection in sea otters (Enhydra lutris); Ziccardi MH et al.; OBJECTIVE: To validate a luciferase bioassay, which is based on a recombinant mouse hepatoma cell line, for the detection of exposure to petroleum in mustelid species . ANIMALS: 122 American mink (Mustela vison) and 15 sea otters (Enhydra lutris) . PROCEDURES: Mink were exposed to Bunker C fuel oil or Alaska North Slope crude oil externally as a single exposure or internally via low dose concentrations in their ration for 6 months . Serum samples were analyzed for cytochrome P450 1A1 induction by quantification of luciferase activity in the bioassay . Mink liver specimens were also evaluated for cytochrome P450 1A1 induction by quantification of ethoxyresorufin-o-deethylase activity . Serum collected from exposed and unexposed sea otters was also analyzed using the luciferase bioassay . RESULTS: Serum samples from mink externally exposed to petroleum had significantly increased luciferase activities at 1 week after exposure . Serum samples taken at later time points or from mink exposed to either product in the ration did not cause significant luciferase induction . Samples from otters exposed to petroleum had significantly higher luciferase induction as compared with samples from otters not exposed to petroleum at 2 and 8 years after the spill . Cytochrome P450 1A1 activity in liver specimens collected from mink that were internally exposed through diet was significantly increased at the conclusion of our study . CONCLUSION AND CLINICAL RELEVANCE: The luciferase bioassay is a sensitive and specific method for determining recent exposure to petroleum in mink . The lack of luciferase activity in serum samples collected from mink greater than 1 week after experimental exposure was likely attributable to lower overall petroleum exposure in our trial, compared with natural exposures.

Mol Cell Proteomics, 2002 May, 1(5), 376 - 86
Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics; Ong SE et al.; Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures . Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins . Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3) . We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate . Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied . Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3 . We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation . Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2 . SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system.

Infect Immun, 2002 Aug, 70(8), 4747 - 9
Phase variation analysis of Coxiella burnetii during serial passage in cell culture by use of monoclonal antibodies; Hotta A et al.; Antigenic changes in Coxiella burnetii Nine Mile strain phase I during serial passages in cell culture were analyzed with three groups of monoclonal antibodies (MAbs) against lipopolysaccharide . The MAbs of group 1 did not react with organisms that were passaged over five times, and the MAbs of group 2 did not react with organisms that were passaged over eight times . The MAbs of group 3 reacted with organisms passaged up to 15 times but did not react with phase II cells . These results suggest that C . burnetii could be differentiated into four phase states during phase variation.

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