Cell cultures

Cell Immunol, 1998 Nov 25, 190(1), 23 - 32
Human CD34(+) thymocyte maturation: pre-T and NK cell differentiation on neonatal thymic stromal cell culture; Sarun S et al.; The development of a simple assay for studying human T and NK lymphopoiesis from CD34 progenitors is of great interest . T and NK cells arise from a common CD34(+) immature precursor . While T cell maturation is dependent on interactions and cytokines provided by thymic stromal cells . NK maturation does not require the thymic microenvironment and primarily takes place extrathymically . In addition to models using a mouse thymic microenvironment, in vitro assays based on coculture on human fetal thymic stroma have been described . As an alternative source of fetal thymic tissue we studied the capacity of neonatal thymic epithelial cell enriched stroma to support T and NK cell differentiation . While in the fetal-based assays on NK cells were observed under the conditions used for T cell differentiation, neonatal stroma can generate CD3/TcR+ as well as CD56(+)3(-)8(+)NKR-P1(+)NK cells from both CD34(+)1(-) and CD34(+)1(+) thymocyte precursors . However, following acquisition of CD3/TcR, T-lineage cells disappeared from the culture after 2-3 weeks as a consequence of the outgrowth of the NK cells . These CD56(+)3(-) NK cells appeared to be functionally immature as they required incubation with IL2 or IL15 to lyse K562 target cells . Our data offer a simple and reliable assay for studying the reconstitution potential of T and NK cell progenitors on a monolayer of thymic epithelial cells .

Neuroendocrinology, 1998 Nov, 68(5), 345 - 54
Dexamethasone rapidly regulates TRH mRNA levels in hypothalamic cell cultures: interaction with the cAMP pathway; Perez-Martinez L et al.; The biosynthesis of thyrotropin-releasing hormone (TRH) in the hypothalamic paraventricular nucleus (PVN) is subject to neural and hormonal regulations . To identify some of the potential effectors of this modulation, we incubated hypothalamic dispersed cells with dexamethasone for short periods of time (1-3 h) and studied the interaction of this hormone with protein kinase C (PKC) and PKA signaling pathways . TRH mRNA relative changes were determined by the RT-PCR technique . One hour incubation with 10(-10)-10(-4) M dexamethasone produced a concentration-dependent biphasic effect: an inhibition was observed on TRH mRNA levels at 10(-10) M, an increase above control at 10(-8)-10(-6) M and a reduction at higher concentrations (10(-5)- 10(-4) M) . The stimulatory effect of 10(-8) M dexamethasone on TRH mRNA was essentially independent of new protein synthesis, as evidenced by cycloheximide pretreatment . Changes in TRH mRNA levels were reflected by enhanced TRH cell content . Incubation with a cAMP analogue (8-bromo-cAMP, 8Br-cAMP) or with a PKC activator (12-O-tetradecanoylphorbol-13-acetate, TPA) increased TRH mRNA levels after 1 and 2 h, respectively . An increase in TRH mRNA expression was observed by in situ hybridization of dexamethasone or 8Br-cAMP-treated cells . The interaction of dexamethasone, PKA and PKC signaling pathways was studied by combined treatment . The stimulatory effect of 10(-7) M TPA on TRH mRNA levels was additive to that of dexamethasone; in contrast, coincubation with 10(-3) M 8-Br-cAMP and dexamethasone diminished the stimulatory effect of both drugs . An inhibition was observed when the cAMP analogue was coincubated with TPA or TPA and dexamethasone . These results demonstrate that dexamethasone can rapidly regulate TRH biosynthesis and suggest a cross talk between cAMP, glucocorticoid receptors and PKC transducing pathways.

Wound Repair Regen, 1998 Jul-Aug, 6(4), 403 - 12
An ependymal cell culture system for the study of spinal cord regeneration; Chernoff EA et al.; Successful regeneration of lesioned adult spinal cord in urodele (caudate) amphibians requires the action of injury-responsive ependymal cells (ependymoglia) . The epithelial-to-mesenchymal transformation of ependymal cells following transection of the salamander spinal cord and the subsequent reformation of an epithelial tube have been described previously . A complete tissue culture model system has now been devised to study mesenchymal ependymal cells, epithelial ependymal cells, and ependymal/neuronal interactions in vitro . Here, we review critical aspects of substrate and growth factor environments required to produce mesenchymal ependymal cells in culture and present the first culture system for epithelial salamander ependymal cells and central nervous system neurons suitable for cell-cell interaction studies . Critical to ependymal epithelialization in culture is the removal of epidermal growth factor and addition of thrombin . Epithelialization occurs on tissue culture plastic as well as on permeable culture substrates . This culture system can now be used to determine the initial trigger for the ependymal response . A preliminary examination of ependymal/neuronal interactions shows that coculture of mesenchymal ependymal cells and central nervous system neurons prolongs survival of the neurons.

Diagn Microbiol Infect Dis, 1998 Oct, 32(2), 111 - 3
Cytospin-enhanced direct immunofluorescence assay versus cell culture for detection of herpes simplex virus in clinical specimens; Sanders C et al.; The reliability of a cytospin-enhanced direct immunofluorescence assay (DFA), using Syva Micro Trak monoclonal antibodies, for detection of herpes simplex virus (HSV) was evaluated by comparing results with those of conventional cell culture . Of 250 clinical specimens, 64 were positive for HSV by cell culture, and 22 were positive by cytospin-enhanced DFA, two of which were negative by culture . The sensitivity, specificity, and positive and negative predictive values for cytospin-enhanced DFA were 31.25, 98.9, 90.9, and 80.7%, respectively . Cytospin-enhanced DFA is not an acceptable alternative to cell culture for detection of HSV.

Matrix Biol, 1998 Oct, 17(5), 361 - 9
Proteoglycan sulfation in cartilage and cell cultures from patients with sulfate transporter chondrodysplasias: relationship to clinical severity and indications on the role of intracellular sulfate production; Rossi A et al.; Mutations in the diastrophic dysplasia sulfate transporter (DTDST) gene have been associated with a family of chondrodysplasias that includes diastrophic dysplasia (DTD), atelosteogenesis type 2 (AO2) and the lethal condition achondrogenesis type 1B (ACG1B) . There is a correlation between the nature of the mutations and the clinical phenotype, but our understanding of the pathophysiology of the disorder, which involves defective sulfation of cartilage proteoglycans, is far from complete . To evaluate the degree of proteoglycan undersulfation in vivo, we have extracted chondroitin sulfate proteoglycans from cartilage of twelve patients with sulfate transporter chondrodysplasias and analyzed their disaccharide composition by HPLC after digestion with chondroitinase ABC . The amount of non-sulfated disaccharide was elevated in patients' samples (controls, 5.5%+/-2.8 (n=10); patients, 11% to 77%), the highest amount being present in ACG1B patients, indicating that undersulfation of chondroitin sulfate proteoglycans occurs in cartilage in vivo and is correlated with the clinical severity . To investigate further the biochemical mechanisms responsible for the translation of genotype to phenotype, we have studied fibroblast cultures of patients with DTD, AO2 and ACG1B, and controls, by double-labelling with {35S}sulfate and {3H}glucosamine . The incorporation of extracellular sulfate, estimated by the 35S/3H ratio in proteoglycans, was reduced in all patients' cells, with ACG1B cells showing the lowest values . However, disaccharide analysis of chondroitin sulfate proteoglycans showed that these were normally sul fated or only moderately undersulfated; marked undersulfation was observed only after addition of the artificial glycosaminoglycan-chain initiator, beta-D-xyloside, to the culture medium . These results suggest that, while utilization of extracellular sulfate is impaired, fibroblasts can replenish their intracellular sulfate pool by oxidizing sulfur-containing compounds (such as cysteine) and thus partially rescue PG sulfation under basal conditions . This rescue pathway becomes insufficient when GAG synthesis rate is stimulated by beta-D-xyloside . These findings may explain why phenotypic consequences of DTDST mutations are restricted to cartilage, a tissue with high GAG synthesis rate and poor vascular supply, and imply that pharmacological therapy aimed at restoring the intracellular sulfate pool might improve PG sulfation in DTD and related disorders.

Curr Opin Biotechnol, 1998 Oct, 9(5), 518 - 21
Expression of heterologous proteins in stable insect cell culture; Pfeifer TA; Stable transformed insect cell lines have been used for producing many highly processed heterologous proteins . Current research has focused on development of new expression and selection systems, and enhancement of vector stability . Defining the variation of modification and processing capabilities between cell lines will further enhance complex protein production from insect cells.

Am J Physiol, 1998 Nov, 275(5 Pt 2), H1673 - 81
Myocardial aerobic metabolism is impaired in a cell culture model of cyanotic heart disease; Merante F et al.; A human pediatric cardiomyocyte cell culture model of chronic cyanosis was used to assess the effects of low oxygen tension on mitochondrial enzyme activity to address the postoperative increase in lactate and decreased ATP in the myocardium and the high incidence of low-output failure with restoration of normal oxygen tension, after technically successful corrective cardiac surgery . Chronically hypoxic cells (PO2 = 40 mmHg for 7 days) exhibited significantly reduced activities for pyruvate dehydrogenase, cytochrome-c oxidase, succinate cytochrome c reductase, succinate dehydrogenase, and citrate synthase . The activity of NADH-cytochrome c reductase was unaffected . Lactate production and the lactate-to-pyruvate ratio were significantly greater in hypoxic cardiomyocytes . Western and Northern analysis demonstrated a decrease in the levels of various mRNA and corresponding polypeptides in hypoxic cells . Thus hypoxia influences mitochondrial metabolism through acute and chronic adaptive mechanisms, reflecting allosteric (posttranscriptional) and transcriptional modulation . Transcriptional downregulation of key mitochondrial enzyme systems can explain the insufficient myocardial aerobic metabolism and low-output failure in children with cyanotic heart disease after cardiac surgery.

Dev Genes Evol, 1998 Dec, 208(10), 595 - 602
Efficiency of cell culture derivation from blastula embryos and of chimera formation in the medaka (Oryzias latipes) depends on donor genotype and passage number; Hong Y et al.; Embryonic stem (ES) cells from early vertebrate embryos only rarely retain their full developmental potential under in vitro culture conditions, but undergo differentiation and lose their ability for chimeric embryogenesis . This is reflected by the fact that the ES cell technology to date could only be fully developed in mice . In the fish Oryzias latipes, the medaka, one ES-like cell line, MES1, has been established which gives rise to a high frequency of somatic chimeras but a low degree of chimerism . Here we have tested the effect of donor genotype and cultivation time on the efficiency of cell culture derivation and on chimera formation . The HB12A, HB32C and HNI strains of medaka most efficiently and reproducibly give rise to blastula-derived cell cultures that produce pigmented chimeras in albino hosts . Seven chimeras grew to male or female adults with normal fertility, although none of them showed obvious donor germline contribution . During prolonged in vitro propagation the frequency of chimeras and the degree of chimerism dropped to a value retained in the long-term cultured MES1 cells . Obviously, genetic factors in host/donor compatibility and physiological changes during prolonged in vitro culture may compromise, but do not abolish, the developmental potential of medaka ES-like cells . Thus, elucidation of conditions that will expand the developmental potential of medaka blastula cell cultures should lead to a further improvement towards establishment of the ES cell technology in medaka.

Mol Hum Reprod, 1998 Oct, 4(10), 985 - 9
Telomerase activity is found in the epithelial cells but not in the stromal cells in human endometrial cell culture; Yokoyama Y et al.; Telomerase activity is associated with the proliferative activity of cells . In the endometrium, telomerase activity is higher in the proliferative phase than in the secretory phase of the menstrual cycle, suggesting that telomerase activity may occur primarily in the glandular epithelial cells . To test this, a dissociated cell culture of the endometrium was performed, and the telomerase activity in each cell fraction was analysed . Telomerase activity was found in all 10 endometrial tissues of the proliferative phase of the menstrual cycle . Both the fragments of epithelial glands and single cells, which were prepared by enzymatic dissociation, showed telomerase activity . In the 7 day cell culture, it was found in nine out of 10 epithelial cell enriched fractions, but in none of the stromal cell enriched fractions . Flow cytometric analysis showed that the epithelial enriched fraction was contaminated with a predominant number of stromal cells, while the stromal cell enriched fraction was comprised mostly of stromal cells with apparent proliferative activity . Our results suggest that telomerase activity of the endometrium occurs primarily in the epithelial cells in the endometrium and that the stromal cells do not express telomerase activity regardless of their potent proliferative activity.

J Cereb Blood Flow Metab, 1998 Nov, 18(11), 1270 - 81
Regulation of a blood-brain barrier-specific enzyme expressed by cerebral pericytes (pericytic aminopeptidase N/pAPN) under cell culture conditions; Ramsauer M et al.; In this study we show that the aminopeptidase N of cerebral pericytes (pAPN) associated with the blood-brain barrier (BBB) is downregulated in pericytic cell cultures . This observation is in accordance with previous data describing comparable in vitro effects for BBB-specific enzymes of endothelial or pericytic origin, such as gamma-glutamyl transpeptidase or alkaline phosphatase . By polymerase chain reaction and in situ hybridization we were able to determine that the down-regulation of pAPN occurs at the posttranscriptional level . The mRNA of pAPN was found to be constitutively expressed even when the protein is no longer detectable . Culturing the pericytes in an endothelial cell-conditioned medium allowed pAPN to be reexpressed . However, the reexpression effect depended largely on the culturing conditions of the pericytes . Although purified pericytes deprived of endothelial cells did not reveal a reexpression effect, pericytes that were kept in contact with endothelial cells were able to acquire a pAPN-positive phenotype, indicating that endothelial cells constitute an essential requirement for the in vitro reexpression of pAPN . Astrocytes, however, were insufficient in exerting any reexpression effect.

Int J Cancer, 1998 Nov 23, 78(5), 636 - 41
Establishment of tumor cell culture (ILA) derived from hamster pancreatic islets treated with BOP; Toshkov I et al.; ILA cells were established from tumors induced by the pancreatic carcinogen N-nitrosobis(2-oxopropyl)amine (BOP) in hamster islets . The proliferation, morphology, karyotype, immunoreactivity with certain antibodies and growth factor secretion of these tumor cells were compared with the same parameters in tumor cells induced by BOP in hamster ductal cells (TAKA-1-BOP) established in a previous study . Minor differences were found in the morphology and ultrastructure of the 2 cell lines . Contrary to TAKA-1-BOP cells, ILA cells did not express cytokeratins 8.13, 13 or 18 but did express DU-PAN-2 and TAG-72, 2 known human pancreatic cancer-associated antigens . No endocrine cell markers were expressed . A significant difference also was found in the chromosomal pattern in that there were more abnormalities and marker chromosomes in ILA cells than in TAKA-1-BOP cells and the Y or X chromosomes were missing in ILA cells . ILA cells produced TGF-alpha, IGF-I, bombesin and gastrin and expressed specific binding sites for hEGF . TGF-alpha secretion from ILA cells was much greater than that from TAKA-1-BOP cells . Our results indicate that pancreatic cancer cells grown in vitro are not a single clone . We conclude that there are some genetic and biological differences between tumors arising from pancreatic duct and islets and that pancreatic ductal adenocarcinomas originating from islets have a profound malignant potential.

Mutat Res, 1998 Nov 9, 419(1-3), 91 - 105
DNA adduct formation in mammalian cell cultures by polycyclic aromatic hydrocarbons (PAH) and nitro-PAH in coke oven emission extract; Topinka J et al.; Mammalian cells in culture were used to study the genotoxic potential of coke oven emissions constituting a complex mixture of chemicals . For this purpose, particle extracts and some polycyclic aromatic and nitroaromatic hydrocarbons (PAH and nitro-PAH) occurring in these mixtures were assayed for DNA adduct formation using the -postlabeling technique . In primary cultures of rat hepatocytes, benzo{a}pyrene (B{a}P), benz{a}anthracene (B{a}A) and benzo{b}fluoranthene (B{k}F) caused DNA adduct levels in the range of 1 adduct/108 nucleotides . 4-Nitropyrene (4-NP), 6-nitrochrysene (6-NC), 3-nitrofluoranthene (3-NF) caused DNA adduct levels that were by one to two orders of magnitude higher . The crude particle extract and its fractions differing in acidity and polarity induced the formation of DNA reactive material within diagonal radioactive zones (DRZ) on the autoradiograms . On a weight base, the neutral aromatic fraction contributed by more than 80% to the total adduct level in hepatocytes . To examine whether the PAH- and nitro-PAH-DNA derived adducts can be further differentiated, hepatocyte cultures were preincubated with 2,3,7,8-tetrachloro-p-dioxin (TCDD) to induce the activity of cytochrome P450 1A1 . TCDD pretreatment strongly increased the levels of PAH-DNA adducts, whereas, the levels of nitro-PAH adducts were markedly decreased . NCI-H322 cells, a human lung tumor cell line derived from Clara cells, exhibited PAH-DNA adduct levels between 10 and 100, and nitro-PAH-DNA adducts at levels between 0.2 to about 30 adducts per 108 nucleotides, respectively . In contrast to hepatocytes, incubations with extractable organic matter (EOM) and the neutral aromatic EOM fraction displayed several distinct spots in the chromatograms of NCI-H322 cells . The major spot was assigned by cochromatography to be identical with the major DNA adduct formed by incubation with B{a}P alone . In V79NH cells, a Chinese hamster lung cell line expressing nitro-PAH activating enzymes, but virtually no cytochrome P450 activity, PAH-derived DNA adducts were not detectable . Nitro-PAH-derived DNA adducts, however, were formed at levels between 10 and 300 adducts/108 nucleotides . The slightly and the moderately polar EOM fraction caused the formation of distinct adduct spots suggesting the occurrence of nitro-PAH in these fractions . GC/MS analyses revealed the presence of twelve PAH in the aromatic fraction, at a total amount of about 10% (w/w), and of four nitro-PAH in the slightly polar and the acidic fraction amounting to about 0.2% (w/w) . In conclusion, our results indicate that PAH and nitro-PAH contribute to the genotoxicity of coke oven emissions . Using DNA adduct analysis in rat hepatocytes (+/-pretreatment with TCDD) and in NCI-H322 and in V79NH cells offers a promising approach to determine the genotoxic activity of PAH and nitro-PAH in any complex environmental samples .

Stroke, 1998 Nov, 29(11), 2426 - 34
Endothelial cell culture from human cerebral cavernous malformations; Baev NI et al.; BACKGROUND AND PURPOSE: The cerebral cavernous malformation (CCM) is a common and frequently unrecognized cause of stroke and epilepsy . It consists of blood-filled caverns lined by endothelial cells (EC) and devoid of mature vessel wall structure . Cultured EC obtained from CCM may express phenotypic and genotypic alterations contributing to CCM pathogenesis . We report the first successful isolation and growth in vitro of primary EC lines from human CCM lesions . METHODS: We developed a procedure for the isolation and growth of EC from human CCM, confirmed their EC origin by a panel of molecular markers, and determined by immunocytochemistry the basic expression patterns of 6 transmembrane receptor protein kinases comparing brain, skin, and CCM primary EC lines grown identically . RESULTS: Several CCM EC lines were established from 2 patients after we treated the excised specimens with 0.3% trypsin/1% EDTA, selective cloning, and growth in MCDB107 containing 0.3 g/L heparin, 0.15 g/L endothelial cell growth supplement, and 15% FBS . The CCM EC showed contact inhibition and a rounded cobblestone appearance . The cells expressed CD31, CD105, von Willebrand factor, and binding sites for Ulex europaeus agglutinin, type 1 and acetylated LDL . They showed low levels of Flt-1, Flk-1, transforming growth factor (TGF)-beta RI, and TGF-beta RII expression but stained strongly with antibodies against Tie-1 and Tie-2 . CONCLUSIONS: Cultured CCM EC retained their endothelial phenotype . Brain, skin, and CCM EC lines did not significantly differ in their staining patterns with antibodies against Flt-1, Flk-1, TGF-beta RI, TGF-beta RII, Tie-1, and Tie-2 . These cell lines will assist in defining molecular phenotype and genotype alterations in association with CCM.

Microbiol Immunol, 1998, 42(9), 591 - 8
Lipopolysaccharide (LPS) stimulates the production of tumor necrosis factor (TNF)-alpha and expression of inducible nitric oxide synthase (iNOS) by osteoclasts (OCL) in murine bone marrow cell culture; Kikkawa I et al.; Osteoclasts (OCL) resorb bone . They are essential for the development of normal bones and the repair of impaired bones . The function of OCL is presumed to be supported by cytokines and other biological mediators, including tumor necrosis factor (TNF)-alpha and nitric oxide (NO) . Bacterial lipopolysaccharide (LPS) is a potent inducer of TNF-alpha and inducible nitric oxide synthase (iNOS), which is the specific enzyme for synthesizing NO from L-arginine . To obtain direct evidence on LPS-induced TNF-alpha production and iNOS expression by OCL, OCL-enriched cultures were prepared by 7-day cocultures of bone marrow cells of adult BALB/c mice and osteoblastic cells (OBs) derived from calvaria of newborn BALB/c mice, and the generation of TNF-alpha and iNOS in OCL stimulated with LPS was examined immunocytochemically . When the cultured cells were stimulated with 100 ng/ml of LPS, OCL clearly showed TNF-alpha and iNOS expression . Without LPS-stimulation, no expression was observed . TNF activity in the culture supernatants of the OCL-enriched cultures in the presence of LPS was also detected by cytotoxic assay that used TNF-sensitive L929 cells . The dentin resorption activity of OCL was estimated by area and number of pits formed on dentin slices, which were covered by the OCL fraction and cultured in the presence or absence of LPS, sodium nitroprusside (SNP; a NO generating compound), N(G)-monomethyl L-arginine acetate (L-NMMA; a competitive inhibitor of NO synthase (NOS)), or LPS plus L-NMMA . Pit formation was obviously inhibited in the presence of SNP and slightly inhibited in the presence of L-NMMA, but it was not affected in the presence of LPS or LPS plus L-NMMA . These findings indicate that OCL produces TNF and expresses iNOS in response to LPS, but the LPS-activation of OCL scarcely affects pit formation by them.

J Control Release, 1998 Dec 4, 56(1-3), 249 - 58
Biocompatibility testing of ABA triblock copolymers consisting of poly(L-lactic-co-glycolic acid) A blocks attached to a central poly(ethylene oxide) B block under in vitro conditions using different L929 mouse fibroblasts cell culture models; Zange R et al.; The biocompatibility of ABA triblock copolymers consisting of poly(L-lactide-co-glycolide) A blocks attached to a central poly(ethylene oxide), (PEO), B block was investigated under in vitro conditions . The ABA triblock copolymer was compared to commercially available Poly(D,L-lactide-co-glycolide) (PLGA) and reference materials in different L929 cell culture models according to the procedure recommended by the International Standard Organization (ISO) . Different preparation methods: namely extraction, indirect contact and direct contact with polymer samples were compared . The extraction method seems to be the most sensitive assay, allowing estimates of IC50 values . ABA and PLGA polymers showed excellent compatibility with L929 fibroblasts with all preparation techniques used.The influence of polymer composition and molecular weight on degradation rate as well as in vitro biocompatibility was then investigated . Changes in pH and osmolarity as well as lactic acid content of the extracts were determined and compared to in vitro degradation data of polymer films in phosphate buffered saline at 37 degreesC evaluating molecular weight (GPC) and massloss (gravimetry) . An acceleration of the degradation rate of the ABA triblock copolymers with increasing PEO content was observed . The in vitro cytotoxicity studies demonstrated that the three ABA polymers were well tolerated by fibroblasts in cell culture . One ABA polymer batch ABA2 showed unusual in vitro cytotoxicity in L929 fibroblasts, possibly related to the molecular weight of the PEO used for this particular batch or residual glycolic acid.Cell culture models for biocompatibility testing of polymers according to ISO are useful as screening models in characterizing biodegradable polymers, but they cannot replace animal testing . The extraction method in combination with the MTT assay allows quantitative ranking of cytotoxic properties with high sensitivity.

Vaccine, 1998 Dec, 16(20), 2031 - 8
Genetic stability of oral polio vaccine prepared on primary monkey kidney cells or Vero cells--effects of passage in cell culture and the human gastrointestinal tract; Chezzi C et al.; The genetic stability of the three Sabin oral poliovaccine (OPV) strains produced on either primary monkey kidney (VK) or Vero cell substrates was compared in vivo and in vitro by measuring the rate at which the bases most strongly associated with attenuation and reversion to neurovirulence (positions 480, 481, and 472 in the 5' non-coding region of Sabin 1, 2 and 3 respectively, and 2034 in VP3 of Sabin 3) reverted during passage of the vaccine strains in the gastrointestinal tract of primary vaccinees and in cell culture . For the in vivo study, the poliovirus excretion patterns of 21 infants receiving OPV produced on either VK or Vero cells were followed for 21 days . No significant differences in either the frequency of excretion or the rate of reversion were observed between the two vaccine groups . The rate of accumulation of revertants during passage in vitro was compared for the three Sabin strains passaged 10 times in either VK or Vero cells . For types 1 and 3, revertants accumulated faster upon passage through VK cells compared with passage through Vero cells . Type 2 appeared to be stable as no revertants were detected in either cell type . Results of this study suggest that the use of Vero as opposed to VK cells as substrate for the manufacture of OPV does not negatively influence the genetic stability of the three Sabin OPV strains in vivo or in vitro.

J Endocrinol, 1998 Oct, 159(1), 103 - 10
Regulation of gonadotrophin secretion by inhibin, testosterone and gonadotrophin-releasing hormone in pituitary cell cultures of male monkeys; Fingscheidt U et al.; The effects of bovine inhibin, testosterone and GnRH on gonadotrophin secretion by primate pituitary cells were characterized in vitro using pituitaries from six male rhesus monkeys and one male cynomolgus monkey . The effect of inhibin on basal secretion of FSH and LH was investigated . Dose-response curves in monkeys and rats were compared . GnRH dose-response curves in the presence and absence of testosterone were also examined in monkeys . In monkey pituitary cells, testosterone at a concentration of 10(-7) M had no effect on LH or FSH secretion . Inhibin suppressed FSH secretion to 50.8% of that of controls with no effect on LH . In rats, FSH secretion was suppressed to 45.0% of that of controls with a median effective dose (ED50, 95% range) of 1.298 (1.064-1.584) U/ml, compared with 1.024 (0.7204-1.455) U/ml in monkeys . In monkey pituitary cells, LH release was stimulated 9.9-fold and FSH 3.3-fold by GnRH . Testosterone had no effect on basal or GnRH-stimulated gonadotrophin release . These results support the view that the pituitary is not the target organ for the negative feedback action of testosterone in the male . In vitro, inhibin is the major regulator of FSH secretion at the pituitary level.

Brain Res Mol Brain Res, 1998 Oct 30, 61(1-2), 121 - 35
Novel synthesis and release of GABA in cerebellar granule cell cultures after infection with defective herpes simplex virus vectors expressing glutamic acid decarboxylase; New KC et al.; The inhibitory amino acid neurotransmitter gamma-aminobutyric acid (GABA) is synthesized from glutamate in a single step by the enzyme glutamatic acid decarboxylase (GAD) . We sought to determine whether viral vectors containing GAD cDNA could be used to enhance synthesis and stimulation-evoked release of GABA in cultures of CNS neurons . For this purpose, we generated double-cassette defective herpes simplex virus (HSV) vectors that expressed one of the two GAD isoforms (GAD65 or GAD67), and Escherichia coli LacZ . Infection of cerebellar granule cell (CGC) cultures with vectors containing GAD cDNA resulted in a significant increase in isoform-specific expression of GAD, synthesis of GABA, and stimulation-evoked GABA release . GAD65 and GAD67 vector-infected neurons exhibited a comparable profile of GABA levels, synthesis and release, as well as GAD protein distribution . In CGCs cultured for 6 days in vitro (DIV), GABA synthesized after vector-derived GAD expression was released by treatment with glutamate or veratridine, but only in a Ca2+-independent fashion . In more mature (10 DIV) cultures, both Ca2+-dependent, K+ depolarization-induced, as well as Ca2+-independent, veratridine-induced, GABA release was significantly enhanced by GAD vector infection . Treatment of CGCs with kainic acid, which destroys most of the GABAergic neurons (<1% remaining), did not prevent vector-derived expression of GAD nor synthesis of GABA . This suggests that defective HSV vector-derived GAD expression can be used to increase GABA synthesis and release in CNS tissue, even in the relative absence of GABAergic neurons . The use of such GAD vectors in the CNS has potential therapeutic value in neurologic disorders such as epilepsy, chronic pain, Parkinson's and Huntington's disease .

In Vitro Cell Dev Biol Anim, 1998 Oct, 34(9), 679 - 84
Modification of materials formed from poly(L-lactic acid) to enable covalent binding of biopolymers: application to high-density three-dimensional cell culture in foams with attached collagen; Zheng J et al.; We describe a method for increasing the hydrophilicity of materials formed from biodegradable polymers and introducing chemical functional groups on their surfaces . Poly(L-lactic acid) was blended with poly(epsilon-CBZ-L-lysine) at an 80:20 ratio . Films of the mixture were prepared and foams were made by solvent casting and salt leaching . Amino groups on the surface of the polymer mixture were deprotected by acid hydrolysis . As an example of the applicability of the technique for attachment of biomolecules, we covalently linked collagen to the deprotected amino groups, creating a surface capable of high density growth of a differentiated cell type (bovine adrenocortical cells) . The method should be generally useful for surface modification of biodegradable polymer materials used in tissue engineering.

Fundam Clin Pharmacol, 1998, 12(5), 517 - 20
Effects of norepinephrine on NMDA-induced neurotoxicity in cerebellar granular cell culture of rat pups; Gepdiremen A et al.; In this study, norepinephrine was tested in 0.1, 1, 10, 25 and 50 microM doses in 100 microM NMDA toxicity on cerebellar granular cell culture of rats . NMDA in 100 microM concentration induced cell death significantly with respect to controls . Death cell population was 1.08 +/- 0.44% in control and 22.15 +/- 2.46% in 100 microM NMDA (P < 0.0001) . None of the norepinephrine concentrations administrated 15 min prior to NMDA was able to reduce death cell scores to control levels . Results were 8.75 +/- 0.83% in 0.1 microM, 7.0 +/- 1.01% in 1 microM, 17.25 +/- 1.31% in 10 microM, 35.5 +/- 1.38% in 25 microM and 17.9 +/- 1.72% in 50 microM norepinephrine plus 100 microM NMDA administrated groups (P < 0.0001 for all with respect to control) . Labetalol, as an alpha and beta blocker in 0.5 microM concentration which was given 15 min prior to norepinephrine was able to block the effects of it . In comparison with 100 microM NMDA administered group, only low doses of norepinephrine reduced the death cell scores significantly (for 0.1 and 1 microM norepinephrine plus NMDA groups; P < 0.0001) . For 10 and 50 microM norepinephrine plus NMDA groups, death cell scores were found statistically insignificant from the NMDA-administered group (P > 0.05 for both) while for the 25 microM norepinephrine plus NMDA group, the death cell score was found to be statistically increased (P < 0.0001).

Biochem Biophys Res Commun, 1998 Oct 20, 251(2), 442 - 8
Comparison of the effects of thyroxine and triiodothyronine on protein turnover and apoptosis in primary chick muscle cell cultures; Nakashima K et al.; Primary chick muscle cells were treated with physiological level of thyroxine (T4) or triiodothyronine (T3) to examine the effects of the hormones on growth, protein turnover, and apoptosis of the cells . Creatine kinase activity, as an index of differentiation, was increased by both T4 and T3 . Even when the conversion from T4 to T3 was blocked by iopanoic acid, T4 increased creatine kinase activity . The rate of protein degradation estimated from {3H} tyrosine release was increased by T3 but not by T4 . DNA cleavage and fragmentation, as indices of apoptosis, were induced by T3 but not by T4 . These results show that T4 stimulates cell differentiation but not protein degradation and apoptosis in primary chick muscle cells, while all events are stimulated by T3 .

J Surg Res, 1998 Nov, 80(1), 35 - 43
Establishment and characterization of primary gallbladder epithelial cell cultures in the prairie dog; Chapman WC et al.; BACKGROUND: The prairie dog has become the established animal gallstone model . This species has a unique propensity to form cholesterol gallstones in response to dietary manipulations . The development of a reliable gallbladder cell culture technique is critical for understanding pathogenic mechanisms of gallstone formation . MATERIALS AND METHODS: Prairie dogs underwent laparotomy and cholecystectomy, followed by initiation of cell cultures . {3H}Thymidine incorporation was used to assess cell growth, and cell lines were assessed using routine histochemical and immunohistochemical staining . RESULTS: Cell yields from prairie dog gallbladders were 4-8 x 10(6) viable cells per animal with viability ranging from 80 to 95% . When plated at 5 x 10(5) cells/cm2, cell clusters, visible within 24 h, coalesced into confluent monolayers within 3-5 days . Cultures remained viable for 6-8 weeks and could be passed for three to four subcultures . Immunohistochemical staining demonstrated a high degree of epithelial purity with immunopositivity for AE1/AE3, and cytokeratin, with no vimentin positivity (mesenchymal antigen) . Intracytoplasmic vacuoles demonstrated positive staining for Alcian blue, periodic acid-Schiff, and mucicarmine and an anti-gallbladder mucin antibody confirmed the presence of the glycoprotein mucin . CONCLUSIONS: This study demonstrates a reliable method for initiation and maintenance of prairie dog gallbladder epithelial cell cultures with a high degree of purity . This technique should allow further studies into the pathogenesis of cholesterol gallstones in this model .

Toxicon, 1998 Nov, 36(11), 1549 - 55
LYS49 phospholipase A2 myotoxins lyse cell cultures by two distinct mechanisms; Fletcher JE et al.; Three Lys49 phospholipase A2 (PLA2) myotoxins from Agkistrodon contortrix laticinctus (ACLMT), Bothrops jararacussu (bothropstoxin-I) and Bothrops asper (myotoxin II) snake venoms are enzymatically inactive on artificial substrates, yet addition of these toxins to cell cultures causes the release of fatty acids derived from the hydrolysis of membrane phospholipids . Bothropstoxin-I treated with p-bromophenacyl bromide is no longer enzymatically active on cell cultures, suggesting the toxin, not tissue PLA2, may hydrolyze the phospholipids . The NB41A3 cell line is sensitive to lysis by ACLMT by two separate mechanisms . The first mechanism is predominant at lower concentrations of ACLMT (0.1-0.5 microM) and over long incubation periods (24 h) with toxin . This mechanism is antagonized by methylprednisolone (MePDN) . The second is predominant at higher concentrations of toxin (1-5 microM) incubated over a short period (1 h) and is not antagonized by MePDN . There is no correlation between enzymatic activity and toxicity at the higher concentrations (5 microM; 1 h) when the enzymatic activity of ACLMT is compared with a noncytolytic PLA2 from Naja naja atra venom (1 microM) . However, over a 24 h period, triglyceride formation relative to fatty acids remaining free is about 10-fold greater for ACLMT (ratio about 40:1) than for the PLA2 from Naja naja atra venom (ratio about 4:1), suggesting the two enzymes act on substrates associated with different cellular compartments under this condition . Therefore, two mechanisms of Lys49 PLA2-induced myonecrosis exist and these are dependent on toxin concentration . The MePDN-sensitive mechanism associated with triglyceride accumulation correlates with myotoxicity.

Br J Cancer, 1998 Oct, 78(8), 1018 - 23
Selective suppression of cytokine secretion in whole blood cell cultures of patients with colorectal cancer; Lahm H et al.; We have investigated the secretion of interferon alpha (IFN-alpha), IFN-gamma, interleukin-1alpha (IL-1alpha), IL-1beta, IL-2 and tumour necrosis factor alpha (TNF-alpha) in whole blood cell cultures (WBCCs) of colorectal cancer patients upon mitogen stimulation . Whereas the values for IL-1beta and TNF-alpha remained virtually unchanged in comparison with healthy control subjects, WBCCs of colorectal cancer patients secreted significantly lower amounts of IFN-alpha (P < 0.005), IFN-gamma (P < 0.0001), IL-1alpha (P < 0.0001) and IL-2 (P < 0.05) . This reduction correlated with the progression of the disease . The total leucocyte and monocyte population were almost identical in both groups . In contrast, a dramatic depletion of lymphocytes was observed in colorectal cancer patients, which affected both lymphocyte counts (P < 0.0005) and their distribution (P < 0.0001) . Our results suggest a selective suppression of cytokines in colorectal cancer patients that is related to tumour burden . Several mechanisms might account for this phenomenon, one of which might be lymphocyte depletion.

Vet Microbiol, 1998 Aug 15, 62(4), 265 - 79
Serological and genetic characterisation of bovine respiratory syncytial virus (BRSV) indicates that Danish isolates belong to the intermediate subgroup: no evidence of a selective effect on the variability of G protein nucleotide sequence by prior cell culture adaption and passages in cell culture or calves; Larsen LE et al.; Danish isolates of bovine respiratory syncytial virus (BRSV) were characterised by nucleotide sequencing of the G glycoprotein and by their reactivity with a panel of monoclonal antibodies (MAbs) . Among the six Danish isolates, the overall sequence divergence ranged between 0 and 3% at the nucleotide level and between 0 and 5% at the amino acid level . Sequence divergences of 7-8%, 8-9% and 2-3% (nucleotide) and 9-11%, 12-16% and 4-6% (amino acid) were obtained in the comparison made between the group of Danish isolates and the previously sequenced 391-2USA, 127UK and 220-69Bel isolates, respectively . Phylogenetic analysis showed that the Danish isolates formed three lineages within a separate branch of the phylogenetic tree . Nevertheless, the Danish isolates were closely related to the 220-69Bel isolate, the prototype of the intermediate antigenic subgroup . The sequencing of the extracellular part of the G gene of additional 11 field BRSV viruses, processed directly from lung samples without prior adaption to cell culture growth, revealed sequence variabilities in the range obtained with the propagated virus . In addition, several passages in cell culture and in calves had no major impact on the nucleotide sequence of the G protein . These findings indicated that the previously established variabilities of the G protein of RS virus isolates were not attributable to mutations induced during the propagation of the virus . The reactivity of the Danish isolates with G protein-specific MAbs were similar to that of the 220-69Bel isolate . Furthermore, the sequence of the immunodominant region was completely conserved among the Danish isolates on one side and the 220-69Bel isolate on the other . When combined, these data strongly suggested that the Danish isolates belong to the intermediate subgroup.

Int J Impot Res, 1998 Sep, 10(3), 165 - 9
Preliminary report on the development and characterization of rabbit clitoral smooth muscle cell culture; Sadeghi-Nejad H et al.; PURPOSE: Scientific model systems for physiological evaluation and investigation of pathophysiologies in clitoral function have been limited . The aim was to develop a New Zealand White rabbit clitoral corpus cavernosum smooth muscle cell culture . METHODS: Clitoral corpus cavernosum erectile tissue was harvested and placed in culture . Clitoral smooth muscle cells which migrated out from explants were grown to confluence and subcultured . Characterizations were performed by morphological and biochemical analyses . RESULTS: The cells exhibited typical morphologic characteristics of smooth muscle cells . Indirect immunofluorescence studies confirmed the presence of a-smooth muscle cell actin . Androgen and estrogen receptors were detected by specific antibodies and binding studies . The cells expressed subtypes of TGF-beta receptors . Treatment with 80 pM TGF-beta 1 24 h resulted in induction and/or increased availability of TGF-beta receptors . CONCLUSIONS: An in-vitro cell culture system using rabbit clitoral smooth muscle cells was developed . These smooth muscle cells retain their biochemical and functional integrity . This in-vitro cell culture system may facilitate studies aimed at understanding the molecular basis of female sexual function.

Comp Biochem Physiol B Biochem Mol Biol, 1998 Jul, 120(3), 507 - 16
The influence of plasma lipoprotein subfractions on 3T3-L1 and human preadipocyte differentiation in cell culture; Stanton LA et al.; 3T3-L1 and human preadipocyte differentiation was significantly (P < 0.001) enhanced by HDL2, LDLII/III and LDLIV . The concentrations of lipoproteins required for maximal differentiation in human preadipocytes were not achieved over the concentration range 50-150 micrograms lipoprotein protein ml-1, whereas maximal differentiation in 3T3-L1 preadipocytes was achieved for all lipoprotein subfractions at approximately 75 micrograms lipoprotein ml-1, a level almost double that required for complete HDL and LDL fractions in 3T3-L1 cells . Despite the enhanced extent of differentiation caused by certain lipoprotein subfractions, the time needed for the conversion process was unaffected . GPDH activity development in both cell types was most pronounced in response to LDLIV, with HDL2 resulting in the lowest activity . In both cell types, the enhancement of differentiation was only evident when the cells were exposed to lipoproteins during the early stage of the program, i.e . before visible formation of lipid droplets.

Mikrobiol Z, 1998 May-Jun, 60(3), 80 - 4
{The anti-adenovirus activity of larifan in a cell culture}; Nosach LN et al.; Larifan, belonging to the number of highly active interferon inducers with a broad antivirus spectrum of action, did not manifest any antivirus effect in vitro in respect to human adenovirus of serotype 2 when it was used to treat the cells before and after the infection.

Prostaglandins Other Lipid Mediat, 1998 Jun, 56(2-3), 183 - 95
Reduced production of PGE2 and PGF2 alpha from decidual cell cultures supplemented with N-3 polyunsaturated fatty acids; Arntzen KJ et al.; A diet rich in n-3 polyunsaturated fatty acid (PUFA) may reduce the intrauterine production of prostaglandins and prolong pregnancy . We tested this hypothesis by assessing the influence of various PUFAs on the spontaneous production of PGE2 and PGF2 alpha from decidual cell cultures . In addition, we assessed prostaglandin and cytokine production stimulated by lipopolysaccharides (LPS) in order to mimic parturition where infection is involved . In both settings, we found that after supplementing with n-3 PUFA, PGE2 and PGF2 alpha were significantly reduced . After supplementing with n-6 PUFA, there was a significant increase in both prostaglandins . Both n-3 and n-6 PUFAs reduced the production of interleukin 1 (IL-1), while n-6 PUFAs reduced TNF production . PUFAs did not influence IL-6 production . Our findings support the hypothesis that dietary n-3 PUFA may prolong pregnancy by reducing intrauterine production of prostaglandins.

Adv Exp Med Biol, 1998, 440, 529 - 35
The mouse hepatitis virus A59 spike protein is not cleaved in primary hepatocyte and glial cell cultures; Hingley ST et al.; Mouse hepatitis virus strain A59 (MHV-A59) produces mild meningoencephalitis and severe hepatitis during acute infection . To determine whether an in vitro system could be established which would mimic in vivo replication of the virus, we examined the ability of MHV-A59 to replicate in primary cultures of hepatocytes derived from C57BL/6 mice . Infection of hepatocytes with MHV-A59 resulted in low levels of replication, with virus remaining cell associated . Maximum viral yield was observed at 24 hours postinfection, while occasional syncytia were observed at 48 hours postinfection . Primary glial cell culture represents a potential in vitro system representing the second main target of MHV-A59, namely the brain . It is known that MHV-A59 produces a productive, but nonlytic infection in these cultures . Since cell-to-cell fusion is associated with the cleavage of S, the observation of little or no syncytia following MHV-A59 infection of both hepatocytes and glial cells prompted us to examine the cleavage of the spike protein (S) by Western blot analysis . The cleavage of S is inefficient in MHV-A59 infected hepatocytes and in glial cells . Furthermore, no cleavage of this protein was detected in liver homogenates from C57BL/6 mice infected with MHV-A59 . These data suggest that cleavage of the MHV-A59 S protein, and by inference cell-to-cell fusion, does not seem to be essential for entry and spread of the virus in vivo and in vitro.

Adv Exp Med Biol, 1998, 440, 451 - 4
Coronavirus MHV-A59 causes upregulation of interferon-beta RNA in primary glial cell cultures; Wang Q et al.; Infection of mice with coronavirus mouse hepatitis virus strain MHV-A59 causes focal acute encephalitis, hepatitis and chronic demyelinating disease . To investigate host interferon (IFN) response to viral infection within the brain, RNA was extracted from A59-or MHV-2- infected and mock-infected primary astrocyte cultures from newborn mice, RT-PCR amplified RNA with primers specific for the various IFNs, transferred to nylon membranes and hybridized with IFN specific digoxigenin-labeled probes . Infection of primary astrocyte cultures from newborn mice with either A59 or MHV-2 caused upregulation of IFN-beta RNA, but not IFN-gamma or IFN-alpha . Thus, brain astrocytes are capable of producing a local IFN-beta response upon infection with MHV . The response of the other IFNs following MHV infection may be derived from inflammatory cells.

Biol Reprod, 1998 Nov, 59(5), 1139 - 42
Cellular specificity of interleukin-1beta-stimulated expression of type-2 prostaglandin H synthase in human amnion cell cultures; Gibb W et al.; Interleukin-1beta (IL-1beta) has been shown in numerous studies to increase prostaglandin output by cultures of human amnion cells . This is due to an increase in the expression of type-2 prostaglandin H synthase (PGHS-2), the inducible form of the enzyme, in these cultures . Amnion consists of an epithelial layer of cells and a subepithelial mesenchymal layer of cells . The purpose of the present study was to determine the cell-type(s) responsible for the IL-1beta-induced PGHS-2 expression in amnion cultures . Amnion was obtained at term after elective Cesarean section or vaginal delivery . Tissues were dispersed with collagenase, and cells were plated in multichamber culture slides and cultured for 7 days in media supplemented with 10% fetal bovine serum . Cell types were characterized with antisera to keratin (epithelial cells) and vimentin (mesenchymal cells) . Cultures contained both cell types, and the proportion of these varied considerably from one culture to another . Cells were treated with various concentrations of IL-1beta for 6 or 24 h and were then fixed in 4% paraformaldehyde . The fixed cells were permeabilized with Triton and examined by immunohistochemistry for PGHS-2 protein using specific antisera, and PGHS-2 mRNA was localized by in situ hybridization using a specific oligonucleotide probe . The cell type(s) expressing PGHS-2 was characterized using double labeling with antisera to keratin (epithelial cell marker) and vimentin (mesenchymal cell marker) . IL-1beta was found to increase expression of immunoreactive PGHS-2 and PGHS-2 mRNA . This increased expression was found to occur only in the vimentin-positive cells and not the epithelial cells . These results highlight the potential importance of the subepithelial cells in the mesenchymal layer of amnion in the formation of prostaglandins during pregnancy and possibly in preterm labor with infection.

Pharmacol Res, 1998 Oct, 38(4), 239 - 42
Response to nimodipine in caffeine-induced neurotoxicity in cerebellar granular cell culture of rat pups; Gepdiremen A et al.; Methylxanthines (theophylline, theobromine and caffeine) are widely used as central nervous system stimulants and caffeine is used in the treatment of apnea in newborns . Plasma therapeutic concentration of caffeine is around 110 microM . Caffeine diffuses the blood brain barrier easily, increasing oxygen consumption in neurones and leading to cell death . In the present study, 4-7-day-old rats were used to obtain cerebellar granular cell cultures . Caffeine was used 50, 150, 250 and 350 microM concentrations and the most toxic dose for it was found to be 350 microM . Death cell scores were 0.9+/-0.63 for control, 1.1+/-0.63 for 50 microM, 0.89+/-0.47 for 150 microM (P>0.05 for both), 3.84+/-0.8 for 250 microM (P=0.024) and 6.2+/-0 . 86 for 350 microM (P=0.001) caffeine concentrations . The role of voltage-dependent calcium channels in caffeine-induced neurotoxicity was tested with the doses of 100 and 200 microM nimodipine 45 min before or after the 350 microM caffeine . Both doses of nimodipine after caffeine administration were found to be ineffective in blocking neurotoxicity . Doses administered 45 min prior to caffeine, reduced death cell score to 0.89+/-0.23 (P=0.000) for 100 microM nimodipine and 2.35+/-0.96 (P=0.000) for 200 microM nimodipine administration into the cultures . A dose-dependent manner of nimodipine in ischemic states is well-known . In the light of these results, nimodipine may be used in the treatment of newborn apneas together with caffeine to prevent neurotoxic side effects of high or repeated doses of it .

J Biotechnol, 1998 Aug 12, 63(2), 85 - 95
Measurement of volumetric (OUR) and determination of specific (qO2) oxygen uptake rates in animal cell cultures; Ruffieux PA et al.; Oxygen is a key substrate in animal cell metabolism . It has been reported that the oxygen uptake rate (OUR) is a good indicator of cellular activity, and even under some conditions, a good indicator of the number of viable cells . The measurement of OUR is difficult due to many different reasons . In particular, the very low specific consumption rate (0.2 x 10(-12) mol cell h-1), the sensitivity of the cells to variations in dissolved oxygen concentration and the difficulty to provide oxygen without damaging the cells are problems which must be taken into account for the development of OUR measurement methods . Different solutions based on an oxygen balance on either the liquid phase or around the entire reactor, and with a variable or stable concentration of dissolved oxygen have been reported . The accuracy of the OUR measurements and the required analytical devices are very different from method to method.

Int J Oncol, 1998 Nov, 13(5), 943 - 9
Effects of all-trans retinoic acid and interferon alpha in peripheral neuroectodermal tumor cell cultures and xenografts; Rosolen A et al.; Peripheral neuroectodermal tumors (PNET) have an unsatisfactory outcome when treated with standard approaches . Among novel treatments, the use of biological response modifiers has rarely been reported in this group of malignancies . We have previously demonstrated that both all-trans retinoic acid (ATRA) and interferon a (IFNa) can inhibit proliferation of human PNET cells and that ATRA can up-regulate IFNa receptor expression in vitro . In this study we evaluated the anti-tumor effects of ATRA and IFNa in PNET cells in vitro and in a human PNET xenograft model, using CHP100 cells . A synergistic inhibitory effect of ATRA and IFNa was observed on CHP100 cells in vitro . On the contrary, a significant inhibition of tumor growth was observed in mice treated with ATRA alone, whereas neither IFNa nor the combination of ATRA and IFNa, reached a statistically significant anti-tumor effect . Histologic examination of tumors revealed the presence of necrosis upon treatment with IFNa, whereas almost no necrosis, but a more differentiated morphology, confirmed by electron microscopy analysis, was associated with the ATRA containing treatments . Taken together these data show an in vitro and in vivo anti-tumor activity of ATRA in human PNET cells, although no synergism of ATRA and IFNa was observed in our xenograft model.

Bioelectromagnetics, 1998, 19(7), 429 - 31
Effect of sinusoidal 50 Hz magnetic field on the testosterone production of mouse primary Leydig cell culture; Forgacs Z et al.; This study evaluated the effect of sinusoidal 50 Hz magnetic field on the basal and human chorionic gonadotropin (hCG)-stimulated testosterone (T) production of 48-h mouse Leydig cell culture . The luteinizing hormone (LH) analog hCG was used to check the T response of the controls and to evaluate the possible effect of the applied magnetic field on the steroidogenic capacity of the exposed cells . Leydig cells were obtained from the testes of 35- to 45-g CFLP mice and isolated by mechanical dissociation without enzyme treatment . The cell cultures were exposed to sinusoidal 50 Hz 100 microT (root mean square) AC magnetic field during the entire time of a 48-h incubation . Testosterone content of the culture media was measured by radioimmunoassay . In cultures exposed to the magnetic field, a marked increase of basal T production was found (P < .05), compared with the unexposed controls, whereas no significant difference was seen between the exposed or unexposed cultures in the presence of maximally stimulating concentration of hCG . These findings demonstrate that sinusoidal 50 Hz 100 microT magnetic fields are able to stimulate the basal T production of primary mouse Leydig cell culture, leaving the steroidogenic responsiveness to hCG unaltered.

J Toxicol Environ Health A, 1998 Oct 9, 55(3), 213 - 24
Effect of Ni2+ on the testosterone production of mouse primary Leydig cell culture; Forgacs Z et al.; This study evaluated the effects of Ni2 on testosterone (T) production of mouse Leydig cells in vitro following an in vivo or in vitro exposure . CFLP mice were subjected to repeated exposure (4 treatments, subcutaneously, every 3 d) to 10, 20 or 40 mg/kg body weight of NiSO4 or 1.0 ml of 0.9% NaCl solution . Depressed human chorionic gonadotropin (hCG)-stimulated T response was seen over a 48-h culture of testicular interstitial cells obtained from the animals exposed to 20 mg/kg or higher dose of NiSO4, while the basal T production remained unaltered . There were no Ni2+-related changes in the body weights or in the weights of testes, epididymides, adrenals, and kidneys . No histopathological alteration was found in the examined organs of NiSO4-treated groups except the dose-dependent tubular lesions in kidney as a result of a specific rather than a general cytotoxic action . To assess the direct effect of Ni2+ on Leydig-cell T production, testicular interstitial cells were cultured with Ni2+ (62.5 to 1000 microM) for 48 h in the presence or absence of maximally stimulating concentration of hCG . Dose-dependent depression in hCG-stimulated T production was seen at 125 microM or higher dose of Ni2+, while basal T production was unaffected . In order to evaluate the time dependency of this effect the cells were cultured for various times in the presence or absence of 250 and 1000 microM Ni2+ . Decreased hCG-stimulated T production was found in the cultures maintained at least for 4 h in the presence of 1000 microM Ni2+, whereas at 250 microM at least 16 h was required to elicit the depression . Cell viability was assessed by a metabolic activity (MTT) assay . The viability of cells was unaltered by 250 microM Ni2+, and only a slight decrease was found even at the end of the 48-h culture period in the presence of 1000 microM Ni2+ . Our results show a dose-related depression in stimulated T production of mouse Leydig cells in culture following either in vivo or in vitro Ni2+ treatment at a dose that does not induce any general toxic or significant cytotoxic action . The data of the time-course study indicate that the effect of Ni2+ on Leydig-cell T production is both time and concentration dependent, and not due to cytotoxicity.

Virology, 1998 Oct 10, 250(1), 205 - 9
Varicella-zoster virus ORF57, unlike its pseudorabies virus UL3.5 homolog, is dispensable for viral replication in cell culture; Cox E et al.; Varicella zoster virus (VZV) encodes five genes that do not have homologs in herpes simplex virus . One of these genes, VZV ORF57, is predicted to encode a protein containing 71 amino acids . Antibody to ORF57 protein immunoprecipitated a 6-kDa protein in the cytosol of VZV-infected cells . Although the homolog of VZV ORF57 in pseudorabies virus, UL3.5, is critical for viral egress and growth in cell culture, VZV unable to express ORF57 replicated to titers similar to those seen with parental virus . Thus VZV ORF57 has a different role in viral replication than its pseudorabies virus homolog.

Pathology, 1998 Aug, 30(3), 286 - 8
Identification of Australian arboviruses in inoculated cell cultures using monoclonal antibodies in ELISA; Broom AK et al.; An ELISA using a panel of specific monoclonal antibodies was developed to identify all alpha and flaviviruses isolated from mosquitoes caught throughout Australia . This technique is sensitive and rapid and is more specific than the traditional methods used to identify flaviviruses . The ability to identify unknown virus isolates from field-caught mosquitoes quickly and accurately improves the efficiency of arbovirus surveillance programs and allows health authorities to give an early warning of an increased health risk from a mosquito-borne virus in a particular region.

Acta Virol, 1998 Apr, 42(2), 65 - 70
Analysis of defective genomes of bombyx mori nucleopolyhedrovirus generated by serial undiluted passage in cell culture; Yanase T et al.; Viral DNA was extracted from cells infected with bombyx mori nucleopolyhedrovirus (BmNPV) D1 strain after 34 serial undiluted passages (P34) . P34 DNA was subjected to restriction analysis and Southern blot hybridisation using standard D1 DNA and P34 DNA of BmNPV as probes . Based on hybridisation profiles, the BmNPV DNA regions retained in the P34 DNA were localised on HindIII and PstI restriction maps . Two regions of BmNPV DNA located at 0-12.8 and 40.2-65.0 map unit (m.u.) were highly conserved in P34 DNA . These regions contained two of three interspersed homologous sequences (ihss), but only one of five homologous regions (hrs) . This suggests that ihss may have an essential role in BmNPV replication.

Brain Res, 1998 Oct 19, 808(2), 270 - 8
Effects of brain endogenous insulin on neurofilament and MAPK in fetal rat neuron cell cultures; Schechter R et al.; We demonstrated the 'de novo' synthesis of insulin within the fetal nervous system in vivo and in vitro . We undertook this study to show a role for brain endogenous insulin within the fetal brain . We used neuron cell cultures (NCC) from 19 days gestational age fetal rat brains incubated in an insulin free/serum free defined medium . The neurons showed the presence of preproinsulin I and II mRNA using polymerase chain reaction and insulin immunoreaction employing peroxidase anti-peroxidase and radioimmunoassay techniques . Using an anti-pan neurofilament antibody (that recognizes non-phosphorylated neurofilaments) neurofilament immunoreaction (NFI) was observed within the neuron body, dendrites and axon . Either insulin antibody or isoproterenol treatment induced the neurites to retract and most of the neurons become round, with NFI confined to the neuron body . The antibody treatments induced the neurons to become hypertrophic and vacuolated . With PD98059 treatment NFI was only observed within the neuron body . The addition of insulin reversed the effects of isoproterenol and PD98059, but not those of the insulin antibody . Treatment with wortmannin had no effect . Western blot analysis showed that the basal level of mitogen activated protein kinase (MAPK) phosphorylation was inhibited by the treatment of the NCC with isoproterenol or trypsin, but was significantly increased by treatment with exogenous insulin, demonstrating that brain endogenous insulin phosphorylated MAPK (p<0.05) . Thus, brain endogenous insulin promotes neurite outgrowth, probably via MAPK and by stimulating neurofilament distribution via this mechanism participates in neuron differentiation .

Plant Physiol, 1998 Oct, 118(2), 419 - 30
Proteasome inhibitors prevent tracheary element differentiation in zinnia mesophyll cell cultures
Woffenden BJ, Freeman TB, Beers EP.
To determine whether proteasome activity is required for tracheary element (TE) differentiation, the proteasome inhibitors clasto-lactacystin beta-lactone and carbobenzoxy-leucinyl-leucinyl-leucinal (LLL) were used in a zinnia (Zinnia elegans) mesophyll cell culture system . The addition of proteasome inhibitors at the time of culture initiation prevented differentiation otherwise detectable at 96 h . Inhibition of the proteasome at 48 h, after cellular commitment to differentiation, did not alter the final percentage of TEs compared with controls . However, proteasome inhibition at 48 h delayed the differentiation process by approximately 24 h, as indicated by examination of both morphological markers and the expression of putative autolytic proteases . These results indicate that proteasome function is required both for induction of TE differentiation and for progression of the TE program in committed cells . Treatment at 48 h with LLL but not clasto-lactacystin beta-lactone resulted in partial uncoupling of autolysis from differentiation . Results from gel analysis of protease activity suggested that the observed incomplete autolysis was due to the ability of LLL to inhibit TE cysteine proteases.

Kidney Int, 1998 Oct, 54(4), 1107 - 16
Transcriptional activation of transforming growth factor-beta1 in mesangial cell culture by high glucose concentration; Hoffman BB et al.; BACKGROUND: Transforming growth factor-beta (TGF-beta) is an important hypertrophic and prosclerotic cytokine in the pathogenesis of diabetic nephropathy . The mechanisms of regulation of the TGF-beta system by high ambient glucose in kidney cells are incompletely defined . This study examined the mechanisms of regulation of TGF-beta1 expression by high glucose in murine mesangial cells (MMCs) in culture . METHODS: MMCs were cultured in either normal (100 mg/dl) or high (450 mg/dl) D-glucose concentration . Total TGF-beta1 protein secretion and bioactivity, mRNA expression and stability, and gene transcription rate were measured; promoter-reporter chloramphenicol acetyltransferase (CAT) assays and electrophoretic mobility shift assay (EMSA) were performed to investigate the presence of putative glucose-response elements . RESULTS: Raising the ambient D-glucose concentration for 72 hours increased TGF-beta1 bioactivity in cell culture medium by 47% and total TGF-beta1 secretion by approximately 90% . Northern analysis demonstrated that the steady-state TGF-beta1 mRNA level was increased nearly twofold after 48 hours of growth in high glucose . This increase was not due to increased stability, as the half-life of the message was approximately five hours in both normal and high glucose conditions . Transcriptional activity of the TGF-beta1 gene (nuclear run-on assay) was increased by 73% in cells grown in high glucose for 24 hours . Transiently transfected MMCs with CAT constructs containing varying lengths of the murine TGF-beta1 promoter demonstrated that high glucose selectively increased the expression of only one of the constructs, pA835 . Sequence inspection revealed the presence of a putative glucose responsive element, CACGTG, within this construct . High glucose in MMC culture for 24 hours increased nuclear protein binding to a probe containing this element when analyzed using EMSA . CONCLUSIONS: High glucose stimulates total TGF-beta1 protein production and bioactivity as well as the steady-state level of TGF-beta1 mRNA . The latter effect is due primarily to stimulation of gene transcription rate rather than message stability . Transcriptional activation by high glucose may involve a region in the TGF-beta1 promoter containing a putative glucose-response element.

J Virol, 1998 Nov, 72(11), 8597 - 604
Reovirus growth in cell culture does not require the full complement of viral proteins: identification of a sigma1s-null mutant; Rodgers SE et al.; The reovirus sigma1s protein is a 14-kDa nonstructural protein encoded by the S1 gene segment . The S1 gene has been linked to many properties of reovirus, including virulence and induction of apoptosis . Although the function of sigma1s is not known, the sigma1s open reading frame is conserved in all S1 gene sequences determined to date . In this study, we identified and characterized a variant of type 3 reovirus, T3C84-MA, which does not express sigma1s . To facilitate these experiments, we generated two monoclonal antibodies (MAbs) that bind different epitopes of the sigma1s protein . Using these MAbs in immunoblot and immunofluorescence assays, we found that L929 (L) cells infected with T3C84-MA do not contain sigma1s . To determine whether sigma1s is required for reovirus infection of cultured cells, we compared the growth of T3C84-MA and its parental strain, T3C84, in L cells and Madin-Darby canine kidney (MDCK) cells . After 48 h of growth, yields of T3C84-MA were equivalent to yields of T3C84 in L cells and were fivefold lower than yields of T3C84 in MDCK cells . After 7 days of growth following adsorption at a low multiplicity of infection, yields of T3C84-MA and T3C84 did not differ significantly in either L cells or MDCK cells . To determine whether sigma1s is required for apoptosis induced by reovirus infection, T3C84-MA and T3C84 were tested for their capacity to induce apoptosis, using an acridine orange staining assay . In these experiments, the percentages of apoptotic cells following infection with T3C84-MA and T3C84 were equivalent . These findings indicate that nonstructural protein sigma1s is not required for reovirus growth in cell culture and does not influence the capacity of reovirus to induce apoptosis . Therefore, reovirus replication does not require the full complement of virally encoded proteins.

Pharmacol Toxicol, 1998 Jul, 83(1), 29 - 35
Growth modulating effects of chlorinated oleic acid in cell cultures; Hostmark AT et al.; Chlorinated fatty acids represent a major fraction of extractable, organically bound chlorine in fish . After dietary intake such fatty acids may be transferred from the mother to the foetus through the placenta, and via breast milk to the child . In the present work we have studied the effect of chlorinated oleic acid on the growth of three widely differing types of cells in culture . Chlorinated oleic acid inhibited growth of Human Microvascular Endothelial Cells (HMVEC), Immortilized Human Kidney Epithelial (IHKE) cells, and human Hepatoma cells (HepG2) . The order of potency was: HMVEC > IHKE > HepG2 . Vitamin E counteracted the inhibitory effect of chlorinated oleic acid on HepG2 cells, but did not significantly affect the fatty acid effect on HMVEC or IHKE . Defatted serum albumin stimulated the growth of HMVEC and IHKE . With HMVEC there was no major interaction between the effect of albumin and chlorinated oleic acid on cell growth . In contrast, with IHKE albumin at low concentration abolished the growth inhibiting effect of chlorinated oleic acid and appreciably counteracted growth inhibition by the fatty acid of HepG2 . We conclude that the growth modulation by chlorinated oleic acid and its interaction with vitamin E and albumin are cell specific.

J Virol Methods, 1998 Sep, 74(1), 47 - 56
Comparative detection of classical swine fever virus in striated muscle from experimentally infected pigs by reverse transcription polymerase chain reaction, cell culture isolation and immunohistochemistry; Thur B et al.; Classical swine fever (CSF) is a highly contagious viral disease, which can be transmitted by CSFV-contaminated swill . In 1993, four CSF outbreaks in Switzerland were caused presumably by feeding pigs with improperly heated swill . The aim of the investigations was to find a suitable method for CSFV detection in striated muscle samples of infected pigs in order to allow routine testing of meat for virus contamination . The sensitivity of virus detection in striated muscle was compared with the detection in target organs . Using reverse transcription polymerase chain reaction (RT-PCR), cell culture isolation and immunohistochemistry on samples from 14 experimentally infected pigs, CSFV was detected in target organs of ten, and in striated muscle of six pigs, respectively . Overall, only 58% of muscle samples from CSFV-positive animals were positive by RT-PCR and 40% by virus isolation in cell culture, whereas the virus was detected in target organs of these pigs . Virus detection from striated muscle was primarily successful in severely diseased animals infected with highly virulent CSFV strains . It is concluded that striated muscle is not suitable for sensitive CSFV detection, and additional organs have to be examined for reliable diagnosis.

Int J Parasitol, 1998 Aug, 28(8), 1293 - 304
Canine neosporosis: clinical signs, diagnosis, treatment and isolation of Neospora caninum in mice and cell culture; Dubey JP et al.; Clinical signs, diagnosis, treatment and isolation of Neospora caninum from two littermate dogs are described . Three of six pups from a Labrador bitch developed paralysis . Neosporosis was diagnosed ante mortem by serological examination in two of the affected pups . At necropsy, tissue cysts were seen in unstained smears and in histologic sections of their brains . Tissue cysts were often thin-walled (approximately 1 micron) but antigenically and ultrastructurally identified as N . caninum . Furthermore, N . caninum (isolates NC-4, NC-5) was isolated in mice and in cell cultures inoculated with neural tissues of these two dogs . Serological diagnosis of neosporosis using a variety of tests is discussed.

Biotechniques, 1998 Sep, 25(3), 482 - 8, 490-2, 494
Selective propagation of retinal pericytes in mixed microvascular cell cultures using L-leucine-methyl ester; Lee CS et al.; Endothelial cell (EC) propagation has been simplified by developing cell-specific selection criteria . Methods commonly used for selectively isolating EC include: (i) differential sieving of disaggregated tissue, (ii) differential plating of cells on extracellular matrices, (iii) lectin affinity isolation of cell populations and (iv) fluorescence-activated cell sorting of cells labeled with a carbocyanine dye of acetylated low-density lipoprotein (DiI-Ac-LDL) . Few criteria for selectively propagating pericytes (PC) are currently available . Nonspecific esterases exhibit a high degree of multiplicity when compared with other mammalian isozymes and may be suitable for the identification and selective propagation of cells of the microvasculature . Evaluation of esterase isotype expression in PC and EC by zymography indicates PC contain alpha-naphthyl acetate and alpha-naphthyl butyrate hydrolyzing esterases as well as dipeptidyl peptidase I, while EC only contain alpha-naphthyl acetate esterase . The cytotoxic response of PC and EC to various amino acid esters is assessed by monitoring vital dye uptake and by light microscopy . Several amino acid esters are cytotoxic to both cell types, whereas 50 mM L-leucine methyl ester (L-Leu OMe) is toxic to EC but not to PC . This amino acid ester is also toxic to mesothelial and retinal pigmented epithelial cells, other common contaminants of PC cultures . Analysis of protein composition by two-dimensional gel electrophoresis indicates that L-Leu OMe does not stimulate expression of stress response proteins in PC . Thus, L-Leu OMe can be utilized to cultivate PC selectively from mixed cell populations.

J Trace Elem Med Biol, 1998 Jul, 12(2), 101 - 8
Chromium determination in osteoblast-like cell culture medium by catalytic cathodic stripping voltammetry with a mercury microelectrode; Morais S et al.; A catalytic cathodic stripping voltammetric procedure for the determination of total chromium in osteoblast-like cell culture medium using a mercury film microelectrode (MFM) was optimised . The method is based on the pre-concentration of the Cr(III)-diethylenetriaminepentaacetic acid (DTPA) complex by adsorption at the potential of-1.00 V (vs . Ag/AgC1) in the presence of 10 x 10(-3) mol/L DTPA, 0.70 mol/L sodium nitrate, 0.04 mol/L sodium acetate and 1.0 x 10(-3) mol/L potassium permanganate at pH 5.9-6.0 . The limit of detection obtained for a 40 s collection time was 2.80 x 10(-10) mol/L of chromium . The results achieved by stripping voltammetry using the MFM were compared to those obtained by atomic absorption spectrometry (AAS) to ensure the reliability of the electrochemical method . This procedure proved to be an alternative to AAS and valuable in biocompatibility studies performed in vitro using osteoblast-like cells.

Brain Res Mol Brain Res, 1998 Oct 1, 60(2), 296 - 300
Differentially expressed genes in hippocampal cell cultures in response to an excitotoxic insult by quinolinic acid; Seidel B et al.; The NMDA-type glutamate receptor agonist quinolinic acid (QA), which causes tissue lesions in the rat brain as well as cell loss in neuronal cultures, is widely used in models of glutamate excitotoxicity . The aim of this study was to evaluate the alterations in gene expression in a primary hippocampal cell culture after exposure to QA . By means of differential mRNA display, we were able to pinpoint as many as 23 bands which appeared to be upregulated after a 6-h treatment with quinolinic acid . The differential expression of 13 cDNAs could be confirmed by dot blot and/or Northern analysis . Of the cDNAs, the p112 regulatory subunit of the 26S proteasome, a PDGF-associated protein and the glia-derived protease nexin PN-1 could be identified . The results provide emphasis to the participation of proteolysis and protease inhibition in neurodegenerative processes .

Neurosci Lett, 1998 Aug 14, 252(2), 123 - 6
Dystrophin deficient myotubes undergo apoptosis in mouse primary muscle cell culture after DNA damage; Sandri M et al.; Apoptosis has been demonstrated to occur in differentiated myocardial muscle, neonatal skeletal muscle and skeletal myoblasts in response to injury . In this report, we studied differentiated normal and dystrophin deficient murine skeletal muscle cell cultures that have been injured by a pulse of cis-platinum (2 h) . Forty-eight hours after DNA damage, dystrophin positive myotubes appeared almost normal though some myoblasts showed DNA fragmentation . On the other hand, dystrophin deficient myotubes presented progressive degeneration via apoptosis detected either by TUNEL or by nuclear morphology . Degeneration of mdx muscle fibers was confirmed by counting both the number of myotubes observed by contrast phase microscopy and myonuclei viewed by immunoreaction for MyoD . A 6-fold decrease in the number of muscle cells was observed in the dystrophin-deficient cell culture compared to the parental culture (P < 0.001) . Direct evidence of degenerating myotubes displaying MyoD- and TUNEL-positive nuclei was obtained . Like myoblasts, differentiated dystrophin deficient myotubes were able to degenerate via apoptosis, showing that mature dystrophin deficient cells are fragile and undergo apoptosis when subjected to a mild injury which would normally be repaired in parental cells.

Hum Reprod, 1998 Aug, 13(8), 2064 - 7
Steroidogenesis in luteinized granulosa cell cultures varies with follicular priming regimen; Lobb DK et al.; During follicular development, a co-ordinated gonadotrophin and endocrine environment is believed to be essential for normal function of the resulting corpus luteum . Whether differences in the gonadotrophins used to promote follicular development can have lasting effects on granulosa cells after they have undergone luteinization and culture, remains to be studied . We measured steroid production under basal and human chorionic gonadotrophin (HCG) stimulation in short and long term cultures of luteinizing granulosa cells obtained from normal ovulatory women undergoing assisted folliculogenesis with either human menopausal gonadotrophin (HMG) or follicle stimulating hormone (FSH) . Basal progesterone and oestradiol production by luteinized granulosa cells obtained from follicles stimulated to develop with FSH was significantly greater than that from HMG derived follicles (P < 0.001) . In short term cultures, treatment with 10 IU HCG caused a 10-fold increase in progesterone release by cells from FSH stimulated follicles, whereas cells of HMG origin produced only 5-fold more progesterone (P < 0.0001) . In cultures that were maintained for 2 weeks, progesterone secretion was reduced, but a similar trend in HCG responsiveness was observed . These experiments demonstrate that the composition of the gonadotrophins used to promote follicular development in vivo leads to differences in granulosa cell steroidogenesis which are evident after luteinization and culture . They additionally support the notion that the environment of follicular development will be reflected in the resulting corpus luteum.

Antiviral Res, 1998 Jul, 39(1), 35 - 46
The antiviral activity of the ribonucleotide reductase inhibitor BILD 1351 SE in combination with acyclovir against HSV type-1 in cell culture; Lawetz C et al.; BILD 1351 SE is a selective peptidomimetic subunit association inhibitor of the herpes simplex virus (HSV) ribonucleotide reductase (RR) with potent antiviral activity both in cell culture assays and animal models of HSV disease . The ability of BILD 1351 SE to inhibit the replication of HSV-1 when used in combination with acyclovir (ACV) for the treatment of HSV infections was investigated in baby hamster kidney cells using a 96-well enzyme-linked immunosorbent assay . The effective concentrations to achieve 50% inhibition (EC50) of virus replication by BILD 1351 SE in serum-starved and non serum-starved cells were 2 +/- 0.9 and 4.1 + 1.6 microM, respectively . The EC50 of ACV under both assay conditions was equal to 2.7 +/- 0.9 microM when tested alone . However, upon addition of BILD 1351 SE, the antiviral activity of ACV was potentiated in a synergistic manner as determined by the isobole method . At a concentration of BILD 1351 SE that produced 30% inhibition of HSV-1 replication, the EC50 of ACV decreased by about 15-fold in confluent cells and 17-fold in serum-starved cells . Similar conclusions were reached when evaluating drug interactions by the median dose-effect . Assuming mutually non-exclusive conditions at a drug ratio of ACV/BILD 1351 SE of 1/2, synergy was demonstrated in confluent cells with a drug enhancement index at EC50 of 14 and a combination index of 0.25 . None of the drug combinations tested showed increased cytotoxicity in comparison with each drug alone . These results are consistent with the expected mode of action of a selective HSV RR inhibitor and support the strategy of combining these inhibitors with ACV for improved therapy of HSV infections.

Proc Natl Acad Sci U S A, 1998 Sep 29, 95(20), 11975 - 80
Inhibition of the p44/42 MAP kinase pathway protects hippocampal neurons in a cell-culture model of seizure activity; Murray B et al.; Excessive release of glutamate and the subsequent influx of calcium are associated with a number of neurological insults that result in neuronal death . The calcium-activated intracellular signaling pathways responsible for this excitotoxic injury are largely unknown . Here, we report that PD098059, a selective inhibitor of the calcium-activated p44/42 mitogen-activated protein kinase (MAP kinase) pathway, reduces neuronal death in a cell-culture model of seizure activity . Dissociated hippocampal neurons grown chronically in the presence of kynurenate, a broad spectrum glutamate-receptor antagonist, and elevated amounts of magnesium exhibit intense seizure-like activity after the removal of these blockers of excitatory synaptic transmission . A 30-min removal of the blockers produced extensive neuronal death within 24 h as assayed by the uptake of trypan blue and the release of lactate dehydrogenase . Phospho-p44/42 MAP kinase immunoreactivity after 30 min of seizure-like activity was present in many neuronal somata and dendrites as well as some synaptic terminals, consistent with both the presynaptic and postsynaptic effects of this pathway . The addition of PD098059 (40 microM; EC50 = 10 microM) during a 30-min washout of synaptic blockers inhibited the phosphorylation of p44/42 MAP kinase and reduced both the trypan-blue staining (n = 13) and the release of lactate dehydrogenase (n = 16) by 73% +/- 18% and 75% +/- 19% (mean +/- SD), respectively . The observed neuroprotection could be caused by an effect of PD098059 on seizure-like events or on downstream signaling pathways activated by the seizure-like events . Either possibility suggests a heretofore unknown function for the p44/42 MAP kinase pathway in neurons.

Proc Natl Acad Sci U S A, 1998 Sep 29, 95(20), 11933 - 8
Effect of vitamin A supplementation on rhodopsin mutants threonine-17 --> methionine and proline-347 --> serine in transgenic mice and in cell cultures; Li T et al.; A therapeutic effect of vitamin A supplementation on the course of photoreceptor degeneration, previously reported for patients with retinitis pigmentosa, was tested in two transgenic mouse models of this disease, each carrying a dominant rhodopsin mutation . The threonine-17 --> methionine (T17M) mutation is a class II rhodopsin mutation, characterized by a thermal instability/folding defect and minimal regeneration with the chromophore . The proline-347 --> serine (P347S) mutation belongs to class I, comprised of a smaller number of mutations that exhibit no recognized biochemical abnormality in vitro . In the present study, each of the two mouse models was fed a diet containing 2.5 mg of vitamin A palmitate (control) or 102.5 mg of vitamin A palmitate (high vitamin A) per kilogram of diet . Dark-adapted, full-field electroretinograms showed that the high vitamin A diet significantly reduced the rate of decline of a-wave and b-wave amplitudes in the T17M mice but had no significant effect on the decline of electroretinogram amplitude in the P347S mice . Correspondingly, histologic evaluation revealed that the treatment was associated with significantly longer photoreceptor inner and outer segments and a thicker outer nuclear layer in the T17M mice but had no effect on photoreceptor morphology in the P347S mice . In a separate series of experiments, the instability defect of the T17M mutant opsin expressed in vitro was partially alleviated by inclusion of 11-cis-retinal in the culture media . These results show that vitamin A supplementation slows the rate of photoreceptor degeneration caused by a class II rhodopsin mutation . Vitamin A supplementation may confer therapeutic benefit by stabilizing mutant opsins through increased availability of the chromophore.

J Neurochem, 1998 Oct, 71(4), 1390 - 5
Ca2+-mediated activation of c-Jun N-terminal kinase and nuclear factor kappa B by NMDA in cortical cell cultures; Ko HW et al.; We examined the possibility that c-Jun N-terminal kinase (JNK) and nuclear factor kappaB (NF-kappaB) might be involved in intracellular signaling cascades that mediate NMDA-initiated neuronal events . Exposure of cortical neurons to 100 microM NMDA induced activation of JNK within 1 min . Activity of JNK was further increased over the next 5 min and then declined by 30 min . Similarly, ionomycin, a selective Ca2+ ionophore, induced activation of JNK . The NMDA-induced activation of JNK was abrogated in the absence of extracellular Ca2+, suggesting that Ca2+ entry is necessary and sufficient for the JNK activation . Immunohistochemistry with anti-NF-kappaB antibody demonstrated nuclear translocation of NF-kappaB within 5 min following NMDA treatment . NMDA treatment also enhanced the DNA binding activity of nuclear NF-kappaB in a Ca2+-dependent manner . Treatment with 3 mM aspirin blocked the NMDA-induced activation of JNK and NF-kappaB . Neuronal death following a brief exposure to 100 microM NMDA was Ca2+ dependent and attenuated by addition of aspirin or sodium salicylate . The present study suggests that Ca2+ influx is required for NMDA-induced activation of JNK and NF-kappaB as well as NMDA neurotoxicity . This study also implies that aspirin may exert its neuroprotective action against NMDA through blocking the NMDA-induced activation of NF-kappaB and JNK.

Histochem Cell Biol, 1998 Sep, 110(3), 231 - 41
Differential expression of CD14, CD36 and the LDL receptor on human monocyte-derived macrophages . A novel cell culture system to study macrophage differentiation and heterogeneity; Wintergerst ES et al.; Macrophages are key players in many aspects of human physiology and disease . It has been hypothesized that in a given microenvironment monocytes differentiate into specific subpopulations with distinct functions . In order to study the role of macrophage heterogeneity in atherogenesis, we established a novel isolation and culture technique for human monocyte-derived macrophages . The present technique does not select for monocyte subpopulations prior to the onset of differentiation . Monocytes were cultured for 2 weeks in the presence of autologous lymphocytes before being plated quantitatively . They differentiated into mature macrophages in terms of morphology, lipid composition, and biological activity . Based on phagocytic activity as well as on the expression of CD14, CD36, and the low-density lipoprotein (LDL) receptor, we have identified macrophage subpopulations that may play distinct roles in atherogenesis . While virtually all adherence-purified monocytes expressed CD14, CD36, and the LDL-R, we characterized three subpopulations of macrophages based on the expression of these antigens: CD36+CD14-LDL-R-(58+/-12%), CD36+CD14+LDL-R+(18+/-5%), the remaining cells being CD36-CD14- LDL-R- . The first two subsets decreased in size during further differentiation (51+/-12% and 8+/-3%, respectively) . Our culture technique may also serve as a good model for studying the implications of macrophage heterogeneity in diseases other than atherosclerosis.

Am J Med Sci, 1998 Sep, 316(3), 162 - 8
Loading paradigms--intentional and unintentional--for cell culture mechanostimulus; Brown TD et al.; Recent interest in the response of cells or tissues to mechanical stimuli has led to the introduction of a variety of laboratory devices designed to deliver quantified mechanical inputs to culture systems . Such devices commonly rely upon distention of a flexible culture substrate, achieved either by direct platen abutment or by transmural pressure differentials . Unfortunately, the substrate distentions in such systems are often unintentionally nonuniform, and typically also induce motions in the overlying liquid nutrient medium--motions which in turn exert unintended reactive stresses upon the culture layer . In order to characterize the nature of these reactive fluid stresses, computer models have been developed for the nutrient medium flow fields (ie, the velocity and pressure distributions) for three established contemporary cell culture mechanostimulus systems . Temporal and spatial distributions of reactive normal and shear stresses are reported for typical duty cycles in these respective instruments.

FEBS Lett, 1998 Aug 21, 433(3), 265 - 8
Apoptosis induced by modified ribonucleosides in human cell culture systems; Meisel H et al.; The in vitro modulation of apoptosis and cell proliferation by modified in comparison with non-modified ribonucleosides was investigated for the first time using peripheral blood lymphocytes, HL-60 cells and Caco-2 cells as human cell culture models . Modulating effects of several ribonucleosides were found in the range of 10(-7)-10(-3) mol/l . The following ribonucleosides induced significant apoptosis of HL-60 cells: adenosine, N6-dimethyladenosine, N6-(2-isopentenyl)-adenosine, N2-dimethylguanosine . A significant apoptotic effect on PBL was found with N6-dimethyladenosine and N6-(2-isopentenyl)-adenosine . N6-Dimethyladenosine, N6-(2-isopentenyl)-adenosine and guanosine had a pronounced inhibitory effect on Caco-2 cell apoptosis . Regarding the known function of ribonucleosides as pathobiochemical marker molecules for cancer, the possibility of a selective apoptotic effect against malignant cells is discussed.

Biochem Pharmacol, 1998 Aug 1, 56(3), 397 - 404
Kappa-opioid potentiation of tumor necrosis factor-alpha-induced anti-HIV-1 activity in acutely infected human brain cell cultures; Chao CC et al.; Opioids have been postulated to play an immunomodulatory role in the pathogenesis of HIV-1 . Synthetic kappa-opioid receptor (KOR) ligands have been found to inhibit HIV-1 expression in acutely infected microglial cell cultures . We recently found that interleukin(IL)-1beta and tumor necrosis factor(TNF)-alpha have antiviral effects in acutely infected mixed glial/neuronal cell cultures . In the present study, we investigated whether selective KOR ligands would exert antiviral effects in acutely infected brain cell cultures . While the KOR ligand trans-3,4-dichloro-N-methyl-N{2-(1-pyrolidinyl)cyclohexyl}benze neaceamide methanesulfonate (U50,488) alone had little anti-HIV-1 activity, this opioid potentiated in a concentration-dependent manner the antiviral activity of TNF-alpha, but not of IL-1beta . The potentiating effect of U50,488 was detected after a 6-hr pretreatment and peaked at 24 hr . The KOR antagonist nor-binaltorphimine completely blocked the potentiating effect of U50,488, suggesting the involvement of a KOR-mediated mechanism . Antibodies to TNF-alpha completely blocked the potentiating effect of U50,488, suggesting a critical role for TNF-alpha . Antibodies to IL-1beta blocked the potentiating effect of U50,488, suggesting that IL-1beta was released following U50,488 treatment, which might contribute to the potentiating effect of U50,488 . These in vitro findings support the notion that synthetic kappa-opioids could be considered as potential adjunctive therapeutic agents in HIV-1-related brain disease.

FEBS Lett, 1998 Sep 4, 434(3), 413 - 6
The iridoid glucoside secologanin is derived from the novel triose phosphate/pyruvate pathway in a Catharanthus roseus cell culture; Contin A et al.; Secologanin is the iridoid building block of the majority of the terpenoid indole alkaloids . In the biosynthesis of secologanin, mevalonate was considered to be the exclusive precursor of isopentenyl diphosphate . After {1-(13)C}glucose feeding to a cell culture of Catharanthus roseus, its incorporation into secologanin was studied by 13C NMR spectroscopy . The data showed that the novel triose phosphate/pyruvate and not the mevalonate pathway was the major route for the biosynthesis of secologanin.

Klin Lab Diagn . 1998 Jul;(7):20.
{A replaceable nonfrictional membrane filter for the prevention of cell culture contamination}; Shchelkanov MIu et al.; A changeable membrane filter preventing the friction between moving parts and the membrane during tight screwing of a culture flask is proposed.

J Control Release, 1998 Apr 30, 53(1-3), 195 - 203
Nasal delivery of peptides: an in vitro cell culture model for the investigation of transport and metabolism in human nasal epithelium; Kissel T et al.; We investigated the transport- and metabolism properties of three peptides in monolayers of human nasal epithelial cells . The effective permeability coefficients of thyrotropin-releasing hormone, met-enkephalin and human recombinant insulin were found to be 4.5, 4.4 and 0.4 x 10(-7) cm/s, respectively . The permeability was inversely proportional to the molecular weight and one order of magnitude lower than in excised nasal mucosa of rabbits . The metabolic cleavage of thyrotropin-releasing hormone (TRH) to the free acid by cytosolic prolyl-endopeptidase was also detected in human nasal cell monolayers, suggesting that ca . 10% of the total amount of TRH is transported via a transcellular pathway . Met-enkephalin is a substrate for aminopeptidases, located on the apical membrane of nasal epithelial cells . Metabolites and enzyme activity are comparable with literature data . Our studies demonstrate that not only morphological, but also functional properties of human nasal epithelial cells are preserved under in vitro conditions . Such a cell culture model based on human nasal cells could be beneficial for the characterization of peptide transport on a cellular level and for investigation of the absorption enhancer mechanism . Further studies are necessary, however, to establish correlations between in vitro permeabilities in cell cultures and nasal drug absorption in animals and humans.

Biochim Biophys Acta, 1998 Sep 16, 1404(3), 314 - 20
Caveat: mycoplasma arginine deiminase masquerading as nitric oxide synthase in cell cultures; Choi JW et al.; We used confluent cultures of dog gallbladder epithelial cells, stimulated by conditioned medium from a culture of human neonatal foreskin fibroblasts, to establish the presence of inducible nitric oxide synthase (NOS, EC 1.14.13.39) . Assay was by conversion of radiolabeled arginine to citrulline . By 4 days after addition of the conditioned medium, a relatively high level of activity was observed . However, further study showed that the enzyme did not require addition of the usual cofactors for maximal activity (NADPH, FAD, FMN and tetrahydrobiopterin) and was stable in the absence of anti-proteolytic agents . Our suspicion that this enzyme might not be NOS but arginine deiminase (EC 3.5.3.6) was confirmed by enzyme purification and by the liberation of ammonia during enzyme reaction . This enzyme, which is absent from primates and virtually confined to single-cell organisms, suggested the presence of Mycoplasma, a common contaminant of cell cultures, and it was subsequently confirmed that the fibroblast culture was a source of Mycoplasma . With the widespread interest in nitric oxide and NOS, and common use of the convenient {3H}arginine assay, there is a considerable danger of the two enzymes being confused . At the very least, it is necessary to check for activity in the absence of added cofactors.

Clin Exp Immunol, 1998 Sep, 113(3), 401 - 6
Tumour necrosis factor (TNF) gene polymorphism influences TNF-alpha production in lipopolysaccharide (LPS)-stimulated whole blood cell culture in healthy humans; Louis E et al.; TNF-alpha is involved in infectious and immuno-inflammatory diseases . Different individuals may have different capacities for TNF-alpha production . This might determine a predisposition to develop some complications or phenotypes of these diseases . The aims of our study were to assess the inter-individual variability of TNF-alpha production and to correlate this variability to a single base pair polymorphism located at position -308 in TNF gene . We studied 62 healthy individuals . TNF-alpha production after LPS stimulation was evaluated using a whole blood cell culture model . The TNF gene polymorphism was studied by an allele-specific polymerase chain reaction . Other cytokines produced in the culture, soluble CD14 concentrations and expression of CD14 on blood cells were also measured . Among the 62 individuals, 57 were successfully genotyped . There were 41 TNF1 homozygotes and 16 TNF1/TNF2 heterozygotes . TNF-alpha production after LPS stimulation of whole blood cell culture was higher among TNF2 carriers than among TNFI homozygotes (929pg/ml (480-1473pg/ml) versus 521 pg/ ml (178-1307 pg/ml); P<0.05) . This difference was even more significant after correction of TNF-alpha production for CD14 expression on blood cells . In conclusion, the single base pair polymorphism at position -308 in the TNF gene may influence TNF-alpha production in healthy individuals.

Dev Biol Stand, 1998, 93, 21 - 9
Raw materials as a source of contamination in large-scale cell culture; Garnick RL; Large-scale cell culture operations for biotechnology products use millions of litres of complex media and gases as well as huge quantities of organic and inorganic raw materials . These raw materials must always be assumed to contain contamination by adventitious agents such as Minute Virus of Mice (MVM) . Genentech has had experience in dealing with two such contaminations . Although the source of these contaminations was not positively identified, there was strong evidence to suggest that the virus entered through a raw material used in cell culture . Analytical methods that can be used to detect the presence of viruses can be used as an early warning system and as part of a strategy to devise barriers against such contamination . Methods such as a polymerase chain reaction (PCR) assay and a cell culture-based infectivity assay have been found to be efficacious in providing early detection of MVM infection . In any contamination, timely interactions with regulatory authorities are vital in minimizing delays in product manufacture.

Biochem Biophys Res Commun, 1998 Sep 8, 250(1), 108 - 14
Nitric oxide response to shear stress by human bone cell cultures is endothelial nitric oxide synthase dependent; Klein-Nulend J et al.; Bone cells, in particular osteocytes, are extremely sensitive to shear stress, a phenomenon that may be related to mechanical adaptation of bone . In this study we examined whether human primary bone cells produce NO in response to fluid shear stress and established by RT/PCR which NOS isoforms were expressed before and after application of shear stress . One hour pulsating fluid flow (PFF; 0.7 +/- 0.02 Pa, 5 Hz) caused a rapid (within 5 min) 2 to 4-fold increase in NO production . NO release was only transiently increased during the first 15 min of exposure to PFF, and remained at control levels during a 1-24 hr postincubation period . In both control and PFF-treated cells, mRNA was easily detected for ecNOS, but not nNOS, and only minimal amounts iNOS were found . mRNA levels for ecNOS increased 2-fold at 1 hr after 1 hr PFF treatment . These results suggest that the rapid production of NO by human bone cells in response to fluid flow results from activation of ecNOS . PFF also leads to an increase in ecNOS mRNA which is likely related to the shear stress responsive element in the promoter of ecNOS .

Anal Biochem, 1998 Aug 15, 262(1), 39 - 44
Development of an assay for the measurement of the surfactant pluronic F-68 in mammalian cell culture medium; Ghebeh H et al.; A colorimetric assay is described for the measurement of Pluronic F-68 in animal cell culture medium . The assay is based on the formation of a complex with cobalt thiocyanate as previously developed for the measurement of Pluronic in liver tissue . To adapt the assay for the lower detection levels required in cell culture medium, the absorbance of the complex was measured at 328 nm . The reproducibility of the assay was improved by washing the complex with ethyl acetate . The addition of a critical volume of ethanol was found to be necessary to reduce the interference caused by unknown components of the culture medium . The assay was linear between concentrations of 0.01 and 0.2% (w/v) Pluronic in serum-free medium . At the lower level of detection of 0.01% (w/v), the coefficient of determination (R2) was 0.998 . The presence of serum in the medium decreased the sensitivity of the assay, which nevertheless was linear from 0.04 to 0.16% (w/v) Pluronic . The sensitivity and precision of the method are appropriate to study the dynamics of Pluronic in large-scale cell cultures in which Pluronic is added to reduce hydrodynamic cell damage .

Can J Microbiol, 1998 Jun, 44(6), 598 - 604
Incidence of enteroviruses in Mamala Bay, Hawaii using cell culture and direct polymerase chain reaction methodologies; Reynolds KA et al.; The consequence of point and nonpoint pollution sources, discharged into marine waters, on public recreational beaches in Mamala Bay, Hawaii was evaluated using virus cell culture and direct reverse transcriptase-polymerase chain reaction (RT-PCR) . Twelve sites, nine marine, two freshwater (one stream and one canal), and one sewage, were assessed either quarterly or monthly for 1 year to detect the presence of human enteric viruses . Water samples were concentrated from initial volumes of 400 L to final volumes of 30 mL using Filterite electronegative cartridge filters and a modified beef extract elution procedure . Cell culture was applied using the Buffalo Green Monkey kidney cell line to analyze samples for enteroviruses . Positive samples were also evaluated by RT-PCR, using enterovirus-specific primers . Levels of RT-PCR inhibition varied with each concentrated sample . Resin column purification increased PCR detection sensitivity by at least one order of magnitude in a variety of sewage outfall and recreational marine water samples but not in the freshwater canal samples . Using cell culture, viable enteroviruses were found in 50 and 17% of all outfall and canal samples, respectively . Samples were positive at beaches 8% of the time . These data illustrate the potential public health hazard associated with recreational waters . Using direct PCR, viruses were detected at the outfall but were not found in any beach or canal samples, in part, owing to substances that inhibit PCR . Therefore, conventional cell culture is the most effective means of detecting low levels of infectious enteroviruses in environmental waters, whereas direct RT-PCR is rendered less effective by inhibitory compounds and low equivalent reaction volumes.

Brain Res Dev Brain Res, 1998 Sep 10, 110(1), 31 - 8
Collagen type IV promotes the differentiation of neuronal progenitors and inhibits astroglial differentiation in cortical cell cultures; Ali SA et al.; In an effort to elucidate the interactions between cells in the developing cortex and their microenvironment, we have employed dissociated cell cultures and immunocytochemistry to analyze the effect of collagen type IV (COL) on the proliferation and differentiation of rat cortical progenitor cells during the period of corticogenesis . COL, present in the proliferative zones throughout the period of neurogenesis, belongs to a group of macromolecular proteins that make up a considerable portion of the extracellular matrix (ECM) . We have shown that this ECM molecule inhibits cell proliferation and glial cell differentiation while promoting neuronal differentiation . We have also demonstrated that COL, when applied to the cultures with basic fibroblast growth factor (bFGF), induces glial cell differentiation while continuing to promote neuronal differentiation . These results indicate that cortical progenitor cells respond differentially to local environmental signals, and that components of the ECM are involved in the regulation of corticogenesis .

Cell Biol Toxicol, 1998 Aug, 14(4), 261 - 6
Sulfur mustard exposure enhances Fc receptor expression on human epidermal keratinocytes in cell culture: implications for toxicity and medical countermeasures; Cowan FM et al.; Sulfur mustard (HD) is a chemical warfare blister agent . The biochemical basis of HD-induced vesication is unknown, and no antidote currently exists . Basal epidermal cells are a major site of HD toxicity in vivo, with inflammation and HD-increased proteolytic activity implicated as factors that contribute to HD pathology . Fc receptors (FcR) bind to the Fc region of antibody to mediate many effector and regulatory functions that can influence inflammatory responses . FcR are found on all types of immune cells and are also expressed on the surface of human keratinocytes . Assay by fluorescent antibodies demonstrated significantly enhanced CD32 (FcRII) and CD16 (FcRIII) on human epidermal keratinocyte (HEK) cell cultures at 8 to 24 h after exposure to HD (50, 100 and 200 micromol/L) . The enhanced CD32 was time- and concentration-dependent and agreed well with the time course of increased proteolysis and cutaneous pathology observed during HD vesication . HD-increased FcR on the surface of HEK might be a mechanism of vesication.

Biomed Sci Instrum, 1997, 33, 514 - 8
Response of human fibroblasts to tantalum and titanium in cell culture; Mostardi RA et al.; The loosening of total joint arthroplasties (TKA) with associated osteolysis has been a persistent problem in orthopaedics . Wear debris from prosthetic devices including Titanium (Ti) is involved in this process . Mechanisms for this osteolytic process are unclear . The purpose of this study was to compare the biological response of Ti and Tantalum (Ta) on retrieved human fibroblasts . Fibroblasts were retrieved from human volunteers and cultured using standard techniques . Twenty-five (25) ml culture flasks were seeded with cells and when reaching confluency four concentrations of Ti and Ta were added . Their mean size was less than 3 microns for both metals and gram weights were 0.0048 . 0.0096, 0.048, and 0.096 gms . After ten (10) days the cells were fixed, stained and photographed . For both Ti and Ta, the lowest concentration had little effect on the cells, while at the two higher concentrations, nearly all of the cell were killed . Since both of the metals tested are considered to be inert with respect to toxicity, these results would suggest that the observed cell death, seen equally for both metals, was due to the size and concentration of the particles and not to the metals tested . Mechanisms are currently being investigated which include mechanical as well as chemical factors.

J Nutr, 1998 Sep, 128(9), 1555 - 61
Caco-2 cell ferritin formation predicts nonradiolabeled food iron availability in an in vitro digestion/Caco-2 cell culture model; Glahn RP et al.; We have adapted an in vitro digestion/Caco-2 cell model to assess Fe availability from foods, by using ferritin formation by Caco-2 cells as an indicator of Fe uptake . Ferritin formation by Caco-2 cells occurs in response to Fe uptake at concentrations of available Fe greater than that of the culture media to which the cells have been adapted . This methodology circumvents the need for using radioactive Fe and thus eliminates the costs and controversies associated with food radiolabeling . To validate this method, we measured ferritin formation in Caco-2 cells exposed to digests containing Fe of relatively high and low availability . Our objective was to determine if ferritin formation would be proportional to Fe uptake and sufficiently sensitive to be an indicator of Fe availability from food digests . Our model uses established in vitro digestion techniques coupled with uptake of Fe by Caco-2 cell monolayers . Measurement of cell ferritin was done by a commercially available RIA . Higher ferritin formation was observed in cells exposed to digests containing FeSO4 plus ascorbic acid vs, digests containing FeSO4 plus citric acid . Additional comparisons of Fe availability from digests of beef, fish, corn and green beans yielded results that demonstrate higher Fe availability (i.e., greater ferritin formation) from beef and fish digests than from digests of corn and green beans . Overall, the results document the promotional effects of ascorbic acid and animal tissue on Fe uptake as measured indirectly by ferritin formation . The results of this study indicate that ferritin formation by Caco-2 cell monolayers is highly sensitive and accurately measures food Fe availability in this in vitro system.

Am J Nephrol, 1998, 18(5), 355 - 8
Effect of L-carnitine and palmitoyl-L-carnitine on erythroid colony formation in fetal mouse liver cell culture; Matsumura M et al.; The administration of L-carnitine to patients undergoing hemodialysis increases hematocrit and improves the response to recombinant human erythropoietin (rhEPO) . This suggests a contribution by carnitine to erythrocyte membrane function or erythropoiesis . We investigated the effect of L-carnitine and palmitoyl-L-carnitine on erythropoiesis by assessing erythroid colony formation in in vitro fetal mouse liver cell cultures . L-Carnitine or palmitoyl-L-carnitine was added with rhEPO to fetal mouse liver cell cultures . Doses of L-carnitine of up to 200 micromol/l to the culture had no effect on colony formation . In contrast, the addition of above 12.5 micromol/l palmitoyl-L-carnitine into the culture increased colony formation significantly . These results suggest that long-chain acyl carnitine may have an effect on erythropoiesis.

Dev Growth Differ, 1998 Aug, 40(4), 403 - 11
Juxtaposition of two heterotypic monolayer cell cultures of chick limb buds; Sato-Maeda M et al.; In chick limb buds, mesenchymal cells of the progress zone (PZ-cells) at different developmental stages segregate one from the other in mixed cell cultures, suggesting they have different cell affinity . In order to learn the possible roles of such differences in the cells, two heterotypic leg PZ-cell populations (cells from stages 25/26 and 20/21) in vitro were juxtaposed to allow them to form the boundary . A method with double cylindrical columns was used to make adjoining monolayer cell cultures . It was shown that heterotypic juxtaposition produced two chondrogenic patterns along the boundary: aggregates of chondrocytes formed by stage 20/21 PZ-cells and a chondrocyte-free band formed by those at stage 25/26 . Juxtaposition of PZ-cells and proximal cells also formed these patterns, while that between cells from anterior and posterior PZ formed indistinct patterns along the boundary . Homotypic PZ-cell juxtaposition did not produce these patterns . The results suggest that different cell affinity has a role in the segmentation of cartilage patterns at a point along the proximodistal axis, as well as a role in retaining cells in one area so as not to be recruited to other condensation areas.

J Neurosci Res, 1998 Sep 1, 53(5), 613 - 25
Uric acid protects neurons against excitotoxic and metabolic insults in cell culture, and against focal ischemic brain injury in vivo; Yu ZF et al.; Uric acid is a well-known natural antioxidant present in fluids and tissues throughout the body . Oxyradical production and cellular calcium overload are believed to contribute to the damage and death of neurons that occurs following cerebral ischemia in victims of stroke . We now report that uric acid protects cultured rat hippocampal neurons against cell death induced by insults relevant to the pathogenesis of cerebral ischemia, including exposure to the excitatory amino acid glutamate and the metabolic poison cyanide . Confocal laser scanning microscope analyses showed that uric acid suppresses the accumulation of reactive oxygen species (hydrogen peroxide and peroxynitrite), and lipid peroxidation, associated with each insult . Mitochondrial function was compromised by the excitotoxic and metabolic insults, and was preserved in neurons treated with uric acid . Delayed elevations of intracellular free calcium levels induced by glutamate and cyanide were significantly attenuated in neurons treated with uric acid . These data demonstrate a neuroprotective action of uric acid that involves suppression of oxyradical accumulation, stabilization of calcium homeostasis, and preservation of mitochondrial function . Administration of uric acid to adult rats either 24 hr prior to middle cerebral artery occlusion (62.5 mg uric acid/kg, intraperitoneally) or 1 hr following reperfusion (16 mg uric acid/kg, intravenously) resulted in a highly significant reduction in ischemic damage to cerebral cortex and striatum, and improved behavioral outcome . These findings support a central role for oxyradicals in excitotoxic and ischemic neuronal injury, and suggest a potential therapeutic use for uric acid in ischemic stroke and related neurodegenerative conditions.

Cell Tissue Res, 1998 Oct, 294(1), 11 - 25
A novel type of adhering junction in an epithelioid tumorigenic rat cell culture line; Schmelz M et al.; Two major types of plaque-bearing adhering junctions are commonly distinguished: the actin microfilament-anchoring adhaerens junctions (AJs) and the desmosomes anchoring intermediate-sized filaments (IFs) . Both types of junction usually possess the common plaque protein, plakoglobin, whereas the other plaque proteins and the transmembrane cadherins are mutually exclusive . For example, AJs contain E-, N-, or P-cadherin in combination with alpha- and beta-catenin, vinculin and alpha-actinin, whereas in desmosomes, desmogleins and desmocollins are associated with desmoplakin and one or several of the plakophilins (PP1-3) . Here we describe a novel type of adhering junction comprising proteins of both AJs and desmosomes and the tight junction (TJ) plaque protein, ZO-1, in a newly established, liver-derived tumorigenic rat cell line (RMEC-1) . By immunofluorescence microscopy, cell-cell contacts are characterized by mostly continuous-appearing lines which are usually resolved by electron microscopy as extended arrays of closely spaced small plaque subunits . These plaque-covered regions are positive for plakoglobin, alpha- and beta-catenin, the arm-repeat protein p120, vinculin, desmoplakin and protein ZO-1 . They are positive for E-cadherin in cultures early on in passaging, but tend to turn negative for all known cadherins in densely grown cultures . On immunoblotting SDS-PAGE-separated proteins from dense-grown cell monolayers, "pan-cadherin" antibodies have reacted with a band at approximately 140 kDa, identified as N-cadherin by peptide fingerprinting of the immunoprecipitated protein, which for reasons not yet clear is modified or masked in immunolocalization experiments . The exact histological derivation of RMEC-1 cells is not known . However, the observations of several endothelial markers and the fact that all cells are rich in IFs containing vimentin and/or desmin, while only subpopulations also reveal IFs containing CKs 8 and 18, is suggestive of a mesenchymal, probably endothelial origin . We discuss the molecular relationship of this novel type of extended junction with other types of adhering junctions.

Am J Physiol, 1998 Sep, 275(3 Pt 1), G393 - 401
Helicobacter pylori-infected human antral primary cell cultures: effect on gastrin cell function; Richter-Dahlfors A et al.; Although Helicobacter pylori infection increases gastrin secretion, it is unknown whether this is a direct effect or requires activation of the immune system . We developed an H . pylori-infected human primary antral epithelial cell culture model to address this question . This culture protocol favors growth of H . pylori, and infected cultures could be maintained for up to 48 h . These cultures were enriched for gastrin (10-40%), somatostatin (2-5%), and gastric mucin (60-80%) cells but did not contain immunocytes . Bacterial attachment occurred in a random manner within 2 h of infection, although bacterial density was lower than in sections from infected patients . After 24 or 48 h, the bacterial microcolonies were similar in size to those seen in vivo, and at 24 h ultrastructural studies demonstrated well-developed pedestal formation underlying the bacteria . Coculture with H . pylori increased basal but not stimulated gastrin secretion at all time points >2 h . In conclusion, a newly developed cell culture model has been used to characterize the interactions between H . pylori and normal human antral epithelial cells.

Int Immunol, 1998 Aug, 10(8), 1017 - 26
Induction of Th2 cell differentiation in the primary immune response: dendritic cells isolated from adherent cell culture treated with IL-10 prime naive CD4+ T cells to secrete IL-4; Liu L et al.; A number of observations indicate that exposure to IL-4 is essential for the priming of Th2-type effector T cells and that exposure to IL-12 is essential for the priming of Th1-type effector T cells . However, the initial source of IL-4 in the early immune response has not been clearly identified . Dendritic cells (DC) are the most potent antigen-presenting cells (APC) in priming naive T cells . In this report, we show that DC exposed to IL-10 may play an important role in the priming of IL-4-secreting cells in the early immune response . DC isolated from splenic adherent cell cultures treated with rIL-10 (IL-10-DC) primed naive ovalbumin (OVA)-TCR transgenic T cells to secrete IL-4 upon re-stimulation with OVA and splenic APC . By contrast, DC isolated from rIL-12, rIL-4 or control treated cultures induced almost exclusively Th1-type effector T cells . IL-4 secretion was detected in the primary cultures of IL-10-DC plus naive CD4+ T cells and the priming of IL-4-secreting T cells by IL-10-DC was dependent on endogenous IL-4 production in the priming culture since anti-IL-4 neutralizing antibody completely abrogated the priming of IL-4-secreting cells . Anti-B7-2 but not anti-B7-1 inhibited the ability of IL-10-DC to prime T cells to secrete IL-4 . Furthermore, the ability of IL-10 DC to prime for IL-4-secreting T cells was closely related to the down-regulation of CD40 ligand-mediated IL-12 p70 production by DC in the primary cultures and was markedly reduced by adding exogenous IL-12 to the priming cultures . Thus, our findings indicate that early immunologic events that drive Th2 differentiation involve the effects of IL-10 on DC.

Nucleic Acids Res, 1998 Sep 15, 26(18), 4301 - 3
Selection of primary cell cultures with cre recombinase induced somatic mutations from transgenic mice; Zeh K et al.; Deletion of genes in defined cell types has been achieved using a combination of gene targeting techniques and the Cre- lox P recombination system . Here we present a method to selectively isolate genetically altered primary cell cultures based on the permanent activation of a drug-resistance gene by the Cre recombinase . Transgenic mice were generated harboring a dormant form of the hygromycin resistance gene . This mouse line was crossed with mice carrying a constitutive Cre gene and an endogenous floxed allele . Primary fibroblasts established from triple transgenic embryos displayed not only hygromycin resistance but also recombination of the endogenous floxed allele . These results prove the potential of this approach.

Virology, 1998 Sep 1, 248(2), 382 - 93
A point mutation in the human cytomegalovirus DNA polymerase gene selected in vitro by cidofovir confers a slow replication phenotype in cell culture; Cihlar T et al.; In cell culture, cidofovir (CDV) was used to select a human cytomegalovirus (HCMV) strain with decreased drug susceptibility . The genotypic characterization of this virus revealed a single base substitution resulting in a K513N amino acid alteration in the viral DNA polymerase (UL54) . Performed in parallel, the selection of HCMV for replication in the presence of ganciclovir (GCV) selected an M460V substitution in the phosphotransferase (UL97), as well as a K513N/V812L double substitution in DNA polymerase . Neither of the two DNA polymerase mutations has been previously identified in HCMV drug-resistant strains . To precisely elucidate their role in drug resistance, corresponding recombinant mutant viruses were generated by recombination of nine overlapping viral DNA fragments . The K513N recombinant virus showed 13- and 6.5-fold decreased susceptibility to CDV and GCV in vitro, respectively, compared with the wild-type recombinant virus . Mutation V812L was associated with a moderate (2-3-fold) decrease in susceptibility to CDV, GCV, foscarnet, and adefovir . A multiplicative interaction of the K513N and V812L mutations with regard to the profile and level of drug resistance was demonstrated in recombinant virus expressing both mutations . In vitro replication kinetic experiments revealed that the K513N mutation significantly decreased HCMV replication capacity . Consistent with this finding, the K513N mutant DNA polymerase exhibited reduced specific activity in comparison with the wild-type enzyme and was severely impaired in its 3'-5' exonuclease function . Unexpectedly, the K513N mutant enzyme showed no decrease in susceptibility to CDV-diphosphate or GCV-triphosphate . However, the K513N mutation decreased the susceptibility to CDV and GCV of the oriLyt plasmid replication in the transient transfection/infection assay, suggesting that the DNA replication of the K513N mutant virus is less sensitive to the corresponding inhibitors .

Biomaterials, 1998 Jul, 19(14), 1303 - 7
Radiopaque poly(2-hydroxyethyl methacrylate) particles containing silver iodide complexes tested on cell culture; Horak D et al.; Silver iodide complexes have been used as an effective radiopacifying agent to prepare radiopaque poly(2-hydroxyethyl methacrylate) (poly(HEMA)) particles . Incorporation of silver iodide complexes inside the poly(HEMA) particles was achieved by first swelling particles in potassium iodide solution and precipitating the silver iodide complexes using a 30 wt% solution of silver nitrate . The dry particles contained 15 wt% of silver iodide complexes . Particles were easily monitored using a standard imaging technique based on X-ray absorption . Toxicity of the particles has been determined in in vitro experiments on a cell culture . As no inhibition of growth of cells surrounding the particles was observed, they can be considered non-toxic.

In Vitro Cell Dev Biol Anim, 1998 Jul-Aug, 34(7), 561 - 7
Growth potential of Rana tigerina skin lipids in cell cultures; Sai KP et al.; This study involves the investigation of the lipid composition of the skin of Rana tigerina which has a significant healing capacity . The results indicated that the lipid extract enhanced keratinocyte and fibroblast cell proliferation progressively and were found to be much more efficient in comparison to agents known to cause cell proliferation and to be anti-inflammatory such as hydrocortisone . Cell proliferation was dose dependent and suppression occurred only at very high doses . {3H}thymidine incorporation studies produced the same results . Because proliferation, migration, and differentiation of the basal cells is essential for initiation and progression of wound healing, any agent enhancing their proliferation would hasten the healing process . This paper therefore aims at elucidating the effect of composition of the total lipid extract confirming the efficacy of frog skin in wound healing and thereby providing an understanding of the natural mechanism of healing.

Acta Histochem, 1998 Jul, 100(3), 297 - 307
Mouse Leydig cell culture on microcarriers; Kmicikiewicz I et al.; Culturing cells on microcarriers maximizes the ratio of surface area to culture medium volume . The aim of this study was to show microcarrier culture as a useful tool for improvement of steroidogenic activity in mouse Leydig cells . Freshly isolated cells were grown on Sephadex microcarriers, Cytodex 3, and gelatin beads (gelaspheres) that were uncoated, coated with collagen or coated with fibronectin . The cells were cultured in control or LH-supplemented medium . The effect of LH on testosterone and estradiol secretion was studied with appropriate radioimmunoassays . The activity of hydroxysteroid dehydrogenases (3 beta-HSD, 17 beta-HSD) was evaluated histochemically . Leydig cells growing on microcarriers formed colonies . The strongest response to luteinizing hormone stimulation was observed on gelatin beads coated with fibronectin and collagen . In cells growing on a fibronectin layer, very strong activity of 3 beta-HSD and 17 beta-HSD and an increase in steroid hormone levels were observed . Basal and LH-stimulated testosterone and estradiol secretion were all much higher in the microcarrier than in the monolayer culture system . We conclude that Leydig cells maintain differentiated functions more efficiently on collagen- and fibronectin-coated gelasperes than on Sephadex microcarriers and that uncoated gelaspheres are less efficient than coated ones . Pure gelaspheres are unsuitable for improvement of the hormonal and enzymatic activity of Leydig cells.

Cell Tissue Res, 1998 Sep, 293(3), 407 - 18
Effect of synthetic peptides derived from SCO-spondin conserved domains on chick cortical and spinal-cord neurons in cell cultures; Monnerie H et al.; SCO-spondin is a newly identified protein, strongly expressed in the subcommissural organ (SCO), an ependymal differentiation of the brain . When secreted into the cerebrospinal fluid at the entrance to the Sylvian aqueduct, it condenses and forms Reissner's fiber . Several conserved domains have previously been characterizedin SCO-spondin, e.g., thrombospondin type 1 repeats (TSRs), low-density lipoprotein receptor (LDLr) type A repeats, and epidermal-growth-factor-like domains, which are potent sites of protein-protein interaction . To clarify the role of this protein on neuronal development, we have tested the effect of oligopeptides, the sequences of which include highly conserved amino acids of TSRs, LDLr type A repeats and a potent site of attachment to proteoglycan, on cortical and spinal-cord neurons in primary cell cultures . One of these peptides (WSGWSSCSRSCG), corresponding to a SCO-spondin TSR sequence, markedly increases adhesivity and neuritic outgrowth of cortical neurons and induces an opposite effect on cortical and spinal-cord neuronal aggregation . These effects are specific, as no response is observed with the scrambled sequence of this peptide . Another peptide (WGPCSVSCG) is only slightly active on adhesivity and neuritic outgrowth of cortical neurons and has no effect on spinal-cord neurons . Peptides derived from other conserved domains of SCO-spondin are not effective under our experimental conditions . Thus, SCO-spondin may be responsible for at least a part of the effects previously observed on neuronal cells cultured in the presence of Reissner's fiber . In addition, SCO-spondin seems to interfere with neuronal development and/or axonal guidance during ontogenesis of the central nervous system in modulating side-to-side interactions and neuritic outgrowth.

Blood, 1998 Sep 1, 92(5), 1598 - 607
Proliferation in monocyte-derived dendritic cell cultures is caused by progenitor cells capable of myeloid differentiation; Cavanagh LL et al.; Dendritic cells (DC) can be generated by culture of adherent peripheral blood (PB) cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) . There is controversy as to whether these DC arise from proliferating precursors or simply from differentiation of monocytes . DC were generated from myeloid-enriched PB non-T cells or sorted monocytes . DC generated from either population functioned as potent antigen-presenting cells . Uptake of {3H}-thymidine was observed in DC cultured from myeloid-enriched non-T cells . Addition of lipopolysaccharide or tumor necrosis factor-alpha led to maturation of the DC, but did not inhibit proliferation . Ki67(+) cells were observed in cytospins of these DC, and by double staining were CD3(-)CD19(-)CD11c-CD40(-) and myeloperoxidase+, suggesting that they were myeloid progenitor cells . Analysis of the starting population by flow cytometry demonstrated small numbers of CD34(+)CD33(-)CD14(-) progenitor cells, and numerous granulocyte-macrophage colony-forming units were generated in standard assays . Thus, production of DC in vitro from adherent PB cells also enriches for progenitor cells that are capable of proliferation after exposure to GM-CSF . Of clinical importance, the yield of DC derived in the presence of GM-CSF and IL-4 cannot be expanded beyond the number of starting monocytes .

J Gen Virol, 1998 Aug, 79 ( Pt 8), 1983 - 7
Bovine herpesvirus type 1 glycoprotein H is essential for penetration and propagation in cell culture; Meyer G et al.; Bovine herpesvirus type 1 (BHV-1) glycoprotein H (gH) is a structural component of the virion which forms a complex with glycoprotein gL . To study the role of BHV-1 gH in the virus infectious cycle, a gH null mutant was constructed in which the gH coding sequences were deleted and replaced by the Escherichia coli lacZ cassette . The BHV-1 gH null mutant was propagated in trans-complementing MDBK cells, stably transfected with plasmid pMEP4 containing the BHV-1 gH gene under the control of the inducible mouse metallothionein promoter . Experiments with the BHV-1 gH null mutant showed that gH is essential in the infectious cycle of the virus and is specifically involved in virus entry and cell-to-cell spread . The lack of infectivity of virions devoid of gH is not due to a defect in attachment . Moreover, PEG-induced fusion of virions to target cells provides evidence that BHV-1 gH is required for virion penetration.

Proc Soc Exp Biol Med, 1998 Sep, 218(4), 390 - 7
In vitro attenuation of nitric oxide production in C6 astrocyte cell culture by various dietary compounds; Soliman KF et al.; Excessive nitric oxide (NO) production in the brain has been correlated with neurotoxicity and the pathogenesis of several neurodegenerative diseases . NO production from neuroglial cells surrounding neurons contributes significantly to the pathogenesis of these diseases . The suppression of NO production in these cells may be beneficial in retarding many of these disorders . The present study was designed to evaluate the capacity of dietary-derived polyphenolic compounds, flavonoids, crude extracts, oils, and other food constituents in suppressing the release of NO from lipopolysaccharide (LPS)/gamma-interferon (IFN-gamma) stimulated C6 astrocyte cells . In this experiment, 61 compounds were tested, and 36 showed significant suppressive effects of NO production . The results indicate that the following compounds exhibited a dose-dependent suppressive effect of NO production with an IC50 less than 10(-3) M: quercetin, (-)-epigallocatechin gallate, morin, curcumin, apigenin, sesamol, chlorogenic acid, fisetin, (+)-taxifolin, (+)-catechin, ellagic acid, and caffeic acid . Compounds, which reduce NO production at less than 300 ppm, include milk thistle, silymarin, grapenol, and green tea . These results demonstrate a possible value for dietary compounds to inhibit the excessive production of NO.

Vaccine, 1998 Oct, 16(17), 1656 - 9
Human diploid cell culture rabies vaccine (HDCV) and purified chick embryo cell culture rabies vaccine (PCECV) both confer protective immunity against infection with the silver-haired bat rabies virus strain (SHBRV); Dietzschold B et al.; The demonstration of extensive differences in the antigenic makeups of the silver-haired bat rabies virus (SHBRV) and canine rabies virus (COSRV) strains raised concerns as to whether current licensed rabies vaccines are sufficiently protective against SHBRV . NIH mouse protection test results show that both the human diploid cell culture rabies vaccine (HDCV) and the purified chicken embryo cell rabies vaccine (PCECV) protected against lethal infection with SHBRV as well as the canine rabies strain COSRV . However, in this investigation, the potencies of both vaccines in mice were found to be significantly higher for COSRV than for SHBRV . The in vivo protection data are confirmed by in vitro virus neutralizing antibody (VNA) test results which demonstrate that mice immunized with HDCV or PCECV develop significantly higher VNA titres against COSRV than against SHBRV . In contrast, VNA tests of sera from individuals immunized with HDCV or PCECV showed that humans, as opposed to mice, develop significantly higher VNA titres against SHBRV than against COSRV . These data suggest that HDCV and PCECV will protect humans against infection with the silver-haired but rabies virus strain in addition to canine rabies virus strains.

Anim Biotechnol, 1998, 9(2), 101 - 20
Multiple regions of the porcine alpha-skeletal actin gene modulate muscle-specific expression in cell culture and directly injected skeletal muscle; Reecy JM et al.; Transcriptional regulation of the porcine alpha-skeletal actin gene was investigated by comparative transient transfection assays in cultured mammalian cells and by direct DNA injection in skeletal muscle . Intron I sequences were necessary to direct high-level, cell-specific porcine alpha-skeletal actin expression in C2C12 myotubes, but they inhibited transcription in skeletal muscle . A 5' distal sequence (-1929 to -550), had enhancer-like activity in C2C12 myotubes and directly injected muscle, and inhibited transcription in Hela cells . In contrast, a central region (-550 to -388) enhanced basal transcription in directly injected muscle, but not in C2C12 myotubes . A distal regulatory element localized to the 3' untranslated region modulated SV40 promoter activity only in cell culture studies . These results suggest that the intragenic and 3' distal regulatory element may be differentially utilized during differentiation and maturation of skeletal muscle . All three regions decreased SV40 promoter activity in Hela cells, suggesting that they play a role in defining tissue-specific expression of porcine alpha-skeletal actin . Furthermore, different regulatory programs of alpha-skeletal actin expression appear to exist in these two experimental systems.

Cell Mol Life Sci, 1998 Jul, 54(7), 653 - 62
Tailored substrates for studies of attached cell culture; Mrksich M; Substrates for studies of the interactions of attached cells with extracellular matrix components are often prepared by allowing a protein to adsorb from solution onto a glass or polystyrene substrate . This method is simple and effective for many studies, but it can fail in cases that require rigorous control over the structure and composition of adsorbed protein . Self-assembled monolayers formed by the spontaneous ordering of terminally functionalized alkanethiols onto a gold substrate are a class of well-ordered substrates and provide a convenient method for tailoring substrates with ligands, proteins and other groups . Methods that can pattern the monolayers provide a general strategy to create substrates that control the size, shape and spacing of attached cells . This review illustrates recent work that has used these methods of surface chemistry to create tailored substrates for studies in cell biology.

Indian J Ophthalmol, 1998 Mar, 46(1), 37 - 40
Effects of viscoelastic ophthalmic solutions on cell cultures; Madhavan HN et al.; The development of mild but significant inflammation probably attributable to viscoelastic ophthalmic solutions in cataract surgery was recently brought to the notice of the authors, and hence a study of the effects of these solutions available in India, on cell cultures was undertaken . We studied the effects of 6 viscoelastic ophthalmic solutions (2 sodium hyaluronate designated as A and B, and 4 hydroxypropylmethylcellulose designated as C, D, E and F) on HeLa, Vero and BHK-21 cell lines in tissue culture microtitre plates using undiluted, 1:10 and 1:100 dilutions of the solutions, and in cover slip cultures using undiluted solutions . Phase contrast microscopic examination of the solutions was also done to determine the presence of floating particles . The products D and F produced cytotoxic changes in HeLa cell line and these products also showed the presence of floating particles under phase contrast microscopy . Other products did not have any adverse effects on the cell lines nor did they show floating particles . The viscoelastic ophthalmic pharmaceutical products designated D and F have cytotoxic effects on HeLa cell line which appears to be a useful cell line for testing these products for their toxicity . The presence of particulate materials in products D and F indicates that the methods used for purification of the solution are not effective.

J Virol Methods, 1998 Jul, 73(1), 59 - 64
Detection of mumps virus in clinical specimens by rapid centrifugation culture and conventional tube cell culture; Germann D et al.; Conventional tube cell culture was compared with a 2 day and 5 day spin-amplified shell vial indirect immunofluorescence assay for the detection of mumps virus in swabs from the area of Stensen's duct . The sensitivity and specificity of the shell vial assay were 95.9 and 100%, respectively . The shell vial detected 66.3% of the positive cultures within 2 days of inoculation while the first positive results were available by conventional tube cell culture after 3 days (1.6%) reaching 72.4% of all culture positive specimens after 7 days . These data suggest that a centrifugation shell vial indirect immunofluorescence assay may be useful for rapid detection of mumps virus in clinical specimens.

Haematologia (Budap), 1998, 29(1), 41 - 5
Ultrastructural, cell culture and karyotype study of bone marrow in a patient with congenital dyserythropoietic anaemia (CDA)-type III presenting with recurrent still-births; Ghosh K et al.; A 28 year old female patients presented with refractory anaemia since childhood and recurrent still-births at 28-30 weeks of gestation . One still-born child was hydropic at birth . Bone marrow showed characteristic morphological changes of congenital dyserythropoietic anaemia (CDA)-Type III . Electron microscopy showed disruption of the nuclear membrane, spongy appearance of nuclei, stacks of microtubules in intermediate normoblasts and myelin figures in erythroid cells . In vitro culture and karyotype data from the bone marrow of the patient is presented . Recurrent still-births in association with congenital dyserythropoietic anaemia has rarely been reported in the literature.

J Neurosci Methods, 1998 Aug 1, 82(2), 187 - 94
Quantitation of dopamine D2 receptor mRNA in a mesencephalic cell culture using a nonradioactive competitive reverse transcription polymerase chain reaction method; Gross J et al.; Studies on gene expression during differentiation and maturation processes have to cope with determinations of extremely low steady state levels of specific mRNA . Using the experimental model of dopamine D2 receptor (D2R) expression in a primary mesencephalic cell culture we worked out a quantitative reverse transcription polymerase chain reaction method which allows to analyze and quantify mRNA levels of cells present in a few wells of the culture . The method uses an internal cRNA standard which shares both primer binding sites and PCR product length with the target sequence . The amplicons are quantitated in microplates by hybridization with immobilized capture probes that allow for the distinction of internal standard and target sequences followed by the chemiluminescent detection of hybridized DNA . Applying this method the levels of D2 receptor mRNA of the mesencephalic cell culture on day in vitro 1 amounted to about 250 fg/microgram RNA and increased to about 1200 fg/microgram RNA on day in vitro 13-15.

J Chromatogr B Biomed Sci Appl, 1998 Aug 7, 712(1-2), 73 - 82
Quantitative analysis and process monitoring of site-specific glycosylation microheterogeneity in recombinant human interferon-gamma from Chinese hamster ovary cell culture by hydrophilic interaction chromatography; Zhang J et al.; A chromatographic method was developed for quantitative analysis of site-specific microheterogeneity of the two N-linked glycosylation sites in recombinant human interferon-gamma produced from Chinese hamster ovary (CHO) cell culture . After the interferon-gamma was harvested by affinity chromatography, the tryptic digestion was carried out . The two glycopeptide pools, isolated from reversed-phase chromatography of tryptic digestion of interferon-gamma, were subjected to further separation by hydrophilic interaction chromatography . Each peak in the chromatograms was identified by matrix-assisted laser desorption ionization and time-of-flight mass spectrometry (MALDI-TOF-MS) . The overall elution order of the glycopeptides was the following: neutral glycopeptides, monosialylated glycopeptides, bisialylated glycopeptides, trisialylated glycopeptide and tetrasialylated glycopeptides . Based on the integrated peak area for each compound in the chromatograms, the percentage for each glycan was utilized to quantify the glycosylation pattern of the interferon-gamma . Finally, sialylation and antennarity structure percentages at the two glycosylation sites were chosen as the quality indicators in process monitoring of interferon-gamma production from a serum-free suspension-batch CHO culture.

Differentiation, 1998 Jul, 63(3), 115 - 23
Retinoic acid suppresses the osteogenic differentiation capacity of murine osteoblast-like 3/A/1D-1M cell cultures; Cohen-Tanugi A et al.; Bone is a target tissue for action of retinoids though their precise role remains unclear . This study investigated the effects of retinoic acid (RA) on the marrow stromal 3/A/1D-1M osteoblast-like cells, derived from the in vivo transplantation of 3/A/1D-1 chondroprogenitor cells . Long-term treatment with 1 microM RA for 7 weeks induced a marked decrease in bone-like nodule number and ultrastructural alterations in the striation and the size of the collagen fibres . RA at concentrations varying from 10 nM to 3.16 microM had a dose-dependent inhibition effect on alkaline phosphatase (AP) activity with an IC50 of 0.7 microM . Treatment with 1 microM RA for up to 17 days induced a time-dependent inhibition of AP activity, while the beginning of RA treatment (4 or 52 h of culture) produced a differential magnitude of inhibition . These variations were unrelated to modifications of the expression of RAR receptor at the protein level, as assessed by Western blot analysis . Exposure to 1 microM RA for 6 or 24 h administered at day 14 produced an inhibition of AP activity, which reached a maximum after 48 h, with a recovery time of 8 days in both cases . Long-term treatment with RA at 1 microM completely abolished the level of osteocalcin mRNA on both days 12 and 16, as revealed by Northern blot analysis . However, such RA-treated cells retained the constitutive expression of type II procollagen transcripts . These results suggest that RA inhibits several aspects of osteogenic differentiation capacity, a loss of phenotype, which, in association with the maintenance of type II procollagen cartilage-related characteristic, could be a prerequisite step for cellular plasticity.

J Virol, 1998 Sep, 72(9), 7681 - 4
Classical swine fever virus leader proteinase Npro is not required for viral replication in cell culture; Tratschin JD et al.; The sequence encoding the viral leader proteinase Npro was replaced by the murine ubiquitin gene in a full-length cDNA clone of the classical swine fever virus (CSFV) strain Alfort/187 . The recombinant virus vA187-Ubi showed growth characteristics similar to those of the parent vA187-1 virus . At two occasions cells infected with vA187-Ubi exhibited a cytopathic effect and were found to contain a subgenomic viral RNA . This RNA lacked the same viral genes as the subgenomic RNA which has been found in all cytopathogenic CSFV strains analyzed so far, but it maintained the ubiquitin sequence.

J Virol Methods, 1998 Jun, 72(2), 145 - 52
Comparison of immunofluorescence with monoclonal antibodies and RT-PCR for the detection of human coronaviruses 229E and OC43 in cell culture; Sizun J et al.; Human coronaviruses, with two known serogroups named 229E and OC43, cause up to one third of common colds and may be associated with serious diseases such as nosocomial respiratory infections, enterocolitis, pericarditis and neurological disorders . Reliable methods of detection in clinical samples are needed for a better understanding of their role in pathology . As a first step in the design of such diagnostic procedures, the sensitivities and specificities of two viral diagnostic assays were compared in an experimental cell culture model: an indirect immuno-fluorescence assay using monoclonal antibodies and reverse transcriptase-polymerase chain reaction amplification of viral RNA from infected cells . Immunofluorescence detected human coronaviruses in cells infected at a MOI as low as 10(-2) (log TCID50/ml = 4.25 for HCV-229E and 2.0 for HCV-OC43; log PFU/ml = 4.83 for HCV-229E and 1.84 for HCV-OC43) versus 10(-3) (HCV-OC43) or 10(-4) (HCV-229E) for reverse transcriptase-polymerase chain reaction amplification (log TCID50/ml = 1.75 for HCV-229E and 1.5 for HCV-OC43; log PFU/ml = 2.3 for HCV-229E and 1.34 for HCV-OC43) . There were no false positive signals with other human respiratory pathogens: influenza virus, respiratory syncytial virus and adenovirus . Moreover, each assay was coronavirus serogroup-specific . These results demonstrate the potential usefulness of immunofluorescence with monoclonal antibodies and reverse transcriptase-polymerase chain reaction RNA amplification for the rapid detection of human coronaviruses in infected cell cultures . Both methods could be applied to clinical specimens for the diagnosis of human infections.

Neuroscience, 1998 Oct, 86(3), 967 - 76
Circadian rhythms of arginine vasopressin and vasoactive intestinal polypeptide do not depend on cytoarchitecture of dispersed cell culture of rat suprachiasmatic nucleus; Honma S et al.; Dispersed cells of rat suprachiasmatic nucleus were cultured for more than a month with chemically defined medium . Arginine vasopressin and vasoactive intestinal polypeptide in the culture medium showed robust circadian rhythms starting 24 h after the cell dissociation . The two rhythms had similar periods, with a phase-lead of the vasoactive intestinal polypeptide peaks to the arginine vasopressin peak of about 1 h . The two rhythms remained two weeks later, with both peaks appearing at almost the same time, suggesting the synchronization of the two rhythms . Significant differences in cell architecture were detected depending on precoating matrices of culture dishes, which did not affect the circadian rhythms of arginine vasopressin and vasoactive intestinal polypeptide . Antimitotic treatment at the beginning of the culture not only reduced the number, but also changed the type of glial cells developed . The treatment did not interrupt the synchronized arginine vasopressin and vasoactive intestinal polypeptide rhythms until day 31 . Early appearance of circadian rhythms indicates that neural networks in the suprachiasmatic nucleus are not necessary for the synchronous release of arginine vasopressin and vasoactive intestinal polypeptide . Glial proliferation is not essential for the generation, expression and synchronization of arginine vasopressin and vasoactive intestinal polypeptide rhythms in the dispersed suprachiasmatic nucleus cell culture.

Int J Radiat Biol, 1998 Jun, 73(6), 679 - 90
Evaluation of boron neutron capture effects in cell culture using sulforhodamine-B assay and a colony assay; Wittig A et al.; PURPOSE: The purpose of this study was to find an in vitro method for determining the cytotoxicity of boronated drugs as well as their potential suitability for neutron capture therapy . MATERIALS AND METHODS: The survival of human melanoma cells has been determined by a colony assay and the sulforhodamine-B assay after X-irradiation and irradiation with fast d(14) + Be-neutrons using the boronated compound borocaptate sodium (BSH) . The cytotoxic effects of BSH have been studied using both methods . RESULTS: Under well-defined experimental conditions, and after a sufficient amount of time for the expression of radiation damage, the results of the sulforhodamine-B assay are qualitatively comparable with the results of the colony assay . CONCLUSION: The sulforhodamine-B assay is suitable for the screening of compounds for potential use in neutron capture therapy because it is a fast and efficient method that is reproducible and technically advantageous.

Am J Physiol, 1998 Jul, 275(1 Pt 2), R220 - 6
Characterization of a primary cell culture model of the avian renal proximal tubule; Sutterlin GG et al.; Methods have been developed for producing functional, transporting monolayers of avian proximal tubule (PT) cells . A highly homogenous fraction of PT fragments was prepared by enzymatic digestion (collagenase + Dispase) of chick (3- to 5-day-old) kidneys, followed by Percoll gradient centrifugation . The PT fraction was enriched in glucose-6-phosphatase, a proximal enzyme marker, and reduced in specific activity of hexokinase, a distal marker . PT fragments were grown to confluence in serum-free media on collagen-coated permeable filter supports . Electron microscopy of confluent monolayers revealed numerous microvilli and mitochondria, central cilia, and tight junctions, all characteristic of PT cells . gamma-Glutamyltranspeptidase, a proximal brush-border enzyme, showed threefold higher activity on apical than on basolateral sides of the monolayer . The electrophysiological characteristics of monolayers were investigated by voltage-clamp techniques . Monolayers displayed low transepithelial resistances (40-60 Omega . cm2), lumen-negative potentials, and baseline currents of 6-12 microA/cm2 (with or without 5 mM glucose) . Both alpha-methyl-D-glucose (2 mM), a nonmetabolizable hexose, and phenylalanine (2 mM) significantly stimulated short-circuit current when added to the mucosal side of glucose-free monolayers . Phloridzin, a specific inhibitor of Na+-coupled glucose transport, significantly inhibited short-circuit current, as did 10(-5) M amiloride . Monolayers also expressed net secretory transport of urate . This cell culture preparation may provide a useful working model for the study of avian PT transport.

Obes Res, 1998 Jul, 6(4), 299 - 306
Preadipocyte screening by laminin in porcine stromal vascular cell cultures; Yu ZK et al.; OBJECTIVE: To determine if subpopulations of cells in stromal vascular (S-V) cultures could be segregated and separated based on affinity for laminin substratum . RESEARCH METHODS AND PROCEDURES: S-V cells were seeded and allowed to attach for various times; 4 hours was found to be optimal for cell attachment . Cultures were rinsed after 4 hours of seeding, and S-V cells were divided into three subpopulations based on affinity for laminin: (1) cells that did not attach to laminin; (2) cells that had a low affinity for laminin; and (3) cells that had a high affinity for laminin . After 24 hours, cultures were either stained for the AD-3 antigen (a marker for preadipocytes), C/EBP-alpha (a terminal differentiation marker), or C/EBP-delta (an early preadipocyte marker) . Companion cultures were treated with various media for 9 days and stained with oil red-O . RESULTS: Cells with a high affinity for laminin had the highest proportion of AD-3 and C/EBP-alpha positive cells and the highest proportion of fat cells after treatment with insulin +/- dexamethasone . Cells with a low affinity for laminin had the highest proportion of C/EBP-delta cells and the highest proportion of fat cells after treatment with fetal bovine serum+dexamethasone, followed by insulin . DISCUSSION: These results indicate that differentiating preadipocytes adhere to laminin to a much greater degree than do non-preadipocytes . Therefore, laminin-coated dishes can be used to screen S-V cells to produce preadipocyte or fibroblast-enriched S-V cultures.

Pharm Res, 1998 Jul, 15(7), 964 - 71
MDCK cell cultures as an epithelial in vitro model: cytoskeleton and tight junctions as indicators for the definition of age-related stages by confocal microscopy; Rothen-Rutishauser B et al.; PURPOSE: Madin Darby Canine Kidney (MDCK) cells were grown in culture, and age-related morphological changes in the cytoskeleton and tight junction (TJ) network were used to define stages in view of establishing an optimal in vitro model for the epithelial barrier . METHODS: Growth curves and transepithelial electrical resistance (TEER) were determined, and the cytoskeleton (actin, alpha-tubulin, vimentin) and TJ (Zonula occludens proteins ZO1, ZO2) were investigated with immunofluorescent methods by confocal laser scanning microscopy (CLSM) and digital image restoration . RESULTS: TEER measurements indicated that TJ were functional after one day . Values then remained constant . Four morphological stages could be distinguished . Stage I (0-1 day): Sub confluent cultures with flat cells; TJ established after cell-to-cell contacts are made . Stage II (2-6 days): Confluent monolayers with a complete TJ network, which remains intact throughout the later stages . Stage III (7-14 days): Rearrangement in the cytoskeleton; constant cell number; volume and surface area of cells reduced (cobble-stone appearance) . Stage IV (> or = 15 days): Dome formation, i.e . thickening and spontaneous uplifting of the cell monolayer . CONCLUSIONS: Based on the structural characteristics of stage III cell cultures, which are closest to the in vivo situation, we expect them to represent an optimal in vitro model to study drug transport and/or interactions with drugs and excipients.

Antimicrob Agents Chemother, 1998 Aug, 42(8), 1959 - 65
Efficacy of nitazoxanide against Cryptosporidium parvum in cell culture and in animal models; Theodos CM et al.; Nitazoxanide (NTZ), a drug currently being tested in human clinical trials for efficacy against chronic cryptosporidiosis, was assessed in cell culture and in two animal models . The inhibitory activity of NTZ was compared with that of paromomycin (PRM), a drug that is partially effective against Cryptosporidium parvum . A concentration of 10 microg of NTZ/ml (32 microM) consistently reduced parasite growth in cell culture by more than 90% with little evidence of drug-associated cytotoxicity, in contrast to an 80% reduction produced by PRM at 2,000 microg/ml (3.2 mM) . In contrast to its efficacy in vitro, NTZ at either 100 or 200 mg/kg of body weight/day for 10 days was ineffective at reducing the parasite burden in C . parvum-infected, anti-gamma-interferon-conditioned SCID mice . Combined treatment with NTZ and PRM was no more effective than treatment with PRM alone . Finally, NTZ was partially effective at reducing the parasite burden in a gnotobiotic piglet diarrhea model when given orally for 11 days at 250 mg/kg/day but not at 125 mg/kg/day . However, the higher dose of NTZ induced a drug-related diarrhea in piglets that might have influenced its therapeutic efficacy . As we have previously reported, PRM was effective at markedly reducing the parasite burden in piglets at a dosage of 500 mg/kg/day . Our results indicate that of all of the models tested, the piglet diarrhea model most closely mimics the partial response to NTZ treatment reported to occur in patients with chronic cryptosporidiosis.

J Biomater Sci Polym Ed, 1998, 9(7), 765 - 78
Formation of a spherical multicellular aggregate (spheroid) of animal cells in the pores of polyurethane foam as a cell culture substratum and its application to a hybrid artificial liver; Ijima H et al.; Monkey kidney cells (Vero), human embryonic kidney cells (293), human liver cells (PLC/PRF/5), and primary rat, dog, and porcine hepatocytes formed spherical multicellular aggregates (spheroids) in the pores of polyurethane foam which was used as a cell culture substratum . These spheroids of various cell types express high cell activity for a long period . A practical hybrid artificial live support system composed of a multi-capillary polyurethane foam packed-bed type cell culture module including primary hepatocyte spheroids was developed . The success of the system is indicated by an 80% recovery rate in hepatic failure rats which died in control experiments.

Planta, 1998 Apr, 204(4), 490 - 8
Regulation of enzymes involved in anthocyanin biosynthesis in carrot cell cultures in response to treatment with ultraviolet light and fungal elicitors; Glassgen WE et al.; The accumulation of anthocyanins in cell cultures of Daucus carota L . and the enzymes involved in their biosynthesis were investigated under growth in the dark, continuous irradiation with UV light, incubation with elicitors from Pythium aphanidermatum, and elicitor treatment of UV-irradiated cells . Upon UV irradiation, anthocyanin accumulation was strongly enhanced, and the enzymes of the phenylpropanoid and flavonoid pathways, including the "late" enzymes cyanidin galactosyltransferase, cyanidin galactoside xylosyltransferase, cyanidin triglycoside sinapoyltransferase and sinapic acid glucosyltransferase, all showed transient increases in their activities . The time courses of the enzyme activities exhibited successive maxima with an ordered sequence corresponding to their position in the biosynthetic pathway, suggesting a coordinated induction of the entire set of enzymes . The key enzymes phenylalanine ammonia-lyase and chalcone synthase are regulated on a transcriptional level . Incubation of dark-grown carrot cells with fungal elicitors led to a rapid and transient induction of phenylalanine ammonia-lyase corresponding to the formation of 4-hydroxybenzoic acid, but the amount of anthocyanin did not increase and there was no enhancement of any of the enzyme activities which are part of the anthocyanin pathway, including the enzymes catalyzing glycosylation and acylation reactions . Treatment with UV light and elicitors resulted in a rapid induction of the phenylpropanoid pathway, whereas the inducing effect of UV light on the anthocyanin content, on chalcone synthase and on the enzymes catalyzing the final steps of anthocyanin biosynthesis was suppressed . These results indicate a coordinated regulation of the enzymes involved in anthocyanin biosynthesis, an independent inducibility of the phenylpropanoid pathway, and a hierarchy of the different effectors, as shown by the dominating role of the elicitor-signal over the UV stimulus.

Curr Genet, 1998 Jul, 34(1), 67 - 70
Plastid promoter utilization in a rice embryogenic cell culture; Silhavy D et al.; Plastid promoter utilization was characterized in rice by mapping transcript 5'-ends in samples derived from leaves and cultured embryogenic cells . We have found that rbcL, atpB and the rRNA operon are transcribed by the plastid-encoded plastid RNA polymerase (PEP), while clpP is transcribed by the nucleus-encoded plastid RNA polymerase (NEP) in both chloroplasts and the non-green plastids of embryogenic cultured cells . This finding is in contrast to reports on BY2 tobacco, in which NEP promoter activity in cultured cells was enhanced relative to leaves, facilitating identification of NEP promoters which are undetectable in chloroplasts . Therefore, it appears that activation of plastid NEP promoters in rice is not essential for adaptation to cell culture.

Virology, 1998 Jul 20, 247(1), 115 - 24
Herpes simplex virus type 1 VP26 is not essential for replication in cell culture but influences production of infectious virus in the nervous system of infected mice; Desai P et al.; VP26 is the smallest capsid protein of herpes simplex virus type 1 and is encoded by the UL35 open reading frame . It resides on the outer capsid surface, interacting with VP5 in a one to one stoichiometry in the hexons that comprise capsids . A null mutation in the gene encoding VP26 was generated and transferred into the KOS genome . Recombinant viruses were isolated on Vero cells, which indicated that the absence of VP26 was not required for growth of the virus in cell culture . This was confirmed by the characterization of the VP26 null mutant, designated K delta 26Z . The yield of virus from K delta 26Z-infected Vero cells was decreased only twofold relative to wild-type-infected cells, as judged by the burst size . All three types of capsids (A, B, and C) were observed after sedimentation analysis of K delta 26Z-infected cell extracts . These capsids were similar in composition to wild-type capsids except for the absence of VP26 . The mouse ocular model was used to determine if VP26 played a major role in vivo . The yield of the mutant virus relative to wild-type virus was decreased twofold in the eye; however, the mutant virus yields were decreased 30- to 100-fold in the trigeminal ganglia . Reactivation of the mutant virus as determined by cocultivation assays was also reduced . To determine the effect of VP26 on capsid translocation, the VP26 null mutation was transferred into a virus specifiying a thymidine kinase mutation that by itself is transported to the trigeminal ganglia but whose DNA is not replicated in the ganglia . Using quantitative PCR assays the number of viral genomes detected in the ganglia was similar in the presence or the absence of VP26 . Therefore, VP26 does not appear to aid in the translocation of the virus capsid from the mouse eye to the trigeminal ganglia but is important for infectious virus production in the ganglia.

Vaccine, 1998 Aug, 16(13), 1331 - 5
Safety and immunogenicity of a new influenza vaccine grown in mammalian cell culture; Halperin SA et al.; In a phase I safety and immunogenicity study, 112 healthy adult volunteers were randomly allocated to receive a new bivalent (A/Texas/36/91{H1N1-like}, B/Harbin/7/94) split virion influenza vaccine propagated in Madin-Darby Canine Kidney cell culture or an identical vaccine manufactured using currently licensed egg propagated virus technology . Soreness at the injection site was common but generally mild (75% of the cell culture-derived vaccine group and 62.5% of the egg-derived vaccine group; p = not significant) . General reactions were less common; headache was the most frequently reported adverse effect (26.8 and 30.4%, respectively; p = not significant) . Geometric mean haemagglutination inhibition titres post-immunization against the A/Texas strain were 1012 reciprocal dilution in the cell culture-derived vaccine group and 790 in the egg-derived vaccine group; against the B/Harbin strain titres were 420 and 447, respectively (all comparisons, p = not significant) . It is concluded that the cell culture-derived split virion influenza vaccine is safe and immunogenic in healthy adult volunteers.

Biophys J, 1998 Aug, 75(2), 745 - 54
Diffusion and reaction of nitric oxide in suspension cell cultures; Chen B et al.; A reaction-diffusion model was developed to predict the fate of nitric oxide (NO) released by cells of the immune system . The model was used to analyze data obtained previously using macrophages attached to microcarrier beads suspended in a stirred vessel . Activated macrophages synthesize NO, which is oxidized in the culture medium by molecular oxygen and superoxide (O2-, also released by the cells), yielding mainly nitrite (NO2-) and nitrate (NO3-) as the respective end products . In the analysis the reactor was divided into a "stagnant film" with position-dependent concentrations adjacent to a representative carrier bead and a well-mixed bulk solution . It was found that the concentration of NO was relatively uniform in the film . In contrast, essentially all of the O2- was calculated to be consumed within approximately 2 microm of the cell surfaces, due to its reaction with NO to yield peroxynitrite . The decomposition of peroxynitrite caused its concentration to fall to nearly zero over a distance of approximately 30 microm from the cells . Although the film regions (which had an effective thickness of 63 microm) comprised just 2% of the reactor volume and were predicted to account for only 6% of the NO2- formation under control conditions, they were calculated to be responsible for 99% of the NO3- formation . Superoxide dismutase in the medium (at 3.2 microM) was predicted to lower the ratio of NO3- to NO2- formation rates from near unity to <0.5, in reasonable agreement with the data . The NO3-/NO2- ratio was predicted to vary exponentially with the ratio of O2- to NO release rates from the cells . Recently reported reactions involving CO2 and bicarbonate were found to have important effects on the concentrations of peroxynitrite and nitrous anhydride, two of the compounds that have been implicated in NO cytotoxicity and mutagenesis.

Immunol Lett, 1998 May, 62(1), 45 - 9
A novel sensitive assay to define immune status using short-term peripheral blood derived cell culture and dual-color flow cytometry; Zella D et al.; In this study we describe a novel and highly sensitive in vitro system to determine the functionality of immune cells based on short term culture of peripheral blood derived mononuclear cells (PBMCs) and subsequent analysis of cellular proliferation and surface marker expression by automated dual-color flow cytometry . The standardized mild stimuli introduced into the culture system by supplemented medium (containing exogenous interleukin-2 (IL-2), and fetal bovine serum (FBS)) allow a more physiological interaction of the different cell subsets contained in PBMCs (including CD14+ accessory cells) than other methods that are based on potent and harsh cell activators, such as phytohemagglutinin (PHA) or anti-CD3 antibodies . Measurement of T-cell proliferation and cell surface marker (CD3, C25, CD26, CD71, HLA-DR) analysis revealed that activation response capacity in our assay depends on both the status of the obtained cells and their ability to interact in culture with CD14+ cells . This in vitro assay proved to be very sensitive in detecting changes in the status of T-cell activation and proliferation capacity, and avoid the use of radioactive reagents.

J Cell Biochem, 1998 Aug 1, 70(2), 252 - 67
Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity; Hatton JP et al.; The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes . However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist . Although systems such as the European 'Biorack' provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight . Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the 'Biorack' facility aboard the Space Shuttle . The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatant in-flight), injection port, and supernatant collection chamber . The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers . Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures . Possible operations include stimulation of cultures with activators, separation of cells from supernatant, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange . Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately . We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground-based investigations simulating the conditions expected in the flight experiment . Several parameters including cell concentration, time between cell loading and activation, and storage temperature on cell survival were examined to characterise cell response and optimise the experiments to be flown aboard the Space Shuttle . Results indicate that the objectives of the experiments could be met with delays up to 5 days between cell loading into the hardware and initial in flight experiment activation, without the need for medium exchange . Experiment hardware of this kind, which is adaptable to a wide range of cell types and can be easily interfaced to different spacecraft facilities, offers the possibility for a wide range of experimenters successfully and easily to utilise future flight opportunities.

Arch Virol, 1997, 142(8), 1521 - 35
The PI capsid region of Theiler's virus controls replication in mouse glial cell cultures; O'Shea H et al.; The GDVII strain of Theiler's virus is virulent . The DA strain is avirulent and can persist and initiate lesions of inflammatory demyelination in the CNS of susceptible strains of mice . Other, resistant strains of mice clear the infection . Replication of the GDVII and DA strains of Theiler's virus and their genetic recombinants R2, R3 and R4 were compared in mixed glial cell cultures derived from the mouse CNS . Differences were observed in the early rate of viral production . These mapped to the P1 capsid region of the viral genome . Viruses with GDVII P1 sequences produced virus and spread more rapidly than viruses with DA P1 sequences . GDVII virus infected greater numbers of cells than DA virus . Both strains of virus rapidly replicated at least to the level of translation in astrocytes (GFAP+), macrophage/microglial cells (F4/80+), oligodendrocytes (O4+) and bipotential precursor (A2B5+) cells . Early in infection many A2B5+ and GFAP+ cells were infected and destroyed . In contrast, O4+ cells were relatively resistant to cell-death . The cultures survived and produced virus over 14 days of study, at which time all 4 cell-type were present in the culture but < 1% of all the cells, the majority of which were O4+, expressed viral protein . Most of these infected O4+ cells retained a healthy morphology with extensive sheets of cytoplasm, suggesting that Theiler's virus infection of mature oligodendrocytes was non-destructive.

Arch Virol, 1997, 142(12), 2421 - 31
Role of the fowlpox virus thymidine kinase gene for the growth of FPV recombinants in cell culture; Scheiflinger F et al.; Fowlpox virus (FPV) insertion plasmids were constructed that, upon integration into the viral genome via in-vivo recombination, inactivate the viral thymidine kinase (tk) gene . Using this approach, no wild-type virus-free stocks of recombinant virus could be obtained . In contrast, either integration of foreign genes into the intergenic region of the intact FPV tk gene and the open reading frame located downstream, or the functional substitution of the inactivated FPV tk gene by an intact vaccinia virus tk gene resulted in the predicted stable recombinants that were free of wild-type virus . Our results suggest that in already highly attenuated poxvirus strains an intact tk gene is essential for efficient growth of the virus in cell culture.

Arch Virol, 1997, 142(12), 2347 - 57
Intracellular localisation of dengue-2 RNA in mosquito cell culture using electron microscopic in situ hybridisation; Grief C et al.; Non-isotopic in situ hybridisation was used at the electron microscope level to determine the localisation of viral RNA in dengue-2 infected mosquito cells at 14, 24, 48 and 72 h post-infection . In situ hybridisation was carried out on sections of dengue-2 infected mosquito cells using a digoxigenin-labelled DNA probe to the envelope protein gene sequence of the virus . Viral RNA was consistently localised over the rough endoplasmic reticulum and the virus-induced smooth membrane structures which form within the endoplasmic reticulum . During the later stages of infection electron-dense areas were observed to develop in close proximity to the smooth membrane structures . Electron microscopic in situ hybridisation showed that these denser areas contained both viral RNA and virus particles . Our results show that in dengue-2 infected mosquito cells the smooth membrane structures are an important site for the concentration of dengue viral RNA and its possible subsequent encapsidation into virus particles.

Vet Rec, 1998 Jun 20, 142(25), 683 - 6
Cell culture-grown putative bovine respiratory torovirus identified as a coronavirus; Cornelissen LA et al.; A putative bovine respiratory torovirus (BRTV) was propagated in bovine fetal diploid lung and human colonic tumour cells, and fringed pleomorphic particles were detected in the culture supernatants by electron microscopy . Antisera directed against a bovine (Breda strain) and equine (Berne strain) torovirus failed to react with BRTV-infected cells in immunofluorescence assays and did not neutralise BRTV . No toroviral RNA was found in the supernatants of infected cells by means of a reverse transcriptase-polymerase chain reaction with torovirus-specific primers . On the other hand, bovine coronavirus-specific antisera and monoclonal antibodies did neutralise the cytopathic effects, and coronaviral antigen was detected in the cultures by immunofluorescence . Furthermore, bovine coronavirus RNA was detected in the supernatants of BRTV-infected cells after nucleic acid amplification . It is concluded that the cytopathic BRTV isolate is a coronavirus.

Ann N Y Acad Sci, 1998 Jun 29, 849, 282 - 92
The development of a semi-automated latex agglutination test for the detection of antibodies to Anaplasma marginale using a cell culture-derived antigen; Rodgers SJ et al.; Serologic diagnosis of anaplasmosis is currently done by the complement-fixation, ELISA, and card agglutination tests . These tests have utilized A . marginale harvested from bovine erythrocytes as antigen which is often contaminated with erythrocyte stroma . We are currently testing A . marginale propagated in a Ixodes scapularis cell line as antigen for serologic tests . In this study, we report the use of the cell culture-derived A . marginale as antigen for development of a rapid, semi-automated latex agglutination test . Diluted serum and latex (polystyrene microspheres), sensitized with cell culture-derived A . marginale proteins, were dispensed into 96-well microtiter plates . An initial reading of light transmission was recorded by a computer-interfaced scanning autoreader . After 30 minutes, the plates were mixed and read a second time, recording the delta % light transmittance . The sensitized latex microspheres (latex) agglutinated in the presence of A . marginale antibodies, thus producing an increase in light transmittance . In preliminary tests, 724/977 of the sera were positive for A . marginale antibodies with an apparent agreement of 83.3% when compared with the complement-fixation test . Sensitization and sera dilution buffers were shown to have a marked effect on the sensitivity and specificity of this assay . Results will be presented on the optimization of buffers and the testing of sera from experimentally and field-infected cattle.

Ann N Y Acad Sci, 1998 Jun 29, 849, 273 - 81
Use of tick cell culture-derived Anaplasma marginale antigen in a competitive ELISA for serodiagnosis of anaplasmosis; Saliki JT et al.; Anaplasma marginale was propagated in a continuous tick cell line and detergent-solubilized infected cells were used as antigen in a competitive ELISA (C-ELISA) for detection of Anaplasma-specific antibody in bovine sera . Positive control sera competed well (> or = 35% inhibition) with an A . marginale-specific monoclonal antibody for binding to this antigen, while negative sera failed to compete (< 35% inhibition) . The C-ELISA was compared to the standard complement-fixation test (CFT) using 2,208 bovine sera . Overall, C-ELISA was more sensitive than CFT (24.9% versus 9.4%), mainly because CFT yielded "suspicious" or "anti-complementary" results in 10.5% of the sera and also failed to identify several vaccinated and carrier cattle that were C-ELISA-positive . The apparent agreement between CFT and C-ELISA was 89.6% and the kappa value was 0.6 . These results show that this C-ELISA would be a suitable replacement of the CFT as the standard test for detection of A . marginale antibody.

Ann N Y Acad Sci, 1998 Jun 29, 849, 253 - 8
Evaluation of Anaplasma marginale from tick cell culture as an immunogen for cattle; Blouin EF et al.; Anaplasma marginale has been propagated and continuously passaged in an Ixodes scapularis cell line . Anaplasma development was characterized and cultures with a high density of rickettsiae were harvested at a predictable rate . Culture-derived A . marginale (CAM) remained infective for cattle and was used effectively as antigen in diagnostic tests with the sensitivity to identify bovine carriers of A . marginale . This study presents results of an initial trial using the CAM as an immunogen for cattle . CAM was mechanically disrupted, frozen at -70 degrees C, and inactivated with beta-propiolactone . Two intact yearling cattle were immunized with CAM and Freund's adjuvant, receiving 4 subcutaneous injections at 3-4 week intervals . Two control yearling cattle received adjuvant and PBS . Serum samples were evaluated by competitive ELISA (C-ELISA) using CAM as antigen and the standard complement fixation test (CFT) . All cattle were subsequently challenged with A . marginale-infected blood from a carrier cow . An additional intact calf was inoculated with live CAM from the same passage and screened by C-ELISA and CFT . Sera collected from immunized cattle were negative or suspicious by CFT throughout the immunization study . The same sera were strongly positive by C-ELISA two weeks after the first injection and throughout the study . All cattle became infected following challenge-exposure with blood, but immunized cattle exhibited longer prepatent periods as well as lower parasitemias and percent reduction of packed cell volumes as compared with the controls . The calf receiving live CAM became infected and underwent a mild clinical reaction with positive C-ELISA and CFT results and did not become clinically ill following blood challenge . This preliminary study suggests that the CAM antigen is highly immunogenic in cattle . Furthermore, the CFT did not identify immunized animals whereas the C-ELISA (using CAM) was highly sensitive for detection of both immunized and infected animals.

Ophthalmic Res, 1998, 30(4), 263 - 70
The effect of minoxidil on keratocyte proliferation in cell culture; McLeod SD et al.; PURPOSE: To determine if minoxidil inhibits keratocyte proliferation in a nontoxic manner . METHODS: Rabbit keratocytes were cultured in Eagle's minimum essential medium supplemented with fetal bovine serum . Minoxidil varying in concentration from 10(0) to 10(3) micrograms/ml was added to the culture medium and incubated for 7 days . The cultures were inspected for morphologic appearance and the cell number was determined at 1, 3 and 7 days after the addition of minoxidil . After 7 days of incubation, minoxidil was withdrawn from the cell culture medium and the cells were examined 3 and 7 days thereafter . In addition, a nonradioactive cytotoxic assay was performed to determine if toxicity is associated with the presence of minoxidil . RESULTS: Minoxidil inhibited keratocyte proliferation in a dose-dependent fashion . 29% of control growth was achieved when keratocytes were cultured for 7 days in 10(3) micrograms/ml, whereas 82% control growth was achieved when keratocytes were cultured in 10(2) micrograms/ml of minoxidil . Intermediate concentrations between 10(2) and 10(3) micrograms/ml produced a linear decline in cell counts in a dose-dependent fashion . The concentration of minoxidil required for 50% control growth at 7 days extrapolated from the dose-response curve was 600 micrograms/ml . Upon withdrawal of minoxidil, cell counts returned to baseline for concentrations of 10(2) micrograms/ml or less . Phase contrast microscopy revealed that the presence of minoxidil was associated with intercellular separation, enlargement of cell bodies and elongated processes . After the withdrawal of minoxidil, the cells in all media reassumed the morphological features of normal keratocytes which included a regular fusiform shape and extensive intercellular contact . The nonradioactive cytotoxic assay revealed the lack of cytotoxicity at all concentrations of minoxidil based on a lack of lactate dehydrogenase release . CONCLUSIONS: Minoxidil inhibits keratocyte proliferation by a nontoxic mechanism . It might be particularly useful for modulating corneal wound healing following excimer laser photorefractive keratectomy.

Growth Dev Aging, 1998 Spring-Summer, 62(1-2), 3 - 12
Growth factor regulation of insulin-like growth factor (IGF) binding proteins (IGFBP) and preadipocyte differentiation in porcine stromal-vascular cell cultures; Richardson RL et al.; The influence of anti-IGF-1 and anti-transforming growth factor beta (TGF-beta) neutralizing antibodies on preadipocyte differentiation and secretion of IGFBPs was examined in serum free porcine stromal-vascular cultures . Cultures were stained for morphological analysis and conditioned media were collected for: TGF-beta determination by ELISA, IGF-1 by RIA, and IGFBP analysis by ligand blotting . After 6 d of treatment, anti-TGF-beta increased fat proportions by 2.7 fold compared to controls . Anti-IGF-1 decreased fat cell proportions by 14-fold . Anti-TGF-beta increased concentrations of IGF-1 5.8-fold and IGFBP-2 and IGFBP-3 by 8- and 7-fold in conditioned media whereas IGFBP-4 decreased 5-fold . Anti-IGF-1 increased concentrations of IGFBP-2 and 3 by 9- and 35-fold, respectively . TGF-beta increased concentrations of IGFBP-1, 2 and 3 by 3-fold, 18-fold and 3-fold, respectively, after 9 d in culture (6 d of treatment) . There was no change in TGF-beta levels in anti-IGF-1 treated cultures compared to controls . Control antibodies and negative controls had no effect . These results provide evidence that endogenously produced IGF-1 and TGF-beta has a major influence on preadipocyte differentiation in serum free media by modulating IGFBP production/secretion.

Biotechnol Appl Biochem, 1998 Jun, 27 ( Pt 3), 181 - 8
Analysis of hybridoma cell culture processes by SDS/gel capillary electrophoresis and matrix-assisted laser desorption ionization-time-of-flight MS; Klyushnichenko V et al.; SDS/gel capillary electrophoresis (SDS/gel CE) and matrix-assisted laser desorption ionization-time-of-flight MS (MALDI-TOF-MS) were employed to analyse changes in the culture broth during batch and continuous cultivation of hybridoma cells . The stability of IgG was analysed by SDS/gel CE and capillary zone electrophoresis (CZE) . The results obtained by the new analytical procedures reflect the changes in the cultivation conditions very well, indicating that these tools can be used to follow animal cell culture processes . A new technique to collect fractions of very small volumes during the CZE separation is described, and the successful off-line coupling of CZE and MALDI-TOF-MS for the analysis of biotechnological processes is demonstrated.

J Clin Endocrinol Metab, 1998 Jul, 83(7), 2429 - 34
Interleukin-1 beta enhances interleukin-1 receptor antagonist content in human somatotroph adenoma cell cultures; Sauer J et al.; In addition to the well-known modulation of immune and inflammatory responses, the interleukin-1 (IL-1) system has been shown to be involved in the regulation of anterior pituitary hormone secretion and growth . We previously demonstrated that IL-1 receptor antagonist (IL-1ra) is expressed in human pituitary adenomas cultured in vitro . In the present study, we investigated the regulation of IL-1ra protein by IL-1 beta (1-100 U/mL) in human somatotroph adenomas (n = 9) cultured for 12-48 h . IL-1 beta significantly enhanced the concentration of IL-1ra dose dependently in the somatotroph adenoma cell lysates, whereas IL-1ra concentrations remained unchanged in the culture supernatants . Furthermore, basal IL-1ra concentrations were significantly higher in the cell lysates compared with the corresponding culture supernatants . The regulation of IL-1ra in somatotroph adenoma cells is different from human cultured monocytes, in which IL-1 beta significantly stimulated IL-1ra secretion into the culture supernatants, and no change of intracellular IL-1ra content was observed . Incubation of the somatotroph adenoma cells with 100 U/mL IL-1 beta did not result in a change of GH concentrations in the culture supernatants . Enhancement of intracellular IL-1ra protein by IL-1 beta may represent a mechanism intrinsic to somatotroph adenoma cells to counterregulate the response to IL-1 beta on hormone secretion or cellular growth.

Tissue Cell, 1998 Apr, 30(2), 226 - 35
Effect of substratum on growth, cell morphology and lactoferrin synthesis and secretion in bovine mammary cell culture; Talhouk RS et al.; The role of extracellular matrix in morphology, growth and lactoferrin synthesis and secretion in bovine mammary cells from a developing gland is poorly defined . In this study, bovine mammary cells from a hormone-primed developing gland were isolated and cultured on plastic, collagen, embedded within collagen, or on EHS-matrix, with the hormones prolactin, insulin, and cortisol in the presence or absence of fetal calf serum . Mammary cells on plastic or collagen spread and formed confluent cells sheets, while those embedded within collagen or on EHS-matrix maintained their acinar-like structure . Histological and ultrastructural analysis of cells showed that cells on plastic and collagen grew in multilayers, while those embedded within collagen or on EHS-matrix lacked any lumen structure . The ultrastructure of cells on different substrata more resembled an undifferentiated phenotype . Mammary cells secreted lactoferrin in increasing concentrations throughout the culture period . The total amount secreted in culture was regulated by extracellular matrix and fetal calf serum . Cells embedded within collagen in serum-free cultures secreted the lowest amounts of lactoferrin (up to 619 ng/ml; day 14), while those on collagen and supplemented with fetal calf serum secreted up to 4920 ng/ml at day 14 . Fetal calf serum induced higher lactoferrin secretion within each substratum on which the cells were cultured . No intracellular accumulation of lactoferrin was noted in cells on plastic or collagen or those embedded within collagen, whereas those on EHS-matrix accumulated more than 500 ng/ml of lactoferrin intracellularly/intracinarly . Furthermore, when cultured on a similar substratum, cells from a developing gland secreted higher lactoferrin than cells from a lactating gland.

Antimicrob Agents Chemother, 1998 Jul, 42(7), 1666 - 70
Antiherpesvirus activities of (1'S,2'R)-9-{{1',2'-bis(hydroxymethyl)cycloprop-1'-yl}methyl}guanine (A-5021) in cell culture; Iwayama S et al.; Antiherpetic activity of (1'S,2'R)-9-({1',2'-bis(hydroxymethyl)cycloprop-1'yl}methyl)guanine (A-5021) was compared with those of acyclovir (ACV) and penciclovir (PCV) in cell cultures . In a plaque reduction assay using a selection of human cells, A-5021 showed the most potent activity in all cells . Against clinical isolates of herpes simplex virus type 1 (HSV-1, n = 5) and type 2 (HSV-2, n = 6), mean 50% inhibitory concentrations (IC50s) for A-5021 were 0.013 and 0.15 microgram/ml, respectively, in MRC-5 cells . Corresponding IC50s for ACV were 0.22 and 0.30 microgram/ml, and those for PCV were 0.84 and 1.5 micrograms/ml, respectively . Against clinical isolates of varicella-zoster virus (VZV, n = 5), mean IC50s for A-5021, ACV, and PCV were 0.77, 5.2, and 14 micrograms/ml, respectively, in human embryonic lung (HEL) cells . A-5021 showed considerably more prolonged antiviral activity than ACV when infected cells were treated for a short time . The selectivity index, the ratio of 50% cytotoxic concentration to IC50, of A-5021 was superior to those of ACV and PCV for HSV-1 and almost comparable for HSV-2 and VZV . In a growth inhibition assay of murine granulocyte-macrophage progenitor cells, A-5021 showed the least inhibitory effect of the three compounds . These results show that A-5021 is a potent and selective antiviral agent against HSV-1, HSV-2, and VZV.

J Neurobiol, 1998 Jul, 36(1), 30 - 40
Regulation of aromatase, 5 alpha- and 5 beta-reductase in primary cell cultures of developing zebra finch telencephalon; Freking F et al.; Sex steroids act on the developing and adult telencephalon of songbirds to organize and activate the neural circuits required for the learning and production of song . Presumably, the availability of active androgens and estrogens to steroid-sensitive neural circuits controlling song is modulated by the local expression of androgen-metabolizing enzymes . Two enzymes, 5 alpha- and 5 beta-reductase, are expressed widely in the songbird telencephalon, as they are in the telencephalons of other avian species . These enzymes convert circulating testosterone (T) into the active and inactive metabolites, 5 alpha- and 5 beta-dihydrotestosterone (DHT), respectively . A third enzyme, aromatase, converts T into estradiol (E2) and is expressed at unusually high levels in several regions of the songbird telencephalon . In many tissues, including the brain, the regulation of expression of one or more of these enzymes can be a critical feature of their ability to control the production of active sex steroids . We have used primary cell cultures to examine factors that might regulate the expression of these enzymes in developing zebra finch telencephalon . Cultures were treated for 0-72 h with sex steroids (T, E2, 5 alpha-DHT, and 5 beta-DHT) or with dibutyryl cAMP . Afterward, activities of aromatase, 5 alpha- and 5 beta-reductase were determined or total RNA was extracted for Northern analysis . Treatments with cAMP increased both aromatase activity and aromatase mRNA levels by 220% . E2 significantly reduced aromatase activity by an average 65%, whereas 5 alpha- and 5 beta-DHT had no effect on aromatase activity . Compared to untreated controls, E2 treatment decreased aromatase mRNA levels by 56% . None of these treatments consistently affected either 5 alpha- and 5 beta-reductase activities . These results suggest that telencephalic E2 may regulate its own synthesis by repression of aromatase expression, whereas factors that upregulate cAMP in the telencephalon can increase the local concentrations of E2.

J Gastroenterol, 1998 Jun, 33(3), 318 - 25
Hydrogen peroxide-induced cellular injury is associated with increase in endogenous fluorescence from rat gastric mucosal epithelial cell culture: A new method for detecting oxidative cellular injury by fluorescence measurement; Matsui H et al.; To develop a new method of detecting cellular injury caused by oxygen radicals, we studied endogenous fluorescence from the cultured cells of a rat gastric mucosal epithelial cell line . Measurement with an ultra-high sensitivity camera-image processor system under an inverted epifluorescence microscope showed that the fluorescence intensity of the cells increased time- and dose-dependently after the addition of hydrogen peroxide (H2O2), an oxygen radical precursor, to the medium . This increase was inhibited by the presence of catalase . Phase-contrast and fluorescence microscopy revealed that the fluorescence was emitted from granular substances in the cytoplasm of the injured cells . The spectral pattern of excitation and emission indicated that the fluorescent substances were flavins . In cell-free experiments, glutathione reductase which has flavin adenine dinucleotide (FAD) at the active site, increased in fluorescence after incubation with H2O2 in the presence of reduced glutathione and glutathione peroxidase . These findings indicate that FAD in the cytoplasm of cells injured by H2O2 increased in endogenous fluorescence according to the extent of injury, and suggest that fluorescence measurement may be a simple method in cellular toxicology to detect oxygen radical-induced injuries.

J Virol, 1998 Aug, 72(8), 6362 - 72
Multiple virulence determinants of foot-and-mouth disease virus in cell culture; Baranowski E et al.; Hypervirulent variants of foot-and-mouth disease virus (FMDV) of serotype C arise upon serial cytolytic or persistent infections in cell culture . A specific mutation in the internal ribosome entry site of persistent FMDV was previously associated with enhanced translation initiation activity that could contribute to the hypervirulent phenotype for BHK-21 cells . Here we report that several hypervirulent FMDV variants arising upon serial cytolytic passage show an invariant internal ribosome entry site but have a number of mutations affecting structural and nonstructural viral proteins . The construction of chimeric type O-type C infectious transcripts has allowed the mapping of a major determinant of hypervirulence to the viral capsid . Tissue culture-adapted FMDV displayed enhanced affinity for heparin, but binding to cell surface heparan sulfate moieties was not required for expression of the hypervirulent phenotype in Chinese hamster ovary (CHO) cells . Virulence was identical or even higher for glycosaminoglycan-deficient CHO cells than for wild-type CHO cells . FMDV variants with decreased affinity for heparin were selected from a high-binding parental population and analyzed . Substitutions associated with decreased heparin binding were located at positions 173 of capsid protein VP3 and 144 of capsid protein VP1 . These substitutions had a moderate effect on virulence for BHK-21 cells but completely abrogated infection of CHO cells . The comparative results with several FMDV isolates show that (i) increased affinity for heparin and alterations in cell tropism may be mediated by a number of independent sites on the viral capsid and (ii) the same capsid modifications may have different effects on different cell types.

Virology, 1998 Jul 5, 246(2), 306 - 16
Varicella-zoster virus ORF61 deletion mutants replicate in cell culture, but a mutant with stop codons in ORF61 reverts to wild-type virus; Cohen JI et al.; Varicella-zoster virus (VZV) ORF61 encodes a phosphoprotein that transactivates VZV promoters . Transfection of cells with cosmid DNAs, including a cosmid with a large deletion in ORF61, resulted in a VZV ORF61 deletion mutant that was impaired for growth in vitro and could be partially complemented by growth in neuroblastoma or osteosarcoma cell lines . Cells infected with the VZV ORF61 deletion mutant expressed normal levels of an immediate-early VZV protein, but had reduced levels of a late protein and showed abnormal syncytia . Carboxy terminal truncation mutants of VZV ORF61 protein have a transrepressing phenotype and inhibit the infectivity of cotransfected wild-type viral DNA . Transfection of cells with cosmid DNAs, including a cosmid with stop codons that should result in an ORF61 truncation mutant expressing a transrepressing protein that retains the RING finger domain, resulted in a viral genome which reverted back to the wild-type sequence . BAL-31 exonuclease was used to produce deletions at the site of the stop codons in ORF61 of the cosmid, resulting in loss of the RING finger domain . Transfection of tissue culture cells with the ORF61 BAL-31 deletion mutants and other cosmid DNAs yielded viable viruses . Thus, while deletion mutants lacking the RING finger domain of ORF61 replicate in cell culture, a mutant with stop codons that retains this domain could not be propagated and reverted to wild-type virus.

Neurosci Lett, 1998 May 15, 247(2-3), 103 - 6
Role of calcium in the activation of erp72 and heme oxygenase-1 expression on depletion of endoplasmic reticulum calcium stores in rat neuronal cell culture; Linden T et al.; Endoplasmic reticulum (ER) calcium pool depletion was induced by 30 min exposure of primary neuronal cells to thapsigargin (Tg), an irreversible inhibitor of ER Ca2+-ATPase . Twelve hours later, erp72 and heme oxygenase-1 (HO-1) mRNA levels were quantified by PCR . Protein synthesis was also measured . Transient Tg exposure of neurons induced a marked rise in mRNA levels (7-fold and a 21-fold increase in erp72 and HO-1 mRNA levels; P < 0.001) . Loading of neurons with the calcium chelator 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetra(acetoxymethyl)ester (BAPTA-AM) prior to thapsigargin treatment had only a minor effect on the Tg-induced rise in gene expression . This small inhibitory effect may result from the severe suppression of protein synthesis caused by BAPTA-AM . The results suggest that the increase in stress gene expression induced by exposure of neurons to Tg is triggered by a decrease in ER calcium activity and not by the corresponding increase in cytoplasmic calcium activity.

Acta Otolaryngol, 1998 Jun, 118(3), 337 - 43
Effects of transforming growth factor-beta1 and basic fibroblast growth factor on proliferation of cell cultures derived from human vestibular nerve schwannoma; Weerda HG et al.; The influence of transforming growth factor-beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF) on growth of cell cultures derived from unilateral vestibular nerve schwannomas was investigated . Cell cultures were initiated from 9 schwannomas and characterized immunocytochemically with antibodies against S-100 and type IV collagen . The effects of TGF-beta1 and bFGF on DNA synthesis in chemically defined serum-free medium were assessed by measuring the incorporation of 5-bromo-2'-deoxy-uridine (BRDU) into cellular DNA . Cell proliferation was evaluated with an electronic cell counter . Reverse transcription polymerase chain reaction (RT-PCR) was performed using oligonucleotide primers specific for TGF-beta1 and TGF-beta2 . TGF-beta1 stimulated DNA synthesis in a dose dependent manner . Maximal stimulation was observed at a concentration of 1 ng/ml, which induced a nearly 2-fold increase in DNA content . This effect was not seen when TGF-beta1 was added in the presence of neutralizing antibodies . In addition, antibodies against TGF-beta1 significantly reduced DNA synthesis in control cultures without supplemented exogenous growth factors . bFGF alone had no significant effects on DNA synthesis . In contrast, when TGF-beta1 and bFGF were added together, the mitogenic response was much greater than produced by TGF-beta1 alone . RT-PCR showed that the cultured cells expressed mRNA for both TGF-beta1 and TGF-beta2 . We hypothesize that TGF-beta1 is an autocrine growth factor for human vestibular nerve schwannomas in culture . A similar mechanism might be involved in the growth of these tumors in situ.

J Infect Dis, 1998 Jul, 178(1), 8 - 15
Limited variability of glycoprotein gene sequences and neutralizing targets in herpes simplex virus type 2 isolates and stability on passage in cell culture; Terhune SS et al.; Nucleotide sequence analyses of polymerase chain reaction-amplified genes were performed to determine whether adaptation of herpes simplex virus type 2 to replication in cultured cells or in internal organs during neonatal disseminated disease results in selection of variants with altered forms of three glycoproteins (gB, gC, or gD) that influence virus entry into cells . No variations in sequence were noted as a consequence of in vitro passage or replication in different organs . Five viruses from different subjects differed with respect to gB, gC, and gD gene sequences, expressing four distinct forms of gB, three of gC, and two of gD . These differences did not confer resistance to neutralization by guinea pig or human antisera from subjects immunized with recombinant gB or gD vaccines and may not be consequential for vaccine development.

J Clin Microbiol, 1998 Jul, 36(7), 2112 - 4
Use of fluorescent-antibody staining of cytocentrifuge-prepared smears in combination with cell culture for direct detection of respiratory viruses; Doing KM et al.; Over a 3-year period, 1,003 respiratory samples were collected and examined for selected respiratory viruses with cytocentrifuged prepared smears stained with fluorescently labeled antibodies (IFA) in conjunction with cell culture . IFA results were compared with results obtained by cell culture . Viruses were isolated or detected by direct means in 401 samples . Agreement between culture and IFA was 90%.

Methods Cell Biol, 1998, 57, 187 - 201
Invertebrate cell culture considerations: insects, ticks, shellfish, and worms; Bayne CJ; Establishment of cell lines from insect and arachnid invertebrates has become routine, whereas other invertebrate taxa have been frustratingly unproductive of cell lines . None is available for any marine invertebrate, despite a strong and well-recognized need for cell lines from species that are important in aquaculture, from parasite vectors and intermediate hosts of parasites, from parasites themselves, from certain biomedical models, and from other species that are pests . Drawing on experiences gained attempting to establish cell lines from molluscs and trematodes and on published and ongoing research with diverse invertebrates, this chapter attempts to anticipate the problems that are likely to be encountered in such endeavors and discusses possible solutions . Criteria to be considered in the selection of basic culture media, temperature, pH, and media additives; approaches that have been developed to yield sterile primary cultures; and factors to consider in decisions about feeding schedules, retention of tissue fragments and nonadherent cells, use of heterologous feeder layers, and other variables are described . Suggestions are made concerning means to objectively score the success of tested variables and means to induce cell replication . The chapter ends with notes on conventional means to characterize cell lines and an account of contemporary efforts to immortalize cells by means of genome manipulation . Enduring success with a single molluscan cell line, transient successes with crustacean and helminth cell lines, and promising developments in transgenesis with invertebrates all lead to the hopeful conclusion that the invisible barrier to cell propagation in historically refractory species will soon be a thing of the past.

Methods Cell Biol, 1998, 57, 49 - 65
Cell culture contamination: sources, consequences, prevention, and elimination; Lincoln CK et al.; The subject of the chapter is cell culture contamination . Contamination may enter the cell culture system as a physical, chemical, and/or biological component of the environment . The potential sources and consequences of cell culture contamination are unique to the cell culture system and the contaminant . A basic understanding of cell culture contamination is necessary to appreciate the need to develop and practice standardized cell culture procedures . General sources, consequences, and preventative measures are discussed for physical and chemical contamination based on current technology . Mycoplasmal contamination is the focus of the discussion on biological contamination and its impact on cell cultures . The introduction of other biological contaminants should be controlled by the institution of cell culture management procedures needed to minimize the incidence of mycoplasmal contamination . The need to eliminate the routine use of antibiotics in cell culture systems and institute routine testing to detect contamination is emphasized . More rapid detection of contamination should reduce the incidence of cross-contamination and minimize the consequences of any contamination event.

Kidney Int, 1998 Jul, 54(1), 87 - 98
PAI-1 secretion and matrix deposition in human peritoneal mesothelial cell cultures: transcriptional regulation by TGF-beta 1; Rougier JP et al.; BACKGROUND: Plasminogen activator inhibitor-1 (PAI-1), the main inhibitor of plasminogen activators in plasma and in peritoneum, impairs plasmin formation that is essential for the repair processes of the mesothelium damaged by peritoneal dialysis fluids and peritonitis . The fibrogenetic cytokine transforming growth factor-beta (TGF-beta) displays variable effects on extracellular matrix remodeling enzymes and their inhibitors depending on tissues and cell lines . We previously found an unexpected stimulating effect of TGF-beta 1 on matrix metalloproteinase-9 in peritoneal mesothelial cells . In this study, we analyzed the effects of TGF-beta 1 on PAI-1 production and deposition in extracellular matrix . METHODS: We used primary cultured mesothelial cells and a recently established human peritoneal mesothelial cell line (HMrSV5) . Cell-associated and secreted plasminogen activators and their inhibitors were detected and characterized by substrate gel zymography . PAI-1 was identified by reverse zymography and by Western blotting, and total PAI-1 was measured by ELISA . Secreted and cell-associated PA activity was measured by its ability to activate plasminogen into plasmin, that is, by the release of paranitroaniline from the plasmin synthetic substrate S-2251 . PAI-1 mRNA accumulation was assessed by Northern blot . In vitro nuclear run-on assays were carried out to determine whether TGF-beta 1 had transcriptional effects on PAI-1 expression . Finally, the subcellular distribution of PAI-1 was analyzed by immunofluorescence and by immunogold silver staining . RESULTS: TGF-beta 1 increased PAI-1 antigen in the conditioned media of HMrSV5 cells, in a time- and concentration-dependent manner . This induced a dramatic decrease of free tPA in the cell medium and of membrane-bound uPA, and a parallel increase of high molecular weight PA-PAI complexes . Consequently, secreted and cell-associated plasminogen activator activities were considerably reduced . In primary cultured peritoneal mesothelial cells, TGF-beta 1 also induced PAI-1 secretion and the shift of tPA toward high molecular weight complexes . TGF-beta 1 increased PAI-1 mRNA in a time- and concentration-dependent manner . This effect was at least in part transcriptional since an approximately threefold increase in the rate of PAI-1 gene transcription was observed in nuclei sampled after a four-hour cell exposure to 5 ng/ml TGF-beta 1 . Finally, TGF-beta 1 substantially increased the amount of intracellular and matrix-associated PAI-1 . CONCLUSIONS: These results suggest that excessive TGF-beta 1 stimulated PAI-1 could prevent appropriate peritoneal healing by impairing the degradation of fibrin and of unorganized matrix components, and by interfering with cell migration.

J Biomater Sci Polym Ed, 1998, 9(5), 489 - 505
Periodontal ligament cell culture on the hydrophobic substrate coated with proteins of periodontal ligament fibroblast-conditioned medium; Kinoshita Y et al.; In regenerating periodontal ligament (PDL) around the root of an artificial tooth, an important role is played by some physiologically active substance that promotes adhesion of the cells to the surface of the tooth root and induces cell proliferation and differentiation . In this study, the supernatant of the conditioned medium (CM) of dog periodontal ligament fibroblast (DPLF) was fractionated using an ion exchange chromatography-diethylaminoethyl (IEC-DEAE) column . DPLFs were cultured on hydrophobic dishes coated with each fraction . Cell proliferative activity and alkaline phosphatase (ALPase) activity, including electron microscopic features of the contact surface between the cells and the dish, were investigated . The DPLF-CM was separated by IEC-DEAE column into six fractions . Each fraction promoted an increase in DNA content and ALPase activity of the cultured DPLF, and especially remarkable were fractions 2 and 3 . Fraction 2 at a molecular weight (Mw) of 210, 160, 85, 50 and 22 kD, and fraction 3 at Mw = 21 and 23 kD contained the type of proteins not found in other fractions . Electron microscopic analysis revealed that the cells in the coating group were in close contact with the surface of the dishes and that fine fibers protruding from the cell membrane clinged to the dishes . In the control group, a wide gap between the cells and the dishes was observed . These findings suggest that the DPLF-CM fractions contain specific physiological activating factors that induce proliferation and differentiation as well as cell adhesion of the DPLF cells.

Pharm Res, 1998 Jun, 15(6), 944 - 9
Development of infrared imaging to measure thermogenesis in cell culture: thermogenic effects of uncoupling protein-2, troglitazone, and beta-adrenoceptor agonists; Paulik MA et al.; PURPOSE: Although the effects of thermogenic agents in cell culture can be measured by direct microcalorimetry, only a few samples can be analyzed over several hours . In this report, we describe a robust non-invasive technique to measure real-time thermogenesis of cells cultured in microtiter plates using infrared thermography . METHODS: Yeast were transformed with uncoupling protein-2 (UCP2) or exposed to carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) or rotenone . Adipocytes were exposed to rotenone, FCCP, cycloheximide . troglitazone, or CL316243 . Thermogenesis was measured using infrared thermography . RESULTS: Thermogenesis increased after exposing yeast to the mitochondrial uncoupler, FCCP, or transforming the cells with UCP2 . Further, thermogenesis in adipocytes was stimulated by CL316243, a beta3-adrenoceptor agonist being developed to treat obesity . The protein synthesis inhibitor, cycloheximide, did not inhibit CL316243-mediated thermogenesis . In contrast, the mitochondrial proton transport inhibitor, rotenone, inhibited thermogenesis in yeast and adipocytes . Similarly, the antidiabetic agent, troglitazone, suppressed thermogenesis in adipocytes . Although increased UCP synthesis resulted in increased thermogenesis in yeast, UCP expression did not correlate with thermogenesis in adipocytes . CONCLUSIONS: The results, taken together with the high resolution (0.002 degrees C) and robustness (384-well format) of the approach, indicate infrared-imaging is a rapid and effective method for measuring thermogenesis in vitro.

J Parasitol, 1998 Jun, 84(3), 635 - 7
Complete development of the porcine coccidium Isospora suis Biester, 1934 in cell cultures; Lindsay DS et al.; Development from inoculated sporozoites to unsporulated oocysts of Isospora suis Biester, 1934 is described in a swine testicular (ST) cell line . Sporozoites penetrated ST cells within 1 hr postinoculation (PI) . Development was initially by endodyogeny to produce binucleate type I meronts and type I merozoites . Division by endodyogeny continued during the 13-day observation period and type I merozoites were the developmental stages most abundant at observation periods >3 days PI . Mutinucleate type II meronts and type II merozoites were first observed 7 days PI . Gamonts and oocysts were present 12 days PI . Oocysts did not sporulate in vitro . The ultrastructural features of stages were similar to those that occur in the pig host.

Am J Reprod Immunol, 1998 Jun, 39(6), 399 - 405
Immunodetection of cell adhesion molecules in rat Sertoli cell cultures; Lustig L et al.; PROBLEM: The presence of cell adhesion molecules (CAMs) in Sertoli cells has not been explored extensively . The expression of CAMs involved in cell-matrix and cell-to-cell interactions in Sertoli cell cultures was examined . METHOD OF STUDY: Immunohistochemical and Western blot techniques were applied to rat Sertoli cell cultures using specific antibodies to alpha 3, alpha 5, and alpha 6 integrin subunits; NCAM; and cadherins . RESULTS: Expression of alpha 3 and alpha 6 integrin subunits (mainly laminin receptors) and lack of expression of alpha 5 integrin subunit (fibronectin receptor) was observed in Sertoli cells by immunohistochemistry . These cells also expressed neural CAM (NCAM) and N-cadherin . By Western blot analysis, Sertoli cell extracts reacted with antibodies to alpha 3 integrin subunit revealed a band approximately 130 kDa, whereas no expression of alpha 5 integrin subunit was detected . Cell extracts incubated with antibodies to pan cadherin exhibited a band approximately 120 kDa, whereas bands of 180, 140, and 120 kDa were observed with antibodies to NCAM . CONCLUSION: New data about the expression of receptors for extracellular matrix proteins (alpha 3 and alpha 6 integrin subunits) as well as cell-to-cell adhesion molecules (NCAM and cadherins) are reported in rat Sertoli cell cultures.

Biochem Biophys Res Commun . 1998 Jun 18;247(2):536.
Volume 244, number 1 (1998), in article no . RC988251, "Functional coupling of secretion and capacitative calcium entry in PC12 Cells," by schuichi koizumi and kazuhide inoue, pages 293-297, and in article no . RC978051, "Hydrocortisone reinforces the blood-brain barrier properties in a serum free cell culture System," by dirk hoheisel, thorsten nitz, helmut franke, joachim wegener, ansgar hakvoort, thomas tilling, and hans-joachim galla, pages 312-316:
Hydrocortisone reinforces the blood-brain properties in a serum free cell culture system.
Institut fur Biochemie, Westfalische Wilhelms-Universitat, Munster, GermanyThe increasing number of newly developed drugs demands for functional in vitro models of the blood-brain barrier to determine their brain uptake . Cultured cerebral capillary endothelial cells are considered to be such a model, however in serum containing media they exhibit low electrical resistances and high permeabilities compared to the in vivo situation . Here we report the establishment of a serum-free cell culture model . Withdrawal of serum already caused a twofold increase of transendothelial resistance (TER), which in presence of serum is about 100-150 Omega x cm2 . We tested several supplements and found that hydrocortisone is a potent stimulator for the formation of barrier properties . TERs up to 1000 Omega x cm2 were measured in the presence of physiological relevant hydrocortisone concentrations . In correspondence to the TER increase hydrocortisone decreased cell monolayer permeability for sucrose down to 5x10(-7) cm/s, which is close to the in vivo value of 1.2x10(-7) cm/s and by a factor of five lower compared to cultures without hydrocortisone and in presence of serum.

Plant Physiol, 1998 Jun, 117(2), 585 - 92
Transcriptional down-regulation by abscisic acid of pathogenesis-related beta-1,3-glucanase genes in tobacco cell cultures; Rezzonico E et al.; Class I isoforms of beta-1,3-glucanases (betaGLU I) and chitinases (CHN I) are antifungal, vacuolar proteins implicated in plant defense . Tobacco (Nicotiana tabacum L.) betaGLU I and CHN I usually exhibit tightly coordinated developmental, hormonal, and pathogenesis-related regulation . Both enzymes are induced in cultured cells and tissues of cultivar Havana 425 tobacco by ethylene and are down-regulated by combinations of the growth hormones auxin and cytokinin . We report a novel pattern of betaGLU I and CHN I regulation in cultivar Havana 425 tobacco pith-cell suspensions and cultured leaf explants . Abscisic acid (ABA) at a concentration of 10 micron markedly inhibited the induction of betaGLU I but not of CHN I . RNA-blot hybridization and immunoblot analysis showed that only class I isoforms of betaGLU and CHN are induced in cell culture and that ABA inhibits steady-state betaGLU I mRNA accumulation . Comparable inhibition of beta-glucuronidase expression by ABA was observed for cells transformed with a tobacco betaGLU I gene promoter/beta-glucuronidase reporter gene fusion . Taken together, the results strongly suggest that ABA down-regulates transcription of betaGLU I genes . This raises the possibility that some of the ABA effects on plant-defense responses might involve betaGLU I.

J Muscle Res Cell Motil, 1998 May, 19(4), 343 - 51
Expression of lactate dehydrogenase, myosin heavy chain and myogenic regulatory factor genes in rabbit embryonic muscle cell cultures; Barjot C et al.; The expression of myogenic regulatory factors (MRFs), lactate dehydrogenase (LDH) and myosin heavy chains (MyHC), as markers of myogenesis, metabolism and contractility respectively, were investigated during differentiation of rabbit embryonic muscle cells in primary culture . Myf5, MyoD and myogenin mRNAs were abundantly expressed at day 1 of culture . The expression of Myf5 and MyoD mRNA transcripts decreased sharply as myoblasts fused and differentiated into myotubes, whilst myogenin mRNA was maintained throughout the duration of the culture . In contrast, MRF4 mRNA was weakly expressed on day 1 of culture, its expression increased slightly as myoblasts fused and reached a maximum level in 7-day-old cultures containing striated myofibres . The specific activity of LDH increased linearly during myoblast proliferation and fusion . In 7-day-old cultures, LDH-M mRNA (dominant in glycolytic muscles) and LDH-H mRNA (predominant in perinatal and oxidative muscles) represented 38% and 62% of total LDH mRNA respectively . At this stage, immunocytochemical staining with perinatal and adult-type MyHC antibodies showed that embryonic and perinatal MyHC isoforms were expressed in all myotubes, while few of them were stained by type I MyHC antibody . However, none of them expressed adult type II MyHC . The latter results were further supported by RT-PCR analysis of adult-type MyHC mRNA which showed that only the type I MyHC mRNA transcript was expressed . These data were in agreement with those reported in vivo on perinatal rabbit muscles . They differed from those obtained on cultured satellite cells isolated from adult rabbit fast-twitch or slow-twitch muscles which did not express embryonic MyHC, and instead expressed fast- or slow-type MyHC according to their muscle origin . Taken together, these results further suggest that myogenic mononucleated cells express different properties in vitro according to their developmental origin as well as properties related to those of the muscles from which they were isolated.

Biotechnology (N Y), 1995 Jul, 13(7), 692 - 8
Removal of sialic acid from a glycoprotein in CHO cell culture supernatant by action of an extracellular CHO cell sialidase; Gramer MJ et al.; We have directly tested the hypothesis that Chinese hamster ovary (CHO) cell-produced glycoproteins are subject to extracellular degradation by a sialidase endogenous to the CHO cell line . Factors important to understanding the potential for extracellular degradation are addressed including the glycoprotein specificity, subcellular source, mechanism of release, and stability of the sialidase activity . The extracellular CHO cell sialidase apparently originates from the cytosol of the cells, and is released to the cell culture supernatant as a result of damage to the cellular membrane . The extracellular sialidase is active toward a variety of CHO cell-produced glycoproteins, and can hydrolyze sialic acid from the recombinant glycoprotein gp120 in the culture supernatant . While measuring the actual degradation of a glycoprotein by extracellular CHO cell sialidase can be difficult, data presented here suggest that the level of degradation can be estimated indirectly by using a more convenient fluorescent substrate, 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid, to quantify sialidase activity . Degradation by sialidase is minimized through addition of the sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid to the culture supernatant . The results in this study suggest additional potential approaches for minimizing degradation by sialidase, including isolation of a sialidase-deficient CHO cell line.

J Gen Virol, 1998 Jun, 79 ( Pt 6), 1383 - 6
Infection of a chimpanzee with hepatitis C virus grown in cell culture; Shimizu YK et al.; Culture supernatant harvested from Daudi cells, a lymphoplastoid cell line, after 58 days of infection with the H77 strain of hepatitis C virus (HCV), was inoculated into a chimpanzee . HCV RNA, as detected by RT-PCR, first appeared in the serum and liver 5 and 6 weeks, respectively, after inoculation . Peripheral blood mononuclear cells (PBMC) collected on week 7 were also positive for HCV RNA . The major sequences of hypervariable region 1 (HVR1) of the viral genome recovered from the inoculated chimpanzee were the ones which were the majority in the original H77 inoculum and not those which were in the majority in the culture supernatant . Only the sequence recovered from PBMC was the same as the major one found in the cell culture.

Mutat Res, 1998 May 11, 414(1-3), 125 - 9
Factors affecting the genotoxic potency ranking of natural anthraquinones in mammalian cell culture systems; Mueller SO et al.; We had reported that the plant-derived 1,8-dihydroxyanthraquinone derivatives, emodin and danthron, were clearly genotoxic in mouse lymphoma L5178Y cells, whereas chrysophanol was only weakly genotoxic and physcion not at all . Danthron was more potent than emodin . Furthermore, we had found that these compounds bound non-covalently to DNA and inhibited topoisomerase II activity . Interestingly, in these systems emodin was more potent than danthron . This inversion of the ranking prompted us to investigate the underlying mechanism . Since emodin shows a high serum-protein binding affinity, horse serum used as a media-supplement in the mouse lymphoma genotoxicity assays was analyzed for a potential selective scavenging of emodin . Non-covalent DNA-binding in mouse lymphoma L5178Y cells was investigated in the absence or presence of serum . In the presence of 10% serum, the DNA-binding potency of emodin was markedly reduced and was lower than that of danthron . We also applied mutation assays with mouse lymphoma cells and AS52 cells and varied the serum concentration used . In the absence of serum emodin showed slightly higher mutagenicity in AS52 cells than danthron . At reduced serum concentration (0.5%) emodin was strongly cytotoxic to the mouse lymphoma cells . For chrysophanol and physcion, a considerable reduction of the non-covalent DNA-binding potency in intact cells was found when compared to danthron, in concordance with their lower genotoxic potency . Overall, these data support the understanding that the genotoxicity of anthraquinones is, at least in part, mediated by non-covalent DNA-binding .

Environ Res, 1998 Jul, 78(1), 25 - 37
Dissolution of short and long rockwool and glasswool fibers by macrophages in flowthrough cell culture; Luoto K et al.; Dissolution of MMVF (man-made vitreous fibers) by macrophages has previously been studied utilizing cell cultures in wells . A new, more dynamic method has been developed to explore the effects of macrophages on MMVF dissolution . In this method, the culture medium flows through a membrane on which the macrophages and fibers are placed . The dissolution of short and long rockwool and glasswool fibers was investigated in the present study by macrophages by assessing the dissolution of Si (silicon), Fe (iron), and Al (aluminium) from the fibers . Dissolution of these elements usually increased as a function of time . Generally, the dissolution of elements from the fibers in the flowthrough culture exceeded that observed with the culture in wells system . The dissolution of glasswool fibers was greater in medium than in cell culture, whereas the opposite was true for rockwool fibers . Dissolution of Si was greater from glasswool than from rockwool fibers, while the opposite was true for Fe and Al . Macrophages that had phagocytized fibers in flowthrough culture contained Si, and there were also precipitations with Si in the samples . The fibers in the flowthrough culture also exhibited surface changes such as breakings, pittings, etching, and peeling . The short rockwool fibers tended to fracture more than short glasswool fibers, while long glasswool fibers were more extensively broken than short glasswool fibers . The results with this new, dynamic, flowthrough culture method with macrophages demonstrate that this method provides valuable information on the abilities of macrophages to dissolve MMVF leading to subsequent morphological changes of fibers.

J Neuroimmunol, 1998 May 15, 85(2), 111 - 21
Cytokine and chemokine production in HSV-1 latently infected trigeminal ganglion cell cultures: effects of hyperthermic stress; Carr DJ et al.; The establishment of a primary trigeminal ganglion (TG) cell culture latently infected with herpes simplex virus type 1 (HSV-1) has been useful in studying stress-induced reactivation of the latent virus . However, the immune profile of this culture system prior to and after stress has never been established . In the present manuscript, cytokine and chemokine production were measured in primary cultures of TG cells obtained from uninfected and HSV-1 latently infected mice . Supernates from TG cell cultures contained detectable interleukin (IL)-6 but not IL-1beta, IL-2, IL-10, interferon (IFN)-gamma or tumor necrosis factor (TNF)-alpha as determined by ELISA . The basal level of IL-6 in uninfected TG cell cultures was 20.5 +/- 2.3 ng/ml, whereas latently infected TG cells produced significantly less IL-6 (12.1 +/- 1.9 ng/ml) . Supernates from TG cell cultures also contained detectable levels of C-10, MCP-1 and eotaxin but little to no MIP-1alpha, MIP-1beta, or MIP-2 . While there were no differences in the basal level of MCP-1 and eotaxin in TG cell cultures from HSV-1-infected and uninfected mice, C10 levels were significantly higher in TG cultures originating from infected mice compared to uninfected ones (5.86 +/- 0.61 ng/ml compared to 1.18 +/- 0.16 ng/ml) . Hyperthermic stress (43 degrees C, 180 min), which induces reactivation of latent HSV-1 from TG cell cultures, significantly reduced IL-6 and C-10 levels from both uninfected and latently infected TG cell cultures . However, there was no correlation between cytokine/chemokine levels and HSV-1 reactivation . Immunofluorescent studies showed TG cell cultures contained 10% MAC-3+ staining cells (macrophage specific) but no dendritic cells . By comparison, cells from freshly isolated TG contained 6% positive dendritic cells but < 1% MAC-3 + cells . Both in vivo and in vitro TG consisted of a low percentage of CD3+ and CD8+ cells . Hyperthermic stress (43 degrees C for 3 h) eliminated the lymphocyte population as determined by RT-PCR . Whereas no spontaneous reactivation has been reported in mice, spontaneous reactivation occurred in 4.5% (10/220) of TG cell cultures surveyed over a 20 day period . Collectively, the dichotomy between HSV-1 replication and reactivation comparing the in vitro and in vivo HSV-1 latency models may reside, in part, to the differences in the levels of cytokines, chemokines and immune cell populations within the microenvironment of the in vitro and in vivo TG.

J Eukaryot Microbiol, 1998 May-Jun, 45(3), 344 - 6
Effect of conditioned media from chicken and turkey intestinal cell cultures on invasion by sporozoites of three species of avian coccidia; Augustine PC et al.; The effect of conditioned media from cultures of turkey and chicken intestinal cells on cellular invasion by sporozoites of avian Eimeria species was examined in vitro . Media conditioned by the growth of cells from the ceca, mid-intestine (area of the yolk stalk diverticulum), and duodenal loop were examined for their ability to enhance invasion . Conditioned medium from cultures of turkey cecal cells significantly enhanced invasion by the turkey coccidia Eimeria adenoeides, by 2.4-fold, and E . meleagrimitis, by 2.2-fold, as compared with invasion in the presence of control medium . Conditioned medium from mid-intestinal cell cultures enhanced invasion by the two coccidial species by 2.0- and 2.1-fold, respectively . The enhancement occurred with conditioned media from early (1) as well as later (11) passages of cells . This suggests that the enhancing factor was produced by fibroblast-like cells, the predominant cell type at both early and late passages, and not by epithelial-like cells that had disappeared by the first or second passage . Additionally, conditioned media from cultures of chicken cecal and duodenal loop cells significantly enhanced invasion by the turkey cecal coccidium, E . adenoeides, (1.7- and 1.6-fold, respectively) . This was less enhancement than was caused by the turkey cell conditioned media . Heat treatment (56 degrees C for 45 min) of conditioned media failed to alter the effect on invasion . Neither the turkey or chicken cecal cell media nor conditioned media from any other chicken intestinal cell cultures enhanced invasion by E . tenella, the chicken cecal coccidium . Although morphologically dissimilar when they were first plated, the gross appearance and growth of the turkey and chicken cells when conditioned media was collected was comparable.

J Magn Reson Imaging, 1998 May-Jun, 8(3), 687 - 9
Preclinical assessment of hepatocyte-targeted MR contrast agents in stable human liver cell cultures; Reimer P et al.; Much effort has been expended in the search for hepatocyte-specific MR contrast agents to improve the detection and characterization of liver tumors . The purpose of this study was to establish human hepatocyte cell cultures to preclinically assess hepatocyte-targeted magnetopharmaceuticals . Cultured human hepatocytes were sandwiched between two layers of collagen preserving both hepatocyte function and morphology over prolonged period of time . Cultures (n = 37) were subsequently used to test different fluorescinated MR contrast agents . Plain and rhodaminated monocrystalline iron oxide particles (MION and MION-rh) and asialoglycoprotein-receptor-specific rhodaminated asialofetuin coupled to MION (MION-ASF-rh) were prepared . Competition experiments of these agents were performed with D(+)-galactose to study the specificity of galactose-mediated cell uptake . To assess the impact of cell integrity on cell uptake, functional experiments with CCl4 were performed . Normal cell cultures showed significantly higher fluorescence light emission after incubation with hepatocyte-directed ASF-MION-rh than after incubation with MION-rh . Competition experiments of ASF-MION-rh with galactose showed a dose-dependent decrease of calibrated fluorescence light emission . Cell cultures treated with CCl4 demonstrated a dose-dependent significant reduction of calibrated fluorescence light emission, indicating reduced uptake of ASF-MION-rh . Our data demonstrate that stable human hepatocyte cell cultures can be used to preclinically assess novel magnetopharmaceuticals . Different contrast agents may be directly compared to each other and may accelerate their preclinical design . Because the assay can be applied to cells from any species, it may represent an ideal test system before clinical trials of new cell-directed MR contrast agents.

Biotechnol Prog, 1998 May-Jun, 14(3), 434 - 41
Variation of stoichiometric ratios and their correlation for monitoring and control of animal cell cultures; Zeng AP et al.; The stoichiometry of animal cell cultures is examined with respect to its variation and suitability for process monitoring and control . In addition to the two often used stoichiometric ratios, i.e., lactate yield from glucose (Lac/Glc) and ammonium yield from glutamine (NH4+/Gln), five other less well characterzied ones, i.e., ammonium yield from the total consumption of amino acids (NH4+/TAA), consumption of total amino acids to glutamine (TAA/Gln), essential amino acids to glutamine (EAA/Gln), glutamine to glucose (Gln/Glc), and oxygen to glucose (OUR/Glc), are also considered . A comparison of a number of cell lines including hybridoma, BHK, and CHO cells under a wide range of experimental conditions revealed that all the cell lines have similar patterns of variation of stoichiometry . In steady states of continuous culture, Lac/Glc and Gln/Glc are primarily determined by the residual glucose concentration while TAA/Gln and EAA/Gln correlate well with the residual glutamine concentration . Ammonium formation not only is a function of glutamine concentration but also is affected by the consumption of other amino acids, particularly at low residual glutamine concentrations . NH4+/TAA turned out to be a more suitable parameter to describe the ammonium formation . Large variations of all these stoichiometric ratios are found under conditions of relatively low residual concentrations of glucose and glutamine (both ca . < 0.2-0.5 mM) . Above these concentrations the stoichiometric ratios are relatively constant and are independent of the cell lines . Thus, the correlations for these stoichiometric ratios may be directly used to control the nutrient concentration at low levels which are otherwise on-line difficult to determine . A stoichiometric equation is also derived for oxygen consumption . It is found that the metabolism of amino acids can significantly contribute to the consumption of oxygen . A correlation is obtained for OUR/Glc which may be used for the monitoring and control of mammalian cell cultures.

J Neural Transm Suppl, 1998, 52, 239 - 50
Properties and functions of tissue-bound semicarbazide-sensitive amine oxidases in isolated cell preparations and cell cultures; Lyles GA et al.; The demonstration of semicarbazide-sensitive amine oxidase (SSAO) activity in some freshly-dispersed cell preparations and in particular types of cells grown in culture, provides increasing opportunities for investigating the importance of SSAO in various aspects of cellular function . Assays of benzylamine and methylamine metabolism in homogenates of cultured cells have established clearly that SSAO is expressed in rat and pig vascular (aortic) smooth muscle cells, as well as in rat non-vascular (anococcygeus, trachea) smooth muscle, brown and white adipocytes . However, to date little or no SSAO activity has been detected in cultures of human vascular smooth muscle cells grown from blood vessels (e.g . umbilical artery) known to contain the enzyme, and the reason for this is not yet apparent . However, those cell cultures expressing SSAO are offering useful experimental models for studying biochemical and toxicological consequences upon cellular function which may result from the metabolism of various aromatic and aliphatic amines suggested to be possible physiological and xenobiotic substrates of the enzyme.

J Neural Transm Suppl, 1998, 52, 93 - 8
A cell culture model of cerebral ischemia as a convenient system to screen for neuroprotective drugs; Ekblom J et al.; Aggregation cultures of rat brain were exposed to a combination of anoxia and hypoglycaemia for 30 minutes . Thereafter, the release of lactate dehydrogenase into the cell culture medium was monitored up to 4 days as a measure of cell damage after the ischemic insult . Some cultures were treated with different concentrations of deprenyl or tolcapone, selective inhibitors of monoamine oxidase B and catechol-O-methyltransferase, respectively . After 1 day in culture, the release of lactate dehydrogenase was significantly reduced in cultures treated with deprenyl (at 1 nM . 100 nM, and 10 microM), as well as in cultures treated with 1 nM or 100 nM tolcapone; 10 microM of tolcapone, on the other hand, resulted in a toxic effect on the cell aggregates . No differences in the release of lactate dehydrogenase into the medium was observed in the aggregates treated with drugs as compared with the control cultures after 2 or 4 days post-ischemia.

J Neural Transm Suppl, 1998, 52, 87 - 91
Modulation of glutamate neurotoxicity in the transformed cell culture by monoamine oxidase inhibitors, clorgyline and deprenyl; Abakumova OYu et al.; Addition of 30mM glutamate to the culture medium decreased growth of rat glioma C6 cells accompanied by a decrease of DNA synthesis and an increase of lactate dehydrogenase (LDH) detected in the conditioned medium . The presence of 1 microM deprenyl attenuated the glutamate effect on cell growth only during the first 24-48 h incubation and had a minor influence on the glutamate-induced decrease of DNA synthesis . Clorgyline (1 microM) potentiated glutamate-induced DNA synthesis during the first 24 h incubation without significant influence on the cell growth . Deprenyl slightly attenuated the glutamate-induced LDH increase during 24 h incubation but potentiated the glutamate effect at 96 h . Clorgyline decreased the glutamate influence at 24 h and especially 96 h . All these effects were observed in the absence of exogenous monoamines in the culture medium . These results suggest that in transformed cells monoamine oxidase (MAO) inhibitors may influence processes of cell death via MAO-independent mechanisms.

Brain Res Dev Brain Res, 1998 Feb 10, 105(2), 219 - 25
Muscimol-induced death of GABAergic neurons in rat brain aggregating cell cultures; Honegger P et al.; During brain development, spontaneous neuronal activity has been shown to play a crucial role in the maturation of neuronal circuitries . Activity-related signals may cause selective neuronal cell death and/or rearrangement of neuronal connectivity . To study the effects of sustained inhibitory activity on developing inhibitory (GABAergic) neurons, three-dimensional primary cell cultures of fetal rat telencephalon were used . In relatively immature cultures, muscimol (10 microns), a GABAA receptor agonist, induced a transient increase in apoptotic cell death, as evidenced by a cycloheximide-sensitive increase of free nucleosomes and an increased frequency of DNA double strand breaks (TUNEL labeling) . Furthermore, muscimol caused an irreversible reduction of glutamic acid decarboxylase activity, indicating a loss of GABAergic neurons . The muscimol-induced death of GABAergic neurons was attenuated by the GABAA receptor blockers bicuculline (100 microns) and picrotoxin (100 microns), by depolarizing potassium concentrations (30 mM KCl) and by the L-type calcium channel activator BAY K8644 (2 microns) . As compared to the cholinergic marker (choline acetyltransferase activity), glutamic acid decarboxylase activity was significantly more affected by various agents known to inhibit neuronal activity, including tetrodotoxin (1 micron), flunarizine (5 microns), MK 801 (50 microns) and propofol (40 microns) . The present results suggest that the survival of a subpopulation of immature GABAergic neurons is dependent on sustained neuronal activity and that these neurons may undergo apoptotic cell death in response to GABAA autoreceptor activation.

Trop Med Int Health, 1998 May, 3(5), 385 - 90
Cytopathic effects of Blastocystis hominis on Chinese hamster ovary (CHO) and adeno carcinoma HT29 cell cultures; Walderich B et al.; Blastocystis hominis isolates from asymptomatic carriers and symptomatic patients were cultured in vitro, purified from the co-cultivated bacterial flora and tested for cytopathic effects on monolayers of Chinese Hamster Ovary (CHO) cells and Adeno Carcinoma HT29 cells . In the case of the CHO cells, living B . hominis cells and B . hominis cell lysates were able to cause significant cytopathic effects, which were dependent on the concentration of cells employed . Destruction of the cell monolayers was observed to the same extent with patient isolates derived from healthy or symptomatic B . hominis carriers . HT29 cells were less susceptible: B . hominis cells and cell lysates caused only minor effects which were not statistically significant . Culture filtrates of B . hominis exhibited cytopathic potential on CHO and HT29 cells; however, the control which consisted of filtrates from Robinson's cultures in which B . hominis failed to grow showed similar effects, too . Therefore the culture supernatants could not be proven to produce a specific cytopathic effect on CHO and HT29 cells.

Biomed Mater Eng, 1997, 7(6), 369 - 77
Biochemical analysis of the response in rat bone marrow cell cultures to mechanical stimulation; Yoshikawa T et al.; Bone marrow cells obtained from rat femora were subjected to primary culture with 15% fetal bovine serum in the presence of 10(-8) M dexamethasone, and following trypsin treatment 5 days later were seeded on Petriperm dishes which have a flexible bottom . After a 2-day subculture, a cyclic stress consisting of a 1 s stretch (0.3% strain . 0.5 Hz) and a 1 s relaxation for 30 min every day was started . Culture tissue was removed on day 2 of the subculture (immediately prior to start of stimulation), and then on days 5 and 8 (3 and 6 days after the start of stimulation, respectively), at which times dry weight, DNA, alkaline phosphatase (ALP) activity, and bone Gla protein (BGP, osteocalcin) were measured . Both the dry weight and DNA showed a significant increase in the stimulated group by day 8, while the ALP activity showed a significant increase by day 5 . The BGP began to increase in the stimulated group on day 5 in contrast to the control group in which it only increased on day 8 . These results support the contention that mechanical stimulation promotes the differentiation of osteogenic cells and enhances bone formation . Since in this experimental model the acceleration of bone formation by mechanical stimulation can be reproduced in vitro, it is extremely useful for investigating the mechanisms underlying mechanical stimulation.

Eur J Med Res, 1998 Jun 17, 3(6), 288 - 94
Soluble intercellular adhesion molecule 1 (sICAM-1) in bronchoalveolar lavage (BAL) cell cultures and in the circulation of patients with tuberculosis, hypersensitivity pneumonitis and sarcoidosis; Baumer I et al.; Intercellular adhesion molecule-1 (ICAM-1) plays an important role in inflammatory diseases . It is believed that its soluble form (sICAM-1) might be a serum parameter of inflammatory activity with possible relevance in granulomatous disorders . To evaluate this role we measured sICAM-1 by ELISA in serum and shedding of this molecule by BAL cells in patients with granulomatous lung diseases (pulmonary tuberculosis (TB), hypersensitivity pneumonitis (HSP), pulmonary sarcoidosis (PS), and controls) . Serum concentrations of sICAM-1 in patients with TB (496.9 +/- 49.7 ng/ml), with HSP (636.5 +/- 85.9.8 ng/ml), and with PS (588.3 +/- 72.2 ng/ml) were significantly increased compared to controls (275.7 +/- 33.1 ng/ml) . Spontaneous release of sICAM-1 by BAL cells differed among patient groups (TB: 9.3 +/- 1.7; HSP: 17.5 +/- 1.4; PS: 9.7 +/- 1.5 ng/ml), however, exceeding that of controls significantly (3.8 +/- 0.6 ng/ml) . No correlations between the circulating level and the shedding of this molecule by BAL cells were observed within the groups . Significant correlations between serum sICAM-1 and serum tumor necrosis factor alpha (TNFalpha) level were observed in patients with HSP and TB . Kinetic cell culture experiments with BAL cells revealed a dissociation in sICAM-1 shedding and TNFalpha release . After stimulation rapid upregulation of both molecules (5 h) was followed by a cessation of TNFalpha production at 28 h . sICAM-1 shedding, however, was maintained over 2 days . Our results evidence that the circulating pool of sICAM-1, as well as the shedding of this molecule by BAL cells reflect the activity of cells in the inflammatory processes of granulomatous diseases.

Ophthalmic Res, 1998, 30(3), 180 - 8
Antiuveitis and inhibition of fibroblast-like corneal and conjunctival cell cultures by interleukin-1 blockers, CK 125, CK 126 and CK 128; Xuan B et al.; Three new interleukin-1 (IL-1) blockers, CK 125, CK 126 and CK 128, were studied for their effects on IL-1-induced uveitis in rat eyes . They were more potent (at 3-10 mg/kg t.i.d.) than prednisolone (20 mg/kg t.i.d.) in effectively inhibiting posterior uveitis . They were also found to inhibit fibroblast-like corneal cells at 10-300 micrograms/ml concentrations and conjunctival cells at 1-30 micrograms/ml levels . The incorporation of leucine into corneal and conjunctival cells was either stimulated or unaffected by CK 126, indicating that the inhibition of cell growth has nothing to do with the protein synthesis . However, the incorporation of uridine into corneal and conjunctival cells was markedly inhibited by CK 126 at 3-30 micrograms/ml concentrations whereas the incorporation of thymidine into the cells was inhibited at a lesser extent than that of uridine . These results indicate that cell inhibition by CK 126 could be related mainly to the synthesis of mRNA and, to a lesser extent, to DNA synthesis.

Invest Ophthalmol Vis Sci, 1998 Jun, 39(7), 1227 - 32
Protective effects of FK506 against glutamate-induced neurotoxicity in retinal cell culture; Kikuchi M et al.; PURPOSE: To examine the effects of FK506 on glutamate neurotoxicity in cultured retinal neurons . METHODS: Experiments were performed with primary retinal cultures obtained from 17- to 19-day-old rat fetuses . To assess the effects of FK506 and other drugs on glutamate neurotoxicity, cultures were treated with a drug beginning 10 minutes before application of glutamate and continuing during the subsequent 10 minutes of glutamate exposure . The treated cells were then incubated for 1 hour in a drug-free and glutamate-free medium . After a 1-hour incubation, cell viability was quantitatively measured by the trypan blue exclusion method . RESULTS: Brief exposure to glutamate markedly decreased cell viability . FK506 protected against glutamate neurotoxicity in a dose-dependent manner . Rapamycin is a competitive inhibitor of FK506 that binds FK506 binding protein . Simultaneous application of rapamycin and FK506 negated the protective effects of FK506 . Cyclosporin A, which binds and inhibits calcineurin, mimicked the protective effects of FK506 . Treatment with FK506 did not affect the intracellular maximum Ca2+ concentration induced by glutamate application . Although FK506 exhibited protective action against Ca2+ ionophore-induced neurotoxicity, it had no effect on nitric oxide-induced neurotoxicity . Treatment with FK506 reduced the activity of nitric oxide synthase (NOS) . CONCLUSION: FK506 protected against glutamate neurotoxicity by inhibiting NOS activity in cultured retinal neurons.

J Cell Physiol, 1998 Jul, 176(1), 216 - 22
Primary cell cultures from murine kidney and heart differ in endosomal pH; Rybak SL et al.; Endosomal and lysosomal pH values have been determined for many established cultured cell lines of different origins . These cell lines may be grouped into two classes based on observed differences in pH of early (recycling) endosomes . Members of the first class typically have an early endosomal pH of 6.2, whereas members of the second class typically have an early endosomal pH of 5.4 . Because established cell lines may have developed artificial differences in endosomal pH due to extended culture, it remains to be determined if endosomal pH differences exist in vivo and whether they are functionally significant . To address this question, we generated adherent primary explants from mouse kidney (primarily epithelial cells) and heart (primarily fibroblasts and cardiac muscle cells) . Interestingly, enhanced acidification was observed in heart cell endosomes (pH = 5.5) compared with kidney cell endosomes (pH = 6.0) . These results indicate that differences in endosomal pH do not solely arise from extended cell culture and imply that such differences may be important for the proper functioning of different cell types.

J Immunol Methods, 1998 Feb 1, 211(1-2), 159 - 69
Development of an ultrasensitive in vitro assay to monitor growth of primary cell cultures with reduced mitotic activity; Blaheta RA et al.; Primary cell cultures, such as isolated epithelial cells, neuronal cells, or hepatocytes are characterized by a very low mitotic activity . Monitoring of small changes in cell numbers requires staining with a DNA-specific dye with an extremely high sensitivity and a low inter- and intraassay variability . For this purpose, an ultrasensitive in vitro assay has been developed based on the fluorescent nucleic acid stain PicoGreen . PicoGreen has been shown to detect as little as 0.5 ng pure DNA or 10(2) cells (interassay SD < 10%, intraassay SD < 5%) . This is far above the limit of sensitivity of conventional fluorochromes, such as Hoechst 33342 or propidium iodide . To obtain optimum efficacy of PicoGreen, cells were digested with papain for 20 h at 60 degrees C prior to staining . Under these conditions, the slope factor was calculated to be 0.105 relative fluorescence units (RFU)/cell, which is far superior to the slope factor of Hoechst 33342 (0.0137 RFU/cell) or propidium iodide (0.0077 RFU/cell) . Analysis of the blank values revealed a very low autofluorescence of PicoGreen, which is only 1/50th of the autofluorescence of Hoechst 33342 and 1/5th of the autofluorescence of propidium iodide . Additional coating of the culture plates with extracellular matrix proteins to prevent cellular dedifferentiation did not influence the high sensitivity of PicoGreen . In conclusion, the PicoGreen-assay seems to be the method of choice when the growth capacity of primary cell cultures needs to be analyzed with high accuracy.

Virology, 1998 May 25, 245(1), 53 - 64
Transmission and propagation in cell culture of virus produced by cells transfected with an infectious molecular clone of bovine leukemia virus; Inabe K et al.; A full-length molecular clone of bovine leukemia virus (BLV) pBLV-IF with two copies of a long terminal repeat (LTR) was constructed from a previously isolated, covalently closed, circular DNA clone, pB6490, that has one copy of the LTR and the pX region split at an EcoRI site . This molecular clone directed the synthesis of viral proteins and the induction of syncytia in transiently transfected cells . In addition, virus particles were released into the culture medium . Serial passages of transient transfectants also resulted in propagation of BLV . After transfection of five cell lines with linearized pBLV-IF and a neomycin-resistance gene, BLV-producing transfectants were established in cell lines COS-1 and 23CLN that did not form syncytia upon expression of BLV . In HeLa and FLK cells, BLV produced by a stable COS-1 transfectant was transmitted by both cell-free and cell-to-cell infection . Thus, pBLV-IF encoded an infectious provirus that successfully induced primary and secondary infections . This study indicates that the infectious molecular clone and the virus-producing transfectants could be useful for further examination of the biological properties of BLV.

J Surg Res, 1998 Feb 15, 75(1), 35 - 41
Cytotoxic effects of basic FGF and heparin binding EGF conjugated with cytotoxin saporin on vascular cell cultures; Chen C et al.; Vascular smooth muscle cell (SMC) proliferation is an integral component of intimal lesion formation . In this study we compared the mitogenic effects of basic fibroblast growth factor (bFGF) and heparin binding epidermal growth factor (HBEGF) and the cytotoxic effects of bFGF and HBEGF conjugated with plant cytotoxin saporin (SAP) on vascular cell cultures . Human vascular SMCs and endothelial cells were cultured and FGF receptor-1 (FGFR-1) and EGF receptor (EGFR) expression were detected by immunohistochemical staining . Cells were grown in 24-well plates . Variable amounts of testing drugs (bFGF, HBEGF, SAP, bFGF-SAP, or HBEGF-SAP) were added to quadruplicate wells after 24 h . Cells without drugs were used as control . The total number of cells was counted at 72 h using a hemocytometer . The cultured human vascular SMCs and endothelial cells expressed both FGFR-1 and EGFR with predominant perinuclear localization . bFGF and HBEGF demonstrated equally potent mitogenic effects on SMC proliferation . SAP alone showed a limited cytotoxic effect on both SMCs and endothelial cells . bFGF had a more potent effect on endothelial cell proliferation than HBEGF . bFGF-SAP was equally cytotoxic for both SMCs and endothelial cells, while HBEGF-SAP had a more selectively cytotoxic effect on SMCs than on endothelial cells . These data suggest that the mitogenic effects of bFGF and HBEGF and the cytotoxic effects of bFGF-SAP and HBEGF-SAP may both be mediated by their corresponding growth factor receptors . Because of its selective cytotoxic effect on SMCs, HBEGF-SAP may become a more attractive agent for controlling intimal lesion formation.

Tsitologiia, 1998, 40(1), 69 - 75
{Structural differentiation of dissociated skeletal embryonal myocytes of frog under conditions of cell culture}; Nasledov GA et al.; The development of membrane structures, providing E-C coupling, and the contractile apparatus organization were investigated in frog skeletal myocytes cultured for 1 to 10 days in conditions preventing both myocyte division and fusion . Ruthenium red was used to determine the membranous structures being in contact with the extracellular environment . The marked membrane structures (vesicles and short tubules) appeared to be near the cell membrane on the first days of culturing . The increase in the ratio of the surface area of all internal membranous structures, marked by Ruthenium red, to the external membrane area with aging was proven by morphometric calculations, that means a progressive development . Contractile filaments were found near the cell membrane on the first days of development . Bundles of filaments with initial signs of sarcomere organization were observed on the 3rd-4th days, and myofibrils with highly organized sarcomeres occupied the main part of the sarcoplasm on the 6th day of culturing . The triads appeared also on the sixth day, being regularly inserted into the sarcomere structure . Degenerative signs in the myocytes (sarcomere disorganization and T-tubule swelling) were observed on the 8-10th days, but the area occupied by contractile elements was increased . These results show that the myocyte fusion into myotubules is not a necessary condition for either sarcomere formation, or the formation of all membranous structures providing the E-C coupling.

J Clin Laser Med Surg, 1997, 15(2), 83 - 7
Pilot in vitro toxicity study of 5-ALA and Photofrin in microvascular endothelial cell cultures; Chang CJ et al.; Complicated hemangiomas are unique problems in which intervention with the proper laser can be an ideal solution . In this study we evaluated the toxicity of 5-Aminolevulinic acid (5-ALA) and Photofrin using in vitro models . The in vitro toxicity of 5-ALA and Photofrin was examined in a microvascular endothelial cell (MEC) culture system . The measurement of the percentage of MEC killed by various drug concentration using fluorescence viability assay . MEC incubated with 5-ALA at various concentrations for evaluation of dark toxicity showed less than a 50% cell kill . A comparison of different intervals of subcultured MEC showed that the early subculture (3 days after primary culture) is more vulnerable than later subculture (7 days after) . Cells treated with Photofrin at various concentrations exhibited less than 50% cell kill (dark toxicity) . The comparison of different intervals of subculture (3 days and 7 days after primary culture) showed a result similar to that of 5-ALA . All controls showed 0% cell kill . In conclusion, both 5-ALA and Photofrin are capable of destroying human microvascular endothelial cells in vitro . Drug concentrations and the power density for photodynamic therapy should be considered and will be included in our subsequent studies.

J Nat Prod, 1998 May, 61(5), 655 - 7
Isolation, identification, and antioxidant activity of three stilbene glucosides newly extracted from vitis vinifera cell cultures
Waffo Teguo P, Fauconneau B, Deffieux G, Huguet F, Vercauteren J, Merillon JM.
Suspension cultures of Vitis vinifera L . (Vitaceae) produce many hydroxylated stilbene glucosides found in red wine . From these cells, we isolated and characterized glycosylated stilbenes, (Z)-piceatannol (3,5,3',4'-tetrahydroxystilbene) -3-O-beta-d-glucopyranoside (6) and (E)- and (Z)-resveratrol (3,5, 4'-trihydroxystilbene)-4'-O-beta-d-glucopyranoside (2 and 7, respectively), which have not previously reported to be constituents of Vitis vinifera or wine . The ability of these compounds to act as radical scavengers was investigated using 1,1 diphenyl-2-picryl-hydrazyl, a stable free radical . Antioxidant activities were assessed by their capacity to prevent Cu2+-induced lipid peroxidation in human low-density lipoprotein.

Intensive Care Med, 1998 Apr, 24(4), 366 - 8
Sequential production of leukaemia inhibitory factor by blood cell culture in patients with ARDS; Gruson D et al.; OBJECTIVE: Leukaemia inhibitory factor (LIF) is a polyfunctional cytokine integrated in cytokine networks and its concentration has been shown to be elevated in bronchoalveolar lavage fluid of patients with the acute respiratory distress syndrome (ARDS) . The aim of our study was to evaluate the production of LIF by culturing blood cells from patients with ARDS . PATIENTS: 8 patients with ARDS, 8 patients with pneumonia and 5 healthy subjects . MEASUREMENTS AND RESULTS: The blood samples were taken on day 1 after onset of ARDS . LIF was measured, in the cell-free supernatant, with an enzyme-linked immunosorbent assay after 24 h, 48 h and 72 h of blood cell culture . LIF was detectable in some patients in the ARDS group: at i) at 24 h and 48 h: in 2 patients ii) at 72 h in 4/5 patients (140 +/- 231 pg/ml) . Only in the 4 patients in whom LIF was measured at 72 h was ARDS associated with the multiple organ dysfunction syndrome . Furthermore, among the 5 patients with ARDS who subsequently died, 4 had a detectable LIF . CONCLUSIONS: We have observed that LIF was produced only in ARDS, but not in all patients . The production of LIF seems to be a good indicator of the severity of ARDS . These preliminary results must be confirmed by a larger study.

Toxicol Lett, 1998 Feb, 94(3), 217 - 25
Effect of cadmium(II) on the extent of oxidative DNA damage in primary brain cell cultures from Pleurodeles larvae; Calevro F et al.; Compounds of cadmium(II) are well-known human and animal carcinogens . Furthermore, they affect development . growth and brain functions at subacute environmental concentrations in experimental animals . We investigated the potential of cadmium(II) to induce oxidative DNA damage in brain cell cultures obtained from larvae of Pleurodeles waltl . As indicators of DNA lesions typical of oxygen free radicals, we determined the frequencies of DNA strand breaks and of DNA base modifications recognized by the bacterial formamidopyrimidine-DNA glycosylase (Fpg protein) . DNA strand breaks were generated in a dose-dependent manner at concentrations of 1 microM and greater . In contrast, no significant increase in Fpg-sensitive sites was observed under our experimental conditions . However, the repair of Fpg-sensitive DNA lesions induced by visible light was slightly diminished at 1 microM and inhibited completely at 10 microM of cadmium(II), while the closure of DNA strand breaks was not affected . Our results show that, although cadmium is not able to induce oxidative DNA base modifications in larval brain cells directly, its capability to generate DNA strand breaks and to interfere with the repair of oxidative DNA damage could explain the early life stage neurotoxicity of this metal.

Arzneimittelforschung, 1998 Apr, 48(4), 423 - 6
Innovative and economic potential of mammalian cell culture; Werner RG; Innovations for economic optimization of manufacturing processes of mammalian cell culture processes address new expression systems, optimized cell culture media and feeding systems, economy of scale, efficient harvest systems for viable cell separation, redesign of downstream processing and reduction of the overall number of quality control assays . A very efficient expression system in Chinese hamster ovary cells is the NEOSPLA expression system yielding 60-100 micrograms monoclonal antibodies per cell and day . Efficient supplements in nutrient feeding are insulin and amino acids which contribute to a high extent to the productivity of the mammalian cell culture process . Large scale manufacturing processes lower cost of goods by reduction of turn around cost for cleaning and steaming in place, media preparation, number of batches for annual campaign, in-process and quality control . In downstream processing the number of process steps and the step yield are responsible for the economics . Process control systems in a computer assisted manufacturing plant increase success rate, reduces man power and minimizes shifts . In the innovative process also alternative technologies such as transgenic animals should be considered to improve the economy of the manufacturing processes.

Vestn Ross Akad Med Nauk, 1998, (3), 29 - 32
{Elaboration of live measles vaccine technology on human embryo lung diploid cell culture L-68}; Nechaeva EA et al.; Measles predominates among childhood droplet infections in many countries . Immunization of all human beings sensitive to this infection is the only radical measure in controlling measles . The quality of a vaccine is primarily determined by the properties of the virus strains and cell cultures and technology of production . Now live measles vaccine is produced in or country on the basis of fibroblasts from Japanese quail embryo . The production of live measles vaccine in the primary cell cultures has a number of drawbacks caused by the nonstandard pattern of the substrate and the probability of contamination . The use the certified human diploid cells deposited in liquid nitrogen in sufficient quantities is promising . The authors have elaborated a new technology of live measles vaccine production by using the Leningrad-16 virus strain on the basis of attested L-68 diploid cell culture from the human fetal lung . Experimental batches of vaccine were obtained and attested in accordance with the present requirements for immunobiological products.

In Vitro Cell Dev Biol Anim, 1998 Jan, 34(1), 1 - 8
Cell cross-contamination in cell cultures: the silent and neglected danger; Markovic O et al.; Cell cross-contamination in cell cultures is a common problem during cell culturing and use . Contamination invalidates research results, compromises the comparison of results between laboratories, reduces reproducibility required in industrial production of cell lines, and may lead to unusable therapeutic products . The problem can be solved by increasing the awareness of its seriousness and by introducing regular quality control of cell cross-contamination in every laboratory where cells are grown and used.

Biochem Biophys Res Commun, 1998 May 8, 246(1), 199 - 204
Osteoclast differentiation factor (ODF) induces osteoclast-like cell formation in human peripheral blood mononuclear cell cultures; Matsuzaki K et al.; We have reported that osteoclast differentiation factor (ODF) expressed on the plasma membrane of osteoblasts/ stromal cells is a ligand for osteoclastogenesis inhibitory factor (OCIF) . A genetically engineered soluble form of ODF (sODF) induced osteoclast-like multinucleated cells (OCLs) in the presence of M-CSF in mouse spleen cell cultures . Osteoblasts/stromal cells were not required in this process . To elucidate the mechanism of human osteoclastogenesis, human peripheral blood mononuclear cells (PBMCs) were cultured for 7 days with sODF and human M-CSF in the presence or absence of dexamethasone . Treatment of human PBMCs with sODF together with M-CSF induced OCLs, which expressed tartrate-resistant acid phosphatase and vitronectin receptors, produced cAMP in response to calcitonin, and formed resorption pits on dentine slices . OCLs were also formed from the adherent cell population of human PBMCs . Dexamethasone was required for human OCL formation in culture of whole PBMCs but not in culture of the adherent cell population . OCL formation was strongly inhibited by OCIF simultaneously added . These results clearly indicate that like in mouse osteoclastogenesis, ODF is a critical factor for human osteoclastogenesis . The present study also indicates that OCIF acts as a naturally occurring decoy receptor for ODF in inhibiting signal transduction in human osteoclast formation.

J Biomed Mater Res, 1998 Jun 15, 40(4), 631 - 9
Novel thermally reversible hydrogel as detachable cell culture substrate; von Recum HA et al.; A novel UV crosslinkable co-polymer of 4-(N-cinnamoylcarbamide)methylstyrene (CCMS) and N-isopropylacrylamide (IPAAm) was partially entrapped in traditional tissue-culture-treated polystyrene and crosslinked by UV light irradiation . Dishes modified by this method showed a change in contact angle with respect to temperature as compared to tissue culture polystyrene controls . Surface chemical analysis indicated that the crosslinked hydrogel does not detach from the surface after successive rinsing in ethanol and water, keeping the cells or cell construct free of unwanted soluble polymer after detachment . Cultures of both bovine endothelium and human retinal pigmented epithelium were confirmed to be able to attach and grow on the polymer-modified surfaces morphologically identical to that on control tissue culture polystyrene surfaces . Corresponding to a change in temperature, these cultures would detach and could be transplanted to another culture surface without functional and structural changes . These results show that the new, photo-crosslinkable hydrogel system can utilize the hydrophobic/hydrophilic change of the surface for cell culture detachment while being permanently applicable to any tissue culture geometry.

Hum Mol Genet, 1998 May, 7(5), 783 - 90
Truncated N-terminal fragments of huntingtin with expanded glutamine repeats form nuclear and cytoplasmic aggregates in cell culture; Cooper JK et al.; Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expanding CAG repeat coding for polyglutamine in the huntingtin protein . Recent data have suggested the possibility that an N-terminal fragment of huntingtin may aggregate in neurons of patients with HD, both in the cytoplasm, forming dystrophic neurites, and in the nucleus, forming intranuclear neuronal inclusion bodies . An animal model of HD using the short N-terminal fragment of huntingtin has also been found to have intranuclear inclusions and this same fragment can aggregate in vitro . We have now developed a cell culture model demonstrating that N-terminal fragments of huntingtin with expanded glutamine repeats aggregate both in the cytoplasm and in the nucleus . Neuroblastoma cells transiently transfected with full-length huntingtin constructs with either a normal or expanded repeat had diffuse cytoplasmic localization of the protein . In contrast, cells transfected with truncated N-terminal fragments showed aggregation only if the glutamine repeat was expanded . The aggregates were often ubiquitinated . The shorter truncated product appeared to form more aggregates in the nucleus . Cells transfected with the expanded repeat construct but not the normal repeat construct showed enhanced toxicity to the apoptosis-inducing agent staurosporine . These data indicate that N-terminal truncated fragments of huntingtin with expanded glutamine repeats can aggregate in cells in culture and that this aggregation can be toxic to cells . This model will be useful for future experiments to test mechanisms of aggregation and toxicity and potentially for testing experimental therapeutic interventions.

J Physiol Pharmacol, 1998 Mar, 49(1), 99 - 110
Effects of acid-degraded products of leminoprazole on acid secretion, mucus secretion and synthesis, and indomethacin-induced damage in cell culture; Takahashi S et al.; We examined the effects of four acid-degraded products of leminoprazole on {1} acid secretion by parietal cells, {2} mucus secretion and synthesis by epithelial cells and {3} indomethacin-induced damage to epithelial cells . These gastric cells were prepared from rabbit stomachs . Upon stimulation with 10 microM histamine, acid secretion by parietal cells was inhibited by leminoprazole, sulfide and 2-(isobutylmethylamino)benzylalcohol . Sulfide also inhibited dibutyryl cyclicAMP (100 microM)-stimulated secretion . However, the inhibitory effects of such compounds were observed only at high concentrations, in comparison with the antisecretory concentrations of leminoprazole . On the other hand, among acid-degraded products, only sulfide enhanced mucus secretion and synthesis by epithelial cells . The stimulatory effects of sulfide were the same as those of leminoprazole . Furthermore, the effects of sulfide as well as leminoprazole were suppressed by NG-nitro-L-arginine methyl ester (L-NAME), and L-arginine, not D-arginine, prevented the inhibition by L-NAME . In contrast, all degraded products failed to protect epithelial cells against indomethacin-induced damage . Overall, these results suggest that only the mucus-elevating effect of administered leminoprazole may be partly due to the stimulatory effects of sulfide derived from leminoprazole on mucus secretion and synthesis by epithelial cells.

J Infect Dis, 1998 May, 177(5), 1290 - 5
Antibody persistence following preexposure regimens of cell-culture rabies vaccines: 10-year follow-up and proposal for a new booster policy; Strady A et al.; Subjects (n = 312) received either the human diploid cell rabies vaccine (HDCV) or the purified Vero cell rabies vaccine (PVRV) according to either two-injection (days 0 and 28) or three-injection (days 0, 7, and 28) primary regimens . They received a booster injection at 1 year . Rabies antibody levels were measured after the primary series and the booster and then each year for the next 10 years . The results confirm the superior long-term immunogenicity of the three-injection over the two-injection protocol . HDCV and PVRV in three doses were equally immunogenic . A booster injection at 1 year provides long-term seroconversion (titer > or = 0.5 IU/mL) . Antibody titers 2 weeks after the 1-year booster allowed prediction of long-term immunity . Good responders, with titers > or = 30 IU/mL, were protected for at least 10 years . An algorithm for differentiation between good responders and poor responders with respect to vaccine booster strategies is proposed.

Tsitol Genet, 1997 Nov-Dec, 31(6), 26 - 34
{The evaluation of the cytotoxicity of preparations with anticarcinogenic action in human cell cultures}; Lukash LL et al.; An approach for determination of cytotoxicity of chemical and biological drugs which combine the estimation of quality and quantity of cells after their treatment are proposed by the authors . The visual control of cellular minicultures permits one: 1) to register specific and pathologic alterations of cell morphology, 2) to trace their development in dynamics under the action of individual or complex drugs and 3) to choose the optimum time for the experiment fixation . The special staining of the treated cells and measurement of the optical density of absorbed histological dye permits one to make a conclusion about the changing of the cells quantity . A combined method of double estimation gives the opportunity to detect the artefacts taking place after staining the cells treated by some drugs and extracts of natural origin in high concentrations.

Baillieres Clin Endocrinol Metab, 1997 Dec, 11(4), 739 - 52
Direct microcalorimetry as a technique in cell cultures; Bottcher H et al.; In vitro studies of energy metabolism in isolated cells contribute to improved knowledge of human energy metabolism under normal and pathological conditions . In every cellular system energy is taken up, metabolized and finally transformed into heat, which is dissipated into the environment . Thus, energy turnover of isolated cells can be assessed by microcalorimetric determination of their heat production . Microcalorimeters of the thermopile heat conduction type facilitate direct physical determination of thermogenesis with a sensitivity of 0.2 microW; 10(4)-10(5) cells being sufficient for one measurement . Peltier elements are sandwiched between the sample and a precisely thermostated heat sink, creating a detectable voltage proportional to the heat production . For adequate interpretation of the results, simultaneous biochemical investigations of relevant metabolic pathways are required . Up to now, numerous studies with blood cells, skeletal and heart muscle cells, hepatocytes, endothelial cells, fibroblasts and adipocytes have been performed in relation to various diseases and under the influence of certain hormones and pharmacological agents.

Curr Opin Biotechnol, 1998 Apr, 9(2), 146 - 51
Advances in hematopoietic stem cell culture; Audet J et al.; Recent advances in our understanding of the earliest stages of hematopoietic cell differentiation, and how these may be manipulated under defined conditions in vitro, have set the stage for the development of robust bioprocess technology applicable to hematopoietic cells . Sensitive and specific assays now exist for measuring the frequency of hematopoietic stem cells with long-term in vivo repopulating activity from human as well as murine sources . The production of natural or engineered ligands through recombinant DNA and/or combinatorial chemistry strategies is providing new reagents for enhancing the productivity of hematopoietic cell cultures . Multifactorial and dose-response analyses have yielded new insight into the different types and concentrations of factors required to optimize the rate and the extent of amplification of specific subpopulations of primitive hematopoietic cells . In addition, the rate of cytokine depletion from the medium has also been found to be dependent on the types of cell present . The discovery of these cell-type-specific parameters affecting cytokine concentrations and responses has introduced a new level of complexity into the design of optimized hematopoietic bioprocess systems.

Am J Clin Nutr, 1998 May, 67(5 Suppl), 988S - 995S
Functional analysis of copper homeostasis in cell culture models: a new perspective on internal copper transport; Harris ED et al.; The movement of copper ions across membrane barriers of vital organs and tissues is a priority topic in nutrition and one for which there continues to be little understanding of the mechanism . Reports of membrane-bound, copper-transporting adenosine triphosphatases (Cu-ATPases) selective for copper ions have brought new focus to the problem and prompted fresh ideas . Using a cell culture model approach, we attempted to learn whether transport into and out of cells depends on a Cu-ATPase . Measurement of transport kinetics in fibroblasts, brain glial cells, neuroblastoma cells, and placental cells showed differences in the rates of copper uptake and response to sulfhydryl reagents . BeWo cells, a human choriocarcinoma placental cell line, behaved as did Menkes fibroblasts by avidly absorbing copper but not releasing copper to the immediate environment . Further tests showed that BeWo cells did not express the transcript for the membrane-bound Cu-ATPase that has been identified with Menkes syndrome . Transcript induction, however, was achieved by growing BeWo cells on porous filters that allowed apical and basolateral surfaces to form . With transcript expression, the cells showed a capacity to release copper into the medium . BeWo cells also synthesized a form of ceruloplasmin whose structure differed from that of the plasma protein and hence may be a product of a different gene . BeWo cells may also express the gene for Wilson disease, thus linking Menkes and Wilson proteins to maternal delivery of copper . We constructed a model in which both ATPases work in concert in a vesicle-based transport mechanism . The vesicle model may help us understand the transport of copper across the placenta and all cells in general.

J Endocrinol, 1998 Mar, 156(3), 441 - 7
Recombinant somatolactin as a stable and bioactive protein in a cell culture bioassay: development and validation of a sensitive and reproducible radioimmunoassay; Calduch-Giner JA et al.; A recombinant somatolactin (SL) obtained by cloning and expression of sole SL cDNA was analyzed and used to develop a sensitive and specific RIA . In contrast to native proteins, which tend to dimerize and aggregate immediately after pituitary isolation, the majority of recombinant sole SL (rsSL) remained as a monomeric protein after long-term storage, as shown by size exclusion chromatography and Western blot . Using rsSL as a tracer and standard in the RIA, the minimum detectable dose and the midrange (ED50) of the assay were 0.15 and 1.8-2.1 ng/ml respectively . Intra-and interassay coefficients of variation were 4.3% and 6.5% at ED50 levels . Recombinant gilthead sea bream GH and recombinant trout GH did not show cross-reactivity, whereas a good parallelism between rsSL standard and serial dilutions of plasma and sole pituitary extracts was observed . In order to demonstrate some biological activity of rsSL, the ability of this recombinant product to prime gilthead sea bream phagocytes for in vitro enhancement of mitochondrial activity was examined by a chromogenic assay . A bell-shape dose-response curve was obtained with a maximum at 50 nM (1.2 micrograms/ml), similar to that reported previously for GH . Therefore, taking together all these data, it appears conclusive that rsSL is a long-term stable protein which retains, at least in part, biological activity, providing a useful tool to clarify the physiological role of fish SL.

Endocrinology, 1998 May, 139(5), 2527 - 34
Thyroid hormone excess increases insulin-like growth factor I transcripts in bone marrow cell cultures: divergent effects on vertebral and femoral cell cultures; Milne M et al.; Thyroid hormones (T3 and T4) regulate bone development, growth, and turnover . Studies have suggested that different skeletal sites respond differently to thyroid hormones . Therefore, we examined the in vitro T3 responsiveness of cells committed to the osteoblast lineage as a function of skeletal location . Bone marrow cells derived from female rat femurs and vertebrae were cultured using conditions that induce osteogenic differentiation . Cells from both sites formed mineralized bone nodules in primary and secondary culture . In femoral cultures, collagen type I (coll I) and osteocalcin (OC) messenger RNA (mRNA) levels increased from the earliest time point examined (day 3) to a maximum on day 12 and thereafter declined to undetectable levels . T3 increased both OC and coll I mRNA, resulting in a continuous expression throughout the culture period . Insulin-like growth factor I (IGF-I) gene expression was detected at very low levels by Northern analysis of femoral total RNA, and T3 only marginally enhanced IGF-I mRNA levels . In vertebral cultures, OC and coll I mRNA levels also increased with time in culture, but remained expressed throughout the culture period . OC and coll I mRNA levels were not markedly altered in response to T3 . In contrast to femoral cells, IGF-I gene expression was easily visualized in Northern blots from untreated vertebral cultures and was markedly increased by the addition of T3 . The continuous presence of T3 (10(-7) M) in the medium for 18 days caused a marked decrease in the number of alkaline phosphatase-positive colonies formed in femoral secondary cultures, but only a slight decrease in the number in vertebral cultures . In addition, short term (6 days) exposure to T3 (10(-7) M) at the beginning of the culture period decreased alkaline phosphatase activity in femoral cultures, but not in vertebral cultures . These findings indicate that there are skeletal site-dependent differences in the in vitro responses of cells of the osteoblastic lineage to thyroid hormone.

Endocrinology, 1998 May, 139(5), 2272 - 7
Effect of 17beta-estradiol on somatostatin receptor expression and inhibitory effects on growth hormone and prolactin release in rat pituitary cell cultures; Djordjijevic D et al.; In the present study, we tested whether 17beta-estradiol (E2)-induced PRL sensitivity to somatostatin-14 (SRIF) involves selective up-regulation of discrete somatostatin receptor subtypes (ssts) in primary cultures of female rat pituitary cells . The efficacy of the endogenous peptide SRIF to inhibit GH and PRL secretion and cAMP accumulation was compared with those of octreotide (OCT), BIM-23052, BIM-23056, and BIM-23268, which have been reported to be relatively selective for rat sst2, sst3, and sst5 . Experiments were performed in steroid-depleted media supplemented or not with 1 nM E2 for 96 h . SRIF, OCT, and BIM-23052 inhibited cAMP accumulation and GH release independently of E2 . In contrast, all three agonists affected PRL release in E2-treated cultures only . Inhibition of cAMP accumulation by SRIF, OCT, and BIM-23052 was enhanced by exposure of cells to E2 . The rank of potency of the agonists, OCT = SRIF > BIM-23052, was similar for GH and PRL inhibition . BIM-23268 was a weak agonist on GH, but not on PRL, secretion . BIM-23056 had no effect on the release of either hormone, but slightly inhibited cAMP formation in E2-treated cells . To verify whether SRIF receptor gene expression correlated with these observations, messenger RNA (mRNA) transcripts corresponding to the five ssts were measured by quantitative RT-PCR in the presence or absence of E2 . Control cells expressed predominantly sst2 and sst3 transcripts; sst1 mRNA was present in moderate amounts, whereas sst4 and sst5 were only weakly expressed . E2 had a differential effect on distinct ssts; it increased mRNA concentrations corresponding to sst2 and sst3, and decreased that of sst1 . These results indicate that sst2 and sst3 receptors are the major somatostatin receptors expressed in the female rat pituitary, and that both of them are positively regulated by estradiol . However, the capacity of lactotropes to respond to SRIF after exposure to E2 seems to depend more upon E2-induced up-regulation of the sst2 than of the sst3 receptor subtype.

Analyst, 1998 Jan, 123(1), 55 - 8
Testing of chelating agents and vitamins against lead toxicity using mammalian cell cultures; Fischer AB et al.; Mammalian cell cultures were used to determine the capacity of antidotes to modify (a) lead uptake, (b) lead toxicity and (c) lead release from cells . The following chelating agents were tested: Na, Ca-ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), nitriloacetic acid, ethylene glycol-bis(aminoethyl)tetraacetic acid (EGTA), D,L-mercaptosuccinic acid (MSA), meso-2,3-dimercaptopropanesuccinic acid (MSA), D,L-2,3-dimercaptopropane-1-sulfonic acid (DMPS), penicillamine (PA), N-acetylpenicillamine (NAPA), and diethylcarbodithioate (DDTC) . The following vitamins were tested: thiamine (B1), riboflavine (B2), pyridoxine (B6), cobalamin (B12) and ascorbic acid (C) . Inhibition of lead uptake was produced by EDTA, EGTA, DMSA, DMPS, MSA, PA, NAPA and vitamins B1, B6 and C, vitamins B2 and B12 being ineffective . The same compounds reduced lead cytotoxicity . Interestingly DDTC and DTPA increased lead uptake, but did not exacerbate lead toxicity . Significant release of lead from preloaded cells was caused by DTPA, NAPA, DMPS and PA, while the other chelators were ineffective.

Haematologica, 1998 Jan, 83(1), 3 - 7
The value of cell cultures for the diagnosis of mixed myelodysplastic/myeloproliferative disorders; Del Canizo MC et al.; BACKGROUND AND OBJECTIVE: Myelodysplastic syndromes (MDS) are a group of disorders characterized by dyshematopolesis in bone marrow (BM) and peripheral blood (PB) cytopenias . In recent years particular attention has been paid to myeloproliferative disorders with dysplastic features or myelodysplastic syndromes that evolve into a myeloproliferative disorder . The present study was designed to analyze patients with MDS but with a normal or increased colony forming capacity, in order to see whether or not cell cultures could contribute to the diagnosis of intermediate MDS-MPD conditions . DESIGN AND METHODS: A total of 80 patients diagnosed as having MDS were included in the study . CFU-GM assay was performed by plating 1 x 10(5) mononuclear cells/mL in IMDM and 0.9% methyl-cellulose containing 10% PHA-LCM . In all cases cultures were run in parallel without PHA-LCM to assess autonomous growth . Cultures were incubated at 37 degrees C in a fully humidified atmosphere with 5% CO2 and scored at day 14 . Cytogenetic analysis was performed according to standard procedures . Short-term cultures of 24 and/or 48 hours were used . RESULTS: Twenty-two patients out of the 80 MDS cases included in the study showed a normal or increased cell growth pattern . Among these 22 patients, eight were diagnosed as suffering from chronic myelomonocytic leukemia (CMML) according to the FAB criteria and were excluded from the present analysis . The remaining 14 cases, which constitute the body of this study, displayed an increased number of clusters and/or colonies, with an altered cluster/colony ratio (anomalous growth) in 10 cases . Autonomous colony formation was present in five of these 14 cases and autonomous cluster growth was seen in all but three of them . In addition, one patient showed endogenous BFU-E growth . Morphological diagnoses were then revised due to this aberrant colony growth pattern: based on actual criteria, 3 patients could have been considered as having a-CML (atypical chronic myeloid leukemia) . Another 6 cases evolved to a more proliferative disorder: 5 to CMML, and one to a-CML . Interestingly, in 3 of these 6 patients the evolution took place concomitantly with an infectious episode . In one additional patient the platelet count increased up to 1000 x 10(9)/L and required treatment with hydroxyurea . INTERPRETATION AND CONCLUSIONS: Our results show that intermediate MDS-MPD cases are relatively common and that in vitro characteristics, i.e . high clonogenic capacity with a high cluster/colony ratio and scanty autonomous growth, in patients showing myelodysplastic features could contribute to an early diagnosis in these cases . It is possible that in some cases an infectious episode, through higher cytokine secretion, contributes to the development of these disorders.

J Physiol, 1998 Jun 1, 509 ( Pt 2), 497 - 506
Release of acetylcholine from embryonic myocytes in Xenopus cell cultures; Fu WM et al.; 1 . Acetylcholine (ACh) is important as the transmitter responsible for neuromuscular transmission . Here we report the non-quantal release of ACh from embryonic myocytes . 2 . Co-cultures of spinal neurons and myotomal muscle cells were prepared from 1-day-old Xenopus embryos . Single channel currents were recorded in the non-innervated myocytes . When the patch pipette was filled with Ringer solution alone, spontaneous single channel currents occurred, which were inhibited by d-tubocurarine (d-Tc) . 3 . The channel conductance appearing in Ringer solution (37.3 pS) was similar to that of an embryonic-type ACh channel (36.9 pS), indicating that ACh is probably released from myocytes in normal Ringer solution . 4 . When the patch pipette was filled with anticholinesterase alone to prevent hydrolysis of ACh released from myocytes, both physostigmine and neostigmine in a concentration-dependent manner increased channel open probability; it was reduced by d-Tc or alpha-bungarotoxin . 5 . Vesamicol and quinacrine, vesicular transporter inhibitors, reduced the channel open probability caused by ACh released from myocytes in the presence of neostigmine or physostigmine . 6 . Intracellular alkalinization with NH4Cl inhibited the ACh release from myocytes, whereas, extracellular alkalinization, brought about by replacing normal Ringer solution, with pH 8.6 Ringer solution enhanced ACh release . 7 . The immunocytochemistry of choline acetyltransferase (ChAT) showed that ChAT exists in both myocytes and neuronal cells but not in fibroblasts . 8 . These results suggest that embryonic myocytes are capable of synthesizing and releasing ACh in a non-quantal manner . Extracellular alkalinization enhanced and intracellular alkalinization inhibited ACh release from myocytes.

Lett Appl Microbiol, 1998 Feb, 26(2), 136 - 9
Galacto-oligosaccharide production using a recycling cell culture of Sterigmatomyces elviae CBS8119; Onishi N et al.; Addition of small amounts of Fe2+, Zn2+, Cu2+ and thiamine-HCL to the culture medium was required for promoting the galactooligosaccharide (Gal-OS)-producing activity of Sterigmatomyces elviae CBS8119, when the concentration of yeast extract in the medium was lowered to 0.1 g l-1 . Galactooligosaccharide production using a recycling cell culture was performed in a medium containing 360 mg ml-1 of lactose supplemented with optimal concentrations of Fe2+ (1.5 mg l-1 of FeSO4 x 7H2O), Zn3+ (15 mg l-1 of ZnSO4 x 7H2O), Cu2+ (0.5 mg l-1 of CuSO4 x 5H2O) and thiamine-HCL (1 mg l-1) . Galacto-oligosaccharide production was maintained at high levels during six cycles of production, with the amount of Gal-OS produced in each cycle being more than 216 mg ml-1 (weight yield of more than 60%.

Int J Dev Neurosci, 1997 Nov, 15(7), 851 - 65
Comparison of glutamate and N-methyl-D-aspartate toxicities on rat mesencephalic primary cell cultures; Mateu G et al.; Excitotoxicities of glutamate and NMDA were studied on primary cultures of rat embryonic substantia nigra . The toxicity of the general neuronal population (identified with neuron specific enolase-NSE) was compared with that of dopaminergic neurons (identified with TH antibodies) . We have shown that there exists a time-dependent toxicity to glutamate in 9 d old cultures in vitro and exposures as short as 5 min are significantly toxic . By comparing the effects of long time exposures (24 h) to NMDA and glutamate, we can show dose-dependent toxicity; however NMDA shows a less marked effect, especially at high doses (> 500-1000 microM) as opposed to less potent lower doses (< 500 microM) . In comparison to the general population of NSE-positive mesencephalic neurons, TH-positive neurons seem to exhibit a similar vulnerability to EAA . The fact that TH-positive neurons are only partially protected against glutamate toxicity by the non-competitive NMDA antagonist TCP indicates that they are more susceptible to non-NMDA mediated neurotoxicity than the general neuronal population.

Anticancer Res, 1998 Jan-Feb, 18(1A), 433 - 9
Growth curves: a method of monitoring established long-term primary human tumor cell cultures for autologous immunotherapy; Rizzo S et al.; Cytomorphologic methods evidentiate tumor cells (TC) and differentiate them from fibroblasts, but are not indicated f for long term cultures . Growth rate and curve shape of TC and autologous fibroblasts cultures were recorded and compared over 4 weeks . Both were morphologically documented ex vivo and after MGG stain . No fibroblast overgrowth was noted in the TC cultures . The cultures showed rather peculiar curves with little variability among TC cultures . The 1st-week rate was higher in the TC cultures (p < .01) . Growth curve evaluation with ex vivo observation of the morphology is- an alternative method in monitoring TC cultures for autologous immunotherapy.

Anticancer Res, 1998 Jan-Feb, 18(1A), 41 - 4
Long-term primary human tumor cell cultures and mixed autologous tumor-lymphocyte cultures for adoptive specific antitumoral immunotherapy; Rizzo S et al.; Anticancer immunotherapy may improve the body's cancer defences, quality of life and survival . Encouraging treatment models have been proposed, namely the non-specific LAK and ALT cell therapies; the relatively specific TIL/CTL cell therapy as well as allogenic cancer vaccines; autologous cancer vaccines . A procedure to prepare an autologous antitumor serum for cancer therapy using ex vivo activated autologous cells is here reported . Peripheral blood lymphocytes obtained by leukaphoresis were cultured with autologous tumor cells in RPM1 1640 and rh-IL-2 for 48 hours at 37 degrees C in a humidified, CO2 enriched atmosphere . The cytokine rich supernatant, as well as the activated cells in medium were frozen, lysed and aliquotted, frozen, and stored at -20 degrees C . The material was used for specific anticancer immunotherapy . Fibroblast-free tumor cells are being maintained in long term culture for future ex vivo sensitization of autologous cells.

Virology, 1998 Apr 10, 243(2), 396 - 405
Recombinant swinepox virus expressing beta-galactosidase: investigation of viral host range and gene expression levels in cell culture; Barcena J et al.; Swinepox virus (SPV) has been proposed as a potential vector for generating recombinant vaccines for swine . However, little is known about important aspects of SPV biology, such as the functionality of SPV promoters or the host range of SPV . Using a transient expression assay, well-characterized vaccinia virus promoters were shown to be active in cells infected with SPV . A recombinant SPV expressing beta-galactosidase (beta-gal) was constructed and characterized . The E . coli LacZ gene was placed under the control of a strong vaccinia synthetic early/late promoter and was inserted by homologous recombination in a noncoding region of the SPV genome . The recombinant SPV expressing beta-gal was used to characterize the host range of the virus by measuring protein expression and virus production in different cell lines . In general, SPV expressed more protein and grew more efficiently than vaccinia virus in porcine cell lines . Surprisingly, the recombinant SPV was able to infect and replicate in several cell lines of nonswine origin . The virus directed regulated early and late gene expression of beta-gal in those cells and formed blue plaques in cell monolayers in the presence of X-gal . Upon infection with the recombinant SPV, there was a significant level of viral replication, and the virus can be serially passaged in some nonswine cell lines . The data presented suggest that despite the strict host tropism of SPV, the virus exhibits a relatively broad host range in cell culture.

Mol Chem Neuropathol, 1998 Feb, 33(2), 99 - 111
Prenatal ethanol exposure enhances glutamate release stimulated by quisqualate in rat cerebellar granule cell cultures; Rhodes PG et al.; Effects of prenatal ethanol exposure on extracellular glutamate accumulation stimulated by glutamate receptor agonists were studied in rat cerebellar granule cell cultures . The prenatal exposure to ethanol was achieved via maternal consumption of a Sustacal liquid diet containing either 5% ethanol or isocaloric sucrose (pair-fed) substituted for ethanol from gestation d 11 until the day of parturition . Neither the basal level of extracellular glutamate nor the increased accumulation of glutamate stimulated by KCl (40 mM) or by ionotropic glutamate receptor agonists, N-methyl-D-aspartate (NMDA) or kainate (KA) (100 microM each), in cells prepared from the ethanol-fed group was significantly different from that in cells prepared from the pair-fed group . Glutamate accumulation stimulated by quisqualate (QA, 100 microM) or by trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD, 250 microM) in the ethanol-fed group was higher than that in the pair-fed group by 116 and 36%, respectively . In the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 100 microM), an ionotropic QA receptor antagonist, the QA-induced accumulation of glutamate in the ethanol-fed group was still higher than that in the pair-fed group . In the presence of MK-801 (5 microM), an antagonist of the NMDA receptor, the enhanced accumulation of glutamate stimulated by either QA or t-ACPD was still observable in the ethanol-fed group as compared to the pair-fed group . Addition of (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG, 500 microM), a selective antagonist of the metabotropic glutamate receptor, abolished the enhanced accumulation of glutamate stimulated by either QA or t-ACPD in the ethanol-fed group . Although immunoblotting of mGluR1 and mGluR2/3 did not show apparent differences between the pair-fed and the ethanol-fed groups, the overall results suggest that the effect of prenatal ethanol exposure was selectively through a pathway mediated by the metabotropic glutamate receptor.

J Neurophysiol, 1998 Mar, 79(3), 1371 - 83
Long-term effects of axotomy on excitability and growth of isolated Aplysia sensory neurons in cell culture: potential role of cAMP; Bedi SS et al.; Crushing nerves, which contain the axons of central sensory neurons, in Aplysia causes the neurons to become hyperexcitable and to sprout new processes . Previous experiments that examined the effects of axonal injury on Aplysia sensory neurons have been performed in the intact animal or in the semi-intact CNS of Aplysia . It therefore has been unclear to what extent the long-term neuronal consequences of injury are due to intrinsic or extrinsic cellular signals . To determine whether injury-induced changes in Aplysia sensory neurons are due to intrinsic or extrinsic signals, we have developed an in vitro model of axonal injury . Isolated central sensory neurons grown for 2 days in cell culture were axotomized . Approximately 24 h after axotomy, sensory neurons exhibited a greater excitability-reflected, in part, as a significant reduction in spike accommodation-and greater neuritic outgrowth than did control (unaxotomized) neurons . Rp diastereoisomer of the cyclic adenosine 3',5'-monophosphorothiate (Rp-cAMPS), an inhibitor of protein kinase A, blocked both the reduction in accommodation and increased neuritic outgrowth induced by axotomy . Rp-cAMPS also blocked similar, albeit smaller, alterations observed in control sensory neurons during the 24-h period of our experiments . These results indicate that axonal injury elevates cAMP levels within Aplysia sensory neurons, and that this elevation is directly responsible, in part, for the previously described long-term electrophysiological and morphological changes induced in Aplysia sensory neurons by nerve crush . In addition, the results indicate that control sensory neurons in culture are also undergoing injury-related electrophysiological and structural changes, probably due to cellular processes triggered when the neurons are axotomized during cell culturing . Finally, the results provide support for the idea that the cellular processes activated within Aplysia sensory neurons by injury, and those activated during long-term behavioral sensitization, overlap significantly.

J Biomed Mater Res, 1998 May, 40(2), 301 - 6
Biocompatibility analysis of different biomaterials in human bone marrow cell cultures; Wilke A et al.; A cell culture system for biocompatibility testing of hip implant materials is described . Human bone marrow cells have been chosen because these cells are in direct contact with the biomaterial after implantation in situ . The sensitivity of this method is evaluated for materials which are already being used as implants in humans and animal, e.g., hydroxyapatite (HA) ceramic, pure titanium, and ultra-high-molecular-weight polyethylene (UHMWPE) . As indicative parameters of biocompatibility primary cell adherence, cell number, cell proliferation, production of extracellular matrix, cell vitality, and cell differentiation are described . After 2 weeks in culture, obvious differences between the biomaterials with respect to the indicative parameters could be observed . Cell numbers were greatest on the HA specimens . In the case of titanium alloys, we observed a decreased number of cells . The production of extracellular matrix was high for the HA ceramics but reduced for titanium specimens . The polymers allowed only a few adherent cells and showed no signs of extracellular matrix production . The results can be correlated astonishingly well to animal experiments and clinical experiences . Therefore, we suggest that this cell culture system seems to be a useful tool for biocompatibility testing of bone implantation materials . It also helps reduce animal experiments . With the help of flow cytophotometry, we analyzed the influence of biomaterials on large numbers of cells with respect to differentiation . There were similar populations of T cells and monocytes on all specimens tested . Extended B-cell and granulocyte populations, however, were observed with titanium and UHMWPE . Most osteocalcin-containing cells adhered to the HA ceramics.

J Biomed Mater Res, 1998 May, 40(2), 244 - 56
Variation of oxide films on titanium induced by osteoblast-like cell culture and the influence of an H2O2 pretreatment; Pan J et al.; Variations of titanium oxide films induced by osteoblast-like cells in a rat calvaria culture system and the influence of an H2O2 pretreatment have been investigated by using X-ray photoelectron spectroscopy and electrochemical impedance spectroscopy . For abraded titanium, the results revealed that phosphate and calcium ions may incorporate into the surface oxide film during the cell culture, forming a precipitate with a Ca/P ratio near that of hydroxyapatite . Oxidized carbon also was found in the surface layer, most likely precipitated hydroxylcarbonated apatite (HCA) . The H2O2 pretreatment of titanium in a phosphate-buffered saline solution results in a 10-fold thickened porous oxide film and large amounts of surface hydroxyl groups as well as a certain amount of phosphate ions inside the oxide film . During the cell culture, the H2O2-treated titanium surface favors the ion incorporation and precipitation of the HCA-like compound, which probably is inlaid into the oxide film . Osteoblast-like cells on the H2O2-treated titanium showed a more active morphology during the initial stage compared with cells on abraded titanium . Moreover, bone-like nodule formation and mineralization appear to be related to the precipitation of the HCA-like compound on the surface . The results are discussed with respect to corrosion resistance, ion incorporation and precipitation of the HCA-like compound on the surface, osseointegration, and bioactivity of titanium implants.

Bone, 1998 Apr, 22(4), 347 - 54
Low-energy laser irradiation stimulates bone nodule formation at early stages of cell culture in rat calvarial cells; Ozawa Y et al.; Although the acceleration of bone regeneration by laser treatment has been reported, the mechanisms of action of laser on bone are unclear . To determine the target cells responsible for the action of laser irradiation and roles of irradiation on these cells during bone formation, we investigated the effects of low-energy laser irradiation at various cell culture stages on cellular proliferation, bone nodule formation, alkaline phosphatase activity, and osteocalcin gene expression, employing rat calvarial cells . Osteoblast-like cells isolated from fetal rat calvariae were irradiated once with a low-energy Ga-Al-As laser (830 nm, 500 mW) at various cell culture stages (days 1-16) . Laser irradiation at early stages of culture significantly stimulated cellular proliferation, ALP activity, and osteocalcin gene expression thereafter . Furthermore, laser irradiation at earlier stages of culture significantly stimulated a greater number (1.7-fold) and larger area (3.4-fold) of bone nodules that had developed in the culture dish on day 21 . However, these effects could not be found by irradiation at a later date . These results suggest that laser irradiation may play two principal roles in stimulating bone formation . One is stimulation of cellular proliferation, especially proliferation of nodule-forming cells of osteoblast lineage, and the other is stimulation of cellular differentiation, especially to committed precursors, resulting in an increase in the number of more differentiated osteoblastic cells and an increase in bone formation . Both bone-formation-stimulating roles may be exhibited by laser irradiation to immature cells only.

Bone, 1998 Apr, 22(4), 341 - 6
Interaction of triiodothyronine with 1alpha,25-dihydroxyvitamin D3 on interleukin-6-dependent osteoclast-like cell formation in mouse bone marrow cell cultures; Schiller C et al.; In mouse bone marrow cultures, the formation of osteoclast-like, that is, tartrate-resistant acid phosphatase-positive (TRAP+) and calcitonin (CT) receptor-positive multinucleated cells (MNCs), induced by 10(-10) to 10(-8) mol/L 1alpha,25-dihydroxyvitamin D3 {1alpha,25(OH)2D3}, could be augmented by triiodothyronine (T3), which alone had no effect on osteoclast-like cell formation . The permissive effect of T3 increased the response to 1alpha,25(OH)2D3 by approximately one order of magnitude . Linear concentration dependence was observed between 10(-11) and 10(-8) mol/L T3 . Importantly, inhibition of prostaglandin synthesis by indomethacin significantly impeded osteoclast-like cell formation by 1alpha,25(OH)2D3 and abrogated the effect of T3 thereon . Basal interleukin-6 (IL-6) production by cultured marrow cells was significantly stimulated by 1alpha,25(OH)2D3 . However, even at an exceedingly high concentration of 20 ng/mL, IL-6 was ineffective in inducing osteoclast-like cell formation . Therefore, any hormonally induced rise in IL-6 release from bone marrow cells could not account for the observed changes in TRAP+ MNC numbers . Nevertheless, the stimulatory effect of 1alpha,25(OH)2D3 on osteoclastogenesis was partially dependent on IL-6 because it could be significantly blocked by a neutralizing monoclonal anti-IL-6 antibody, and to the same extent by a monoclonal anti-IL-6 receptor antibody . Unimpaired signaling through the IL-6/IL-6R system is also a prerequisite for the auxiliary effect of T3 on induction of osteoclast-like cells by 1alpha,25(OH)2D3 . Our data provide evidence that 1alpha,25(OH)2D3 induces osteoclast-like cell formation, at least in part, in an IL-6-dependent mode of action, which is also subject to modulation by T3 . The mechanism of interaction of the two hormones apparently involves joint stimulation of prostaglandin synthesis.

J Nat Prod, 1998 Mar 27, 61(3), 354 - 7
Modified Monoterpenes from Biotransformation of (-)-Isopiperitenone by Suspension Cell Culture of Mentha piperita
Park SH, Kim SU.
The biotransformation of (-)-(4R)-isopiperitenone (1) by suspension cell culture of Mentha piperita yielded three new hydroxylated derivatives, 4-6, and two new epoxidized derivatives, 7 and 8 . (-)-7-Hydroxyisopiperitenone (2) and its glucoside were previously isolated from the culture . The structures of 4-8 were elucidated using spectral methods, and their absolute stereochemistry was established by NMR experiments and correlation with compounds of known configuration.

Cell Tissue Res, 1998 Mar, 291(3), 497 - 505
Loss of myocardial capillary endothelial-cell alkaline phosphatase (ALP) activity in primary endothelial cell culture; Lindner H et al.; Primary cultures of rat myocardial capillary endothelial cells were established and characterized . A range of typical endothelial cell-specific markers were retained in vitro . Cell kinetic studies in confluent endothelial-cell cultures in vitro revealed a roughly 50-fold increase in the proportion of cells in s-phase, indicating a very considerable shortening of cell turnover time, compared to in vivo conditions . Alkaline phosphatase enzyme activity and encoding mRNA are strongly expressed in myocardial capillary endothelial cells in vivo, but were not detectable in vitro . This was true in cell cultures from two strains of rat, which revealed significantly different enzyme expression levels in vivo . In co-cultures of pericytes and endothelial cells, positive ALP enzyme reaction was detected in pericytes, which in vivo show only very weak enzyme reactivity . Treatment of cell cultures with </=10 M retinoic acid had no effect in pure endothelial cell cultures, but did increase ALP expression of pericytes in co-cultures . The observation of a loss of endothelial ALP expression in vitro supports other in vitro as well as our own in vivo observations, indicating a negative correlation of ALP expression and proliferative activity of endothelial cells.

J Neurosci Res, 1998 Mar 15, 51(6), 675 - 81
Cell culture models for reactive gliosis: new perspectives; Wu VW et al.; Reactive gliosis, which occurs in response to any damage or disturbance to the central nervous system, has been recognized for many years, but is still not completely understood . The hallmark is the increased expression of glial fibrillary acidic protein (GFAP), yet studies in GFAP knockout mice suggest that GFAP may not be required for an astrocyte to become hypertrophic . In this review, we describe a series of tissue culture models that have been established in order to address: 1) the biochemical phenotype of reactive astrocytes; 2) the factor and/or cell responsible for induction of gliosis; 3) the mechanisms by which one might block the induction . These models range from cultures of astrocytes, both neonatal and adult, to co-cultures of astrocytes with either neurons or microglia, to organ cultures . None is ideal: each addresses a different set of questions, but taken together, they are beginning to provide useful information which should allow a better understanding of the plasticity response of astrocytes to brain injury.

Regul Pept, 1998 Jan 2, 73(1), 59 - 65
Effects of dexamethasone on the profile of cytokine secretion in human whole blood cell cultures; Franchimont D et al.; EXPERIMENTAL OBJECTIVES: The interaction between the endocrine and immune systems is a very intriguing area . Endogenous glucocorticoids, as end-effectors of the hypothalamo-pituitary-adrenal axis, inhibit the immune and inflammatory responses and are used as immunosuppressive drugs in many inflammatory, autoimmune and allergic diseases . The aims of this study were to investigate the effects of dexamethasone on the profile of cytokine secretion in whole blood cell cultures from healthy subjects and to analyse the gender-related sensitivity to dexamethasone on each cytokine secretion . RESULTS: There was a significant inhibition by dexamethasone (from 1 to 100 nM) on the secretion of monokines (IL-1beta, IL-6, IL-8 and TNF alpha) and lymphokines (IL-2, IL-4, IL-10 and IFN gamma), either after LPS or PHA stimulation (P < 0.01) . Interleukin 4 and IL-10 were less inhibited than IFN gamma (P < 0.05 at 1 nM, P < 0.01 at 10 nM and P < 0.001 from 100 nM to 10 microM) . No gender difference was observed in the rate of inhibition of the secretion of each cytokine . CONCLUSION: This study shows that the inhibition of cytokine secretion by dexamethasone is more marked on Th1-type cytokines than on Th2-type cytokines . These data support the idea that glucocorticoids may induce a shift from the Th1 to Th2 profile of cytokine secretion.

Artif Organs, 1998 Mar, 22(3), 177 - 81
The influence of recombinant human erythropoietin on tumor necrosis factor alpha and interleukin-10 production by whole blood cell cultures in hemodialysis patients; Bryl E et al.; Impaired immunological response in hemodialysis (HD) patients, which leads to inappropriate cytokine production, is partially caused by the hyperstimulation of both T lymphocytes and monocytes/macrophages . Recent data suggest that human recombinant erythropoietin (rhEPO) may have an immunological action . The goal of our study was to estimate the influence of rhEPO treatment on the production of the inflammatory cytokine tumor necrosis factor alpha (TNFalpha) and antiinflammatory cytokin interleukin-10 (IL-10) in 10 HD patients receiving rhEPO for 6 months . The levels of cytokines were measured in the in vitro cultures of whole blood . The level of IL-10 increased in all treated patients during the therapy, and it was accompanied by a transitory decrease of TNFalpha . The results of our studies suggest that rhEPO may reduce the inflammatory process by decreasing production of TNFalpha and increasing production of IL-10.

Clin Chim Acta, 1998 Jan 30, 269(2), 175 - 84
Protein binding of homocysteine and other thiols in HeLa cell cultures after addition of homocysteine and copper ions; Hultberg B et al.; Homocysteine can be viewed as the first risk factor for atherosclerosis, believed to exert its effects through a mechanism involving oxidative damage . Oxygen radicals are known to interact with a variety of macromolecules leading to lipid peroxidation, DNA strand breakage and a variety of changes in proteins, including thiol oxidation . The present study deals with the protein-binding of homocysteine, cysteine and glutathione in a human cell line culture exposed to homocysteine and copper ions in order to elucidate the possible role of homocysteine in cell injury and atherogenesis . It is shown that homocysteine has the highest tendency of the thiols investigated to create disulfide bonds with proteins . The interaction with the protein cysteine thiol groups, which are involved in the function of many enzymes, structural proteins and receptors might disturb many metabolic functions in the cell . This finding might therefore be one reason for the cell-damaging effects of homocysteine . Addition of reduced homocysteine to cell cultures decreased the intra- and extracellular proportions of protein-bound thiols except that of intracellular glutathione . In agreement with the pro-oxidative effects of copper ions, the findings in the present study showed an increase of the protein-bound fractions of all thiols after the addition of copper ions . Another finding is that increased (in the presence of 100 mumol/l of copper ions) or decreased (in the presence of 100-2000 mumol/l of homocysteine) proportions of protein-bound fractions of the thiols in these short-term experiments did not seriously affect the cells since the cell growth was unchanged.

Am J Reprod Immunol, 1998 Mar, 39(3), 217 - 22
Evidence that folliculo-stellate cells mediate the inhibitory effect of Japanese kampo medicine, unkei-to, on growth hormone secretion in rat anterior pituitary cell cultures; Koike K et al.; PROBLEM: Cytokine-induced neutrophil chemoattractant (CINC) was reported to influence anterior pituitary hormone release . We recently found that Unkei-to, one of the Japanese Kampo medicines, stimulated CINC secretion by rat anterior pituitary cells and the pituitary folliculo-stellate (FS)-like cell line (TtT/GF) . Therefore, the effect of Unkei-to on growth hormone (GH) secretion by rat anterior pituitary cells was investigated . METHOD OF STUDY: Dispersed normal anterior pituitary cells, the folliculo-stellate-like cell line TtT/GF, and the GH3 cell were used to test the effect of Unkei-to on GH secretion . In reconstitutive coculture experiments, TtT/GF cells were mixed with GH3 cells at a ratio (TtT/GF cells: GH3 cells) of 1:99 . From this mixture, cells were seeded onto plates at a density of 10(4) cells/well and were cultured for 5 days . The cells were then used in the experiments . RESULTS: Unkei-to at 20 micrograms/ml significantly inhibited GH secretion by normal anterior pituitary cells within 12 hr of incubation . In contrast Unkei-to stimulated GH secretion by GH3 cells in a time- and dose-dependent manner, suggesting that an accessory cell type was involved . To assess the contribution of CINC as a paracrine factor, an experiment using a reconstitutive coculture system was performed, and Unkei-to was found to inhibit GH secretion when GH3 cells were cocultured with TtT/GF cells . The addition of anti-CINC antibody to the reconstitutive coculture system antagonized Unkei-to-inhibited GH secretion . CONCLUSION: CINC, which was secreted from FS cells by Unkei-to, may be responsible for mediating the inhibitory effect of Unkei-to on GH secretion by rat anterior pituitary cells.

J Physiol, 1998 Feb 15, 507 ( Pt 1), 41 - 53
Regulation of acetylcholine release by intracellular acidification of developing motoneurons in Xenopus cell cultures; Chen YH et al.; 1 . The effects of intracellular pH changes on the acetylcholine (ACh) release and cytoplasmic Ca2+ concentration at developing neuromuscular synapses were studied in Xenopus nerve-muscle co-cultures . 2 . Spontaneous and evoked ACh release of motoneurons was monitored by using whole-cell voltage-clamped myocytes . Intracellular alkalinization with 15 mM NH4Cl slightly reduced the frequency of spontaneous synaptic currents (SSCs) . However, cytosolic acidification following withdrawal of extracellular NH4Cl caused a marked and transient increase in spontaneous ACh release . 3 . Another method of cytosolic acidification was used in which NaCl in Ringer solution was replaced with weak organic acids . The increase in spontaneous ACh release paralleled the level of intracellular acidification resulting from addition of these organic acids . Acetate and propionate but not isethionate, methylsulphate and glucuronate, caused an increase in intracellular pH and a marked increase in spontaneous ACh release . 4 . Impulse-evoked ACh release was slightly augmented by intracellular alkalinization and inhibited by cytosolic acidification . 5 . Cytosolic acidification was accompanied by an elevation in the cytoplasmic Ca2+ concentration ({Ca2+}i), resulting from both external Ca2+ influx and intracellular Ca2+ mobilization . In contrast, the increase in {Ca2+}i induced by high K+ was inhibited by cytosolic acidification . 6 . We conclude that cytosolic acidification regulates spontaneous and evoked ACh release differentially in Xenopus motoneurons, increasing spontaneous ACh release but inhibiting evoked ACh release.

J Bone Miner Res, 1998 Mar, 13(3), 354 - 62
Persistence of Ca2+-sensing receptor expression in functionally active, long-term human parathyroid cell cultures; Roussanne MC et al.; An original human parathyroid cell culture model from uremic patients with IIo hyperparathyroidism has been developed, with its main feature being long-term functionally active viability up to 5 months, as assessed by persistent responsiveness to changes of extracellular Ca2+ concentrations ({Ca2+}e) . In addition to the inhibitory effect of increasing {Ca2+}e, increasing extracellular phosphate exerted a biphasic effect on parathyroid hormone (PTH) secretion . The presence of the Ca2+-sensing receptor (CaR), on which depends the response to {Ca2+}e and its persistence, has been demonstrated in our culture system both by direct detection and by inhibition of its activity . CaR protein was detected by Western blot analysis with a specific anti-CaR antibody . CaR gene transcripts have been identified by reverse transcription-polymerase chain reaction analysis . mRNA (by in situ hybridization) and protein (by immunocytochemistry) expression were detected for both CaR and PTH . Adding a specific anti-CaR antibody to the medium induced a marked reduction of low {Ca2+}e-stimulated PTH release, which decreased to levels equivalent to those obtained in high {Ca2+}e medium . The described long-term functionality could be due to several factors, including the clustered cell type of culture yielded by our preparation procedure, the growth characteristics of hyperplastic uremic tissue, and the use of a phosphate-rich medium . The present model, because of its long-term functionality, is a unique tool for the exploration of PTH synthesis and secretion and for studies of parathyroid cell growth in vitro.

Ophthalmic Res, 1998, 30(2), 120 - 5
Cell growth inhibition and DNA incorporation of mitomycin C in cell culture; Takahashi N et al.; The present study was performed to clarify the effects of a 4-min exposure of mitomycin C (MMC) on cell growth, the cell cycle and MMC dose incorporated into DNA, using Chang's cultured human conjunctival cells . A low dose of MMC ranging from 0.00025 to 0.004% showed dose-dependent cytotoxicity when cell growth was active . Fifty percent cell viability was found when cells were treated with 0.001% MMC . A flow cytometer showed that 0.001% MMC inhibited the DNA synthetic phase . After 0.04% MMC was exposed to 3 x 10(6) cells and immediately rinsed, DNA was isolated to measure the dose of MMC detected from DNA . The total amount of DNA was 7 micrograms from which 3 micrograms of MMC was detected by high performance liquid chromatography . The above results revealed that the lowest concentration of MMC which caused 50% cell viability and cell cycle inhibition was 0.001% and that MMC was rapidly incorporated into DNA.

Biochemistry, 1998 Mar 17, 37(11), 3602 - 11
Oligomerization of endogenous and synthetic amyloid beta-protein at nanomolar levels in cell culture and stabilization of monomer by Congo red; Podlisny MB et al.; Amyloid beta-proteins (A beta) are proteolytic fragments of the beta-amyloid precursor protein (beta APP) that are secreted by mammalian cells throughout life but also accumulate progressively as insoluble cerebral aggregates in Alzheimer's disease (AD) . Because mounting evidence indicates that A beta aggregation and deposition are early, critical features of AD leading to neurotoxicity, many studies of A beta aggregation have been conducted using synthetic peptides under generally nonphysiological conditions and concentrations . We recently described the oligomerization of A beta peptides secreted by beta APP-expressing cells at low nanomolar (20-30 ng/mL) levels into sodium dodecyl sulfate- (SDS-) stable oligomers of 6-16 kDa . Here, we extensively characterize this in vitro system and show that the amyloid binding dye, Congo red, acts to markedly decrease oligomer/monomer ratios by stabilizing the 4 kDa A beta monomers (ID50 approximately equal to 3.4 microM) . Addition of radioiodinated synthetic A beta 1-40 to the cultures or to their conditioned media at physiological concentrations (0.25-2.5 nM) reveals that it undergoes progressive aggregation into SDS-stable oligomers of 6-25 kDa during brief (approximately 4 h) incubation at 37 degrees C, and this is inhibitable by Congo red . The level of A beta oligomers can be quantitated in the Chinese hamster ovary (CHO) conditioned medium by size-exclusion chromatography as well as by SDS-polyacrylamide gel electrophoresis (PAGE), and comparison of these two methods suggests that aggregation of A beta into higher molecular weight polymers that are not detectable by SDS-PAGE occurs in the cultures . We conclude that both endogenous and synthetic A beta can assemble into stable oligomers at physiological concentrations in cell culture, providing a manipulable system for studying the mechanism of early A beta aggregation and identifying inhibitors thereof under biologically relevant conditions.

AIDS Res Hum Retroviruses, 1998 Mar 1, 14(4), 347 - 52
Differences in reverse transcriptase activity versus p24 antigen detection in cell culture, when comparing a homogeneous group of HIV type 1 subtype B viruses with a heterogeneous group of divergent strains; Corrigan GE et al.; Failure to detect infection with HIV-1 non-B subtypes in some antibody screening assays has been shown . To date, however, no studies have been published evaluating the capacity of standard tests to quantify replication of divergent HIV-1 in cell culture . Reverse transcriptase (RT) activity and p24 antigen assays are the two methods most commonly used for this purpose . A homogeneous panel of HIV-1 subtype B viruses from northern Italy and a heterogeneous panel of diverse genetic subtypes (A to F and O) from different regions of the world were cultured under identical conditions . A new nonradioactive RT assay was used as a basis for comparison to evaluate the capacity of two p24 assays to quantify viral growth in both panels . Comparison of the p24 amount/RT activity (p24/RT) ratios showed that ratios in the subtype B panel tended to be markedly higher than in the diverse subtype panel . Greatest variation was seen with one of the subtype O isolates, where up to a 400 times lower ratio was obtained compared with the average ratio for the subtype B panel . In addition, one Thai subtype B virus also gave a markedly reduced ratio . Furthermore, comparison between the two p24 assays showed different abilities to detect p24 from different HIV-1 isolates . We discuss limitations for the use of anti-HIV-1 p24 antibodies produced by immunization with subtype B p24 in p24 assays.

Gynecol Obstet Invest, 1998, 45(2), 116 - 20
Cervical dilatation: induction by antigestagens via adhesion molecules . An in vitro examination in endothelial cell cultures; Kemp B et al.; Cervical ripening at term resembles an inflammatory reaction with invasion of activated granulocytes into the cervical stroma . Our objective was to investigate the influence of the antigestagen onapristone alone and in combination with other substances associated with cervical ripening on the expression of the inflammation-associated adhesion molecules ELAM-1, ICAM-1 and VCAM-1 . Human endothelial cell cultures were stimulated with onapristone (200 ng/ml), TNF-alpha (100 U/ml), IL-8 (20 ng/ml) and PGE2 (3 ng/ml) separately and in combination (n = 6) . The expression of adhesion molecules was determined qualitatively and quantitatively by immunofluorescence staining and flow cytometry with statistical evaluation by Kolmogorov-Smirnov analysis . Onapristone upregulated slightly the expression of ELAM-1 (19%) . The costimulation of onapristone and TNF-alpha provoked an additive expression of VCAM-1 (64%) beyond the effect of TNF-alpha alone, while the costimulation of onapristone and PGE2 as well as the combination with IL-8 did not result in an additional stimulatory effect . All results were statistically significant (p < 0.001) . This result supports our hypothesis that onapristone may lead to an increased adhesion of granulocytes to the capillary endothelium thereby initiating cervical ripening.

J Neurosci Res, 1998 Feb 15, 51(4), 454 - 62
Evidence for non-transferrin-mediated uptake and release of iron and manganese in glial cell cultures from hypotransferrinemic mice; Takeda A et al.; Transferrin (Tf) is accepted as the iron mobilization protein, but its role in transport of other metals is controversial . In this study, we used mixed glial cultures from hypotransferrinemic (Hp) mice to determine the dependence of these cells on transferrin for iron and manganese delivery and release . Hp mice have a splicing defect in the transferrin (Tf) gene, resulting in < 1% of the normal plasma levels of Tf . Cellular iron and manganese uptake increases over 24 hr in cultures of normal and Hp glial cells in the presence of standard concentrations of Tf in the media; although total 59iron uptake in the Hp mouse cultures was 2X greater than normal, 54Mn uptake was similar between the two groups . The absence of Tf in the media resulted in a significant increase in 59iron uptake in both normal and Hp glial but did not affect Mn uptake . Elevated Tf (10X normal) in the media reduced both 59iron and 54Mn uptake . Efflux of 59Iron and 54Mn occurred in normal and Hp cultures, indicating the existence of a dynamic exchange of metals, and that intracellular Tf is not necessary for metal release . However, in the absence of Tf in the media, significantly more iron was retained in the cells than if Tf were present in both normal and Hp glial cultures . 54Mn release was minimally affected by extracellular Tf . The data demonstrate that Tf is not required for iron and Mn uptake into glial cells . These data further demonstrate a dynamic metal exchange system for glial cells which is not dependent on intracellular Tf.

Biochem Biophys Res Commun, 1998 Mar 6, 244(1), 312 - 6
Hydrocortisone reinforces the blood-brain barrier properties in a serum free cell culture system; Hoheisel D et al.; The increasing number of newly developed drugs demands for functional in vitro models of the blood-brain barrier to determine their brain uptake . Cultured cerebral capillary endothelial cells are considered to be such a model, however in serum containing media they exhibit low electrical resistances and high permeabilities compared to the in vivo situation . Here we report the establishment of a serum-free cell culture model . Withdrawal of serum already caused a twofold increase of transendothelial resistance (TER), which in presence of serum is about 100-150 omega.cm2 . We tested several supplements and found that hydrocortisone is a potent stimulator for the formation of barrier properties . TERs up to 1000 omega.cm2 were measured in the presence of physiological relevant hydrocortisone concentrations . In correspondence to the TER increase hydrocortisone decreased cell monolayer permeability for sucrose down to 5 x 10(-7) cm/s, which is close to the in vivo value of 1.2 x 10(-7) cm/s and by a factor of five lower compared to cultures without hydrocortisone and in presence of serum.

J Chromatogr A, 1998 Feb 13, 796(1), 165 - 75
Development of a downstream process for the isolation and separation of monoclonal immunoglobulin A monomers, dimers and polymers from cell culture supernatant; Luellau E et al.; The isolation and separation of the molecular variants of monoclonal IgA from cell culture supernatants is possible using several filtration and ion-exchange chromatography steps, followed by size-exclusion chromatography for the actual separation of the molecular variants . The latter step is especially time consuming and laborious . This report presents possible improvements of the procedure . Use of the displacement rather than the elution mode may render the ion-exchange step more productive (higher product concentrations and space-time yield) . For the final separation of the molecular variants, hydroxyapatite (HA) elution chromatography can serve as an alternative to size-exclusion chromatography . By using an optimized, complex phosphate gradient, the IgA dimers can be separated quantitatively from the monomers and higher oligomers . It may in individual cases be necessary to use a size-exclusion polishing step to reach the required final degree of purity, however, the amount of material to be processed is reduced to such an extend by the HA-step, that the overall process is still more productive . Buffer pH and flow-rate as well as the stationary phase material used were additional factors considered during the optimization of the HA elution chromatography . HA-displacement chromatography resulted only in a concentration of the overall IgA fraction, but not in a separation of the molecular forms.

Auris Nasus Larynx, 1998 Jan, 25(1), 25 - 32
Inflammatory mediators and otitis media with effusion . An experimental approach using cell culture; Tan CT et al.; Otitis media with effusion is characterized by the presence of an inflammatory cellular infiltrate of the submucosa and a poor ventilation of the middle ear . This result in hypersecretion of mucus and alteration of the mucociliary clearance, which produce accumulation of fluid and cellular debris in the middle ear . The aim of this work was to investigate whether inflammatory mediators such as prostaglandins and oxygen metabolites modulate the absorptive function of the middle ear epithelium . The data we present demonstrated that: (i) among prostanoids, only prostaglandin E2 modulated the rate of sodium transport; (ii) oxidants had a stimulatory effect on ion transport; (iii) the role of reactive oxygen species was mediated by prostaglandin E2 . This process might be involved in the impairment of the mucociliary clearance.

Trop Anim Health Prod, 1997 Nov, 29(4 Suppl), 109S - 113S
Responses in animals vaccinated with the Theileria annulata (Hisar) cell culture vaccine; Beniwal RK et al.; Bovine tropical theileriosis caused by Theileria annulata is an economically important disease of cattle in India . The disease has assumed paramount importance with the intensification of cross-breeding programmes aimed at enhancing milk production in the country . To control this disease, a cell culture vaccine was developed in this department by continuous passaging of T . annulata (Hisar) schizonts in vitro . Current work in this department has concentrated on the epidemiology of theileriosis: development of the cell culture vaccine for very young calves and pregnant cows; evaluation of serological responses using immunofluorescent antibody (IFA) tests and Enzyme-linked immunosorbent antibody assays (ELISA); studies on the duration of immunity stimulated by the cell culture vaccine; the immune/susceptible status of calves born to vaccinated dams . Results have shown the following . Clinical cases of theileriosis were mainly observed in young calves below two months of age followed by adults in exotic and cross-bred animals . Amongst indigenous animals, only young calves below two months of age suffered from clinical disease . Clinical cases of theileriosis mainly occurred between the months of April to October . The T . annulata schizont cell culture vaccine developed in the department was extensively used in the susceptible calves and pregnant/lactating cows in the field . Sufficiently high antibody titres were detected by both schizont as well as piroplasm antigen using both ELISA and IFAT . The results indicated that the vaccine was safe, potent and effective for all breeds and age groups of cattle under field conditions . ELISA was standardised for T . annulata using three antigens, viz.: soluble piroplasm, soluble schizont and cellular schizont antigens . Comparison of results with IFAT showed that ELISA is more sensitive, objective, reliable and specific as well as less cumbersome than IFAT . Piroplasm, cellular schizont and soluble schizont antigens were found to be suitable for the detection of antitheilerial antibodies as per their order in ELISA . Studies on the duration of immunity stimulated by the T . annulata schizont cell culture vaccine indicated that immunity started waning after six months . Calves born of dams immunised against T . annulata with the cell culture vaccine were found to be fully susceptible to theileriosis soon after birth . This indicated that there was no passive transfer of immunity from dams to their offspring through colostrum.

Trop Anim Health Prod, 1997 Nov, 29(4 Suppl), 98S - 100S
Research on the schizont cell culture vaccine against Theileria annulata infection in Xinjiang, China; Guo G et al.; Theileria annulata infection (TAI) is one of the most serious diseases of cattle in Xinjiang Autonomous Region of Uigur Minority Nationality . It has been recorded in 14 prefectures, except the Tulufan Prefecture, but the enzootic areas are mainly distributed around the Zhunger Basin and Talim Basin . According to the records collected in the ten years before vaccination with the schizont cell culture vaccine was carried out, the average incidence rate of TAI in enzootic areas was 7.22% and the mortality rate was 24% . The milk production of cattle suffering from TAI was sharply decreased, and there were usually abortions in pregnant cows . The incidence rate and mortality rate were greater in high grade cattle, so TAI was a constraint to improving cattle breeds . To control this disease effectively in Xinjiang, researchers at the Xinjiang Academy of Animal Science began to study the schizont cell culture vaccine in 1972 . In 1977 an immortalised cell line was achieved from a primary cell culture starting with white blood cells from cattle suffering from acute TAI caused by an artificial tick bite . The cell culture medium mainly consisted of calf serum, lactalbumin-hydrolysate, Eagles' medium DMEM and three antibiotics . As a vaccine, the above cells were mixed with preserving medium containing gelatin . This paper describes the experiments on the immunological properties of the vaccine carried out in subsequent years . Up to 1996, vaccine doses for 1,186,150 cattle have been produced and sold . This vaccine has had a critical effect on the control of TAI in Xinjiang . Owing to the sharp decrease in the incidence rate and mortality rate of TAI after cattle were vaccinated, the annual economic benefit of the vaccine is at least 1,620,000 yuan.

J Neurosci Res, 1998 Mar 1, 51(5), 602 - 11
Prenatal hippocampal granule cells in primary cell culture form mossy fiber boutons at pyramidal cell dendrites; Grosse G et al.; Mossy fiber boutons are the sites of synaptic signalling between hippocampal granule and pyramidal neurons . We studied the formation and localization of these terminals during development of prenatal hippocampal neurons in primary culture . Using the synaptic vesicle membrane proteins synaptophysin and synaptoporin as markers we observed that both proteins were mainly localized in perikarya and processes of fetal hippocampal neurons during the first days in vitro (DIV) . Following DIV 6 synaptophysin was present in small terminals . After DIV 20 in addition large terminals immunoreactive for synaptophysin and synaptoporin were found, which were identified by electron microscopy as mossy fiber boutons impinging on pyramidal neuron dendrites . Synaptic vesicles and endosomes in the mossy fiber boutons were labeled when incubated with exogenous horseradish peroxidase, indicating that they were competent for exo-endocytosis . Taken together, our data show that hippocampal granule neurons grown in dissociated primary cultures form mossy fiber boutons containing synaptophysin and synaptoporin at pyramidal cell dendrites . Since the composition and the characteristic morphology of mossy fiber boutons formed in vitro is the same as observed in vivo we conclude that their development follows an intrinsic program.

J Med Microbiol, 1997 Aug, 46(8), 711 - 3
Comparison of the Sanofi Diagnostics Pasteur Chlamydia Microplate EIA shortened assay with the original standard assay and cell culture; Chan EL et al.; The new Sanofi Diagnostics Pasteur Chlamydia Microplate EIA shortened assay was evaluated by comparison with the original standard assay and cell culture . A total of 853 paired male and female genital tract specimens was tested with both Sanofi Chlamydia Microplate EIA shortened and standard assays and the results were compared with those of cell culture . For confirmation, a blocking assay run in the shortened format was used . Discrepancies between the three methods were resolved by a direct fluorescent antibody (DFA) test on the EIA samples or the culture retentate, or both . After resolution of discrepant results, the standard assay had a sensitivity, specificity, positive predictive value and negative predictive value of 98.5%, 100%, 100% and 99.9%, respectively . The shortened assay results were 100%, 100%, 100% and 100%, respectively . The shortened assay takes approximately 1.5 h less time than the standard assay and this study demonstrated that they have equivalent sensitivity and specificity . The improvement in turnaround time enables results to be reported on the same day.

Biosci Biotechnol Biochem, 1998 Jan, 62(1), 148 - 50
Molecular cloning and characterization of a cDNA encoding asparagine synthetase from soybean (Glycine max L.) cell cultures; Yamagata H et al.; A cDNA encoding glutamine-dependent asparagine synthetase was isolated from dark-adapted Glycine max cell culture . The deduced amino acid sequence showed 76-89% identity with other plant sequences . The gene for asparagine synthetase is expressed predominantly in shoots as compared to roots of etiolated plants and the level of expression decreases following light treatment, suggesting that the gene expression is down-regulated by light.

Dtsch Tierarztl Wochenschr, 1998 Jan, 105(1), 29 - 31
{Cytopathogenic bovine viral diarrhea viruses induce apoptosis in bovine cell cultures}; Grummer B et al.; The pestivirus bovine viral diarrhea (BVD) virus is the causative agent of Mucosal Disease (MD) of cattle . From persistently infected animals, only the non-cytopathogenic biotype BVD virus can be isolated . Cattle succumbing to MD additionally harbour cytopathogenic BVD virus . While cp BVD virus isolates induce a cytopathic effect (cpe) in susceptible monolayer cell cultures, infection with ncp BVD virus isolates has no visible effect . The purpose of this study was to investigate the correlation between cpe and apoptosis.

Arch Biochem Biophys, 1998 Feb 15, 350(2), 145 - 56
Attenuation of 19-9 antigen secretion in human colorectal carcinoma cell cultures by transfection with cDNA encoding novel ADP-ribosylation factor-like proteins; Eboue D et al.; We have used cDNAs coding for novel ADP-ribosylation factor-like molecules (ARL184 and ARL184Delta) to alter 19-9 antigen glycoprotein secretion in cultured human colorectal carcinoma cells SW1116 by transfection and cloning . This ARL contains a lipophilic N-terminal with an isoleucyl and 3 leucyl residues, 4 functioning consensus sequence GTP binding sites, and 184 total aminoacyl residues . An ARL cDNA was also constructed deleting the codon for the N-terminal glycyl moiety . The resulting cell clones were shown by Northern blots to overexpress ARL mRNA . Electron microscopy-immunocytochemistry also indicated the overexpression of ARL granules subcellularly . Secretion of the tumor-associated 19-9 antigen into apical medium was decreased 3- to 5-fold and the secretion of TCA/PTA precipitable 3H-labeled glycoprotein was decreased by 34% in clone SW1116(ARL184)Delta . Western blot analyses of cell homogenates and media were in agreement with the secretion assays and showed a diminution of 170-200 kDa, 19-9, antigenicity in transfected cells and their media . Apical secretion of 19-9 antigen was diminished 14-fold in cells, SW1116 (ARL184)alpha, transfected with the complete ARL cDNA sequence, suggesting that the glycyl moiety may be required for maximal abatement . However, incorporation of label from {3H}myristate into 22-kDa bands of NP-40 extracts and ARL-antigenic molecules of parent cells was 3-fold greater than that in samples from the two transfectants; thus the transfected cells may not myristylate the overexpressed ARL efficiently . Notwithstanding the N-terminal glycyl moiety undergoing some other modification, we conclude that overexpression of this ARL is sufficient to generate a 19-9-deficient phenotype . These ARLs may eventually disrupt terminal oligosaccharide glycosylation, resulting in an apparent diminished exocytosis of 19-9 glycoprotein carriers by transfected and cloned cells .

Vopr Virusol, 1997 Nov-Dec, 42(6), 254 - 8
{Persistence of hepatitis C virus in newborn mice brain cell culture}; Deriabin PG et al.; A model of chronic infection of primary cultures of suckling mouse brain (SMB) cells actively producing hepatitis C virus (HCV) is developed . Destruction and repopulation of cells was observed for at least 6 months; this phenomenon was paralleled by virus release in culture medium . Persistent HCV contained in SMB cultures induced a cytopathogenic effect in PS, BHK-21, Vero, HAK, and click embryo cell cultures, its infective titers being 10.0-12.0 lg TCD50/0.2 ml . Persistent HCV formed heterogeneous plaques under agar in chick embryo cells . The polymerase chain reaction (PCR) regularly detected the HCV RNA at the stage of cell destruction in the culture fluid of HCV-infected cell cultures . The cytopathogenic activity of persistent HCV was neutralized by anti-HCV positive patients' sera with the neutralization index of 8.0-9.0 lg . The results of persistent HCV neutralization were confirmed by PCR . Immunofluorescence detected virus-specific HCV antigens in 15-40% of infected cells . Hence, the SMB-HCV system realized the cytopathogenic potential of HCV circulating in the blood of patients with hepatitis C . This system is promising for the study of the pathogenesis of HCV infection at a cellular level, for screening for specific and nonspecific antiviral agents, and for preparing native virus-specific proteins and RNA.

J Pathol, 1997 Dec, 183(4), 486 - 93
A novel grid polymerase chain reaction (G-PCR) approach at ultrastructural level to detect target DNA in cell cultures and tissues; Kareem BN et al.; A novel grid polymerase chain reaction (G-PCR) method has been developed to be used at the ultrastructural level and with a high degree of resolution . Samples applied to test the method were fresh cell lines (CaSki, SiHa) and HPV-16 DNA-containing tissues rescued from routine paraffin blocks . The specimens were embedded in Epon-Araldite and/or hydrophilic-resin LRWhite . Ultrathin sections mounted on grids were subjected to G-PCR using an HPV-16-specific primer set . The amplified products were identified by auro-immunohistochemical labelling of the biotinylated nucleotide . The results indicated successful amplification of target DNA in both cell and tissue samples, being confined to the intranuclear region . The negative controls {HeLa cells, isolated mammary carcinoma cell cultures (MCF 7, and T47-D) (ATCC) (U.S.A.), normal thyroid tissue and steroid-producing tumour tissue} failed to exhibit any amplification of the target DNA sequences . The sensitivity of the G-PCR system was evaluated by performing a parallel in situ hybridization (ISH) of serial sections . The signals obtained from G-PCR were more intense than those of ISH and more informative as to the precise subcellular localization of amplicons.

J Neurosci Methods, 1997 Dec 30, 78(1-2), 133 - 7
Cold jet: a method to obtain pure Schwann cell cultures without the need for cytotoxic, apoptosis-inducing drug treatment; Jirsova K et al.; This paper presents a new and gentle method to separate Schwann cells from fibroblasts obtained from foetal rat dorsal root ganglia (DRG) . The method exploits the different growth and adhesion characteristics of fibroblasts and Schwann cells under different experimental conditions such that antiproliferative (cytotoxic) drugs or time-consuming centrifugation is not needed . Standard procedures were used to obtain mixed cultures of Schwann cells, fibroblasts and neurons . After about 5 days further purification of the cells was achieved by exploiting the different responses of Schwann cells and fibroblasts to a temperature shock . Cooling the cells with cold phosphate-buffered saline (PBS), followed by pipetting cold medium directly on top of the cells ('cold jet'), resulted in specific detachment of Schwann cells and neurons, whereas fibroblasts remained securely attached . Schwann cells attached to the surface of new, uncoated culture dishes whereas neurons did not . Two cycles of the cold jet procedure resulted in nearly pure (98-100%) cultures of Schwann cells . Besides being gentle, this method is easy and fast, and because cytotoxic drugs are not used, it does not affect cell survival negatively.

J Bone Miner Res, 1998 Feb, 13(2), 175 - 84
LIF, but not IL-6, regulates osteoprogenitor differentiation in rat calvaria cell cultures: modulation by dexamethasone; Malaval L et al.; Cytokines of the interleukin 6 (IL-6) subfamily are a group of factors produced by osteoblasts and acting through the same transducing element, membrane protein gp130 . We have previously shown that exogenous (added to the culture medium) leukemia inhibitory factor (LIF) inhibits bone nodule formation and expression of osteoblast-associated genes in fetal rat calvaria (RC) cell cultures and that dexamethasone (Dex) increases the ID50 of LIF . To investigate the respective roles of IL-6-related cytokines and receptors in osteprogenitor differentiation, and their regulatory interplay with Dex, we used reverse transcribed polymerase chain reaction, bioassay, and blocking antibody techniques to assess the time courses of LIF, IL-6, LIF transmembrane receptor, IL-6 receptor, and gp130 expression in RC cell cultures grown with and without Dex . The levels of the mRNAs for IL-6, LIF, and gp130 decreased concomitantly with the formation of bone nodules . Dex treatment, which stimulates bone nodule formation, reduced the expression of LIF and IL-6 mRNAs and IL-6 bioactivity in the culture medium . LIF treatment strongly stimulated the expression of IL-6 . Incubation with anti-LIF antibodies increased the number of nodules, while an antibody blocking IL-6 activity had little or no effect on nodule numbers and did not antagonize the action of exogenous LIF, indicating that IL-6 does not mediate the action of LIF in this system . Moreover, although exogenously added IL-6 was active in the cultures as noted by a reduction of nodule mineralization, it had no effect on nodule numbers, i.e., on osteoprogenitor differentiation, in the presence or absence of Dex . In conclusion, IL-6, LIF, and their receptors are expressed throughout the time-course of osteogenesis in RC cell cultures . However, only LIF, but not IL-6, appears to play a significant role in autocrine regulation of osteoblastic differentiation in this system . The antagonist action of Dex on the effects of exogenously added LIF, as well as the bone-promoting action of Dex in RC cell cultures, could be exerted partly through the down-regulation of the expression of endogenous LIF.

Anticancer Res, 1997 Nov-Dec, 17(6D), 4435 - 41
Systematic determination of telomerase activity and telomerase length during the progression of human breast cancer in cell culture models; Imam SA et al.; The purpose of the study was to determine systematically the expression of telomerase activity and the length of telomere repeat arrays by utilizing two different cell culture models that derive from normal individual donors, and probably represent various stages of human breast oncogenesis in cell culture . The models consist of mortal, non-tumorigenic immortal and tumorigenic immortal human mammary epithelial cell (MEC) lines . Using a recently developed polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP) assay, telomerase activity was undetectable in mortal MEC cells . In contrast, the immortal MEC that were nontumorigenic or tumorigenic in immunosuppressed athymic mice, showed telomerase activity . The absence of telomerase activity in mortal and its presence in both non-tumorigenic and tumorigenic immortal cell lines did not reflect their proliferative rate, as demonstrated by the similar pattern and intensity of reactivity of these cell lines with anti-Ki 67 antibody which recognizes a human nuclear cell proliferation--associated antigen . Southern blot analyses of Hinf I-digested genomic DNA hybridized with a (TTAGGG)4 probe revealed arrays of telomeric repeat lengths ranging from 3 to 5, 3.5 to 9, 3.2 to 9 or 3 to 15 kilobase pair (kbp) for mortal, nontumorigenic immortal, and tumorigenic immortal or established MEC lines respectively . These results suggest that telomerase activity and stable telomeric repeat lengths may be a molecular phenotype of the early stages in the progression of breast cancer.

Microbiol Immunol, 1997, 41(12), 947 - 55
Immunological studies of a 21 kDa cellular component efficiently incorporated into rabies virion grown in a BHK-21 cell culture; Sagara J et al.; To investigate cellular components incorporated into the rabies virion, monoclonal antibodies (MAbs) were screened based on their reactivity with additional virion components . Two of the MAbs we prepared recognized a virion-associated 21 kDa polypeptide (referred to as VAP21) from a BHK-21 cell . Since the MAbs precipitated the rabies virion and trypsin digestion eliminated the VAP21 antigen from the virion but alkaline treatment (pH 11) did not, VAP21 seems to be anchored into the viral envelope and exposed on the virion surface . Although quantitative immunoblot analyses indicated an apparently increased concentration of VAP21 in the virion, the ratio of the content of VAP21 to that of viral glycoprotein (G) was several times decreased as compared to the ratio of those in the cell . These data suggest that sorting of VAP21 occurs during the viral budding process on the cell but that it might be inefficient, probably due to a more intimate association of VAP21 with the viral envelope proteins . This assumption seems to be consistent with the results of immunofluorescence studies; that is, VAP21 displayed colocalized distribution with viral envelope antigens in the cell . From these results, it is suggested that VAP21 closely associates with the viral envelope proteins in the cell, and this association might cause passive but relatively efficient incorporation of VAP21 into the virion.

AIDS Res Hum Retroviruses, 1998 Feb 10, 14(3), 255 - 60
A novel mutation (F227L) arises in the reverse transcriptase of human immunodeficiency virus type 1 on dose-escalating treatment of HIV type 1-infected cell cultures with the nonnucleoside reverse transcriptase inhibitor thiocarboxanilide UC-781; Balzarini J et al.; Treatment of wild-type human immunodeficiency virus {HIV-1(IIIB)}-infected cell cultures with the thiocarboxanilide UC-781 under low selective pressure (i.e., 0.01 microg/ml) resulted in the emergence of V106A RT mutant virus . On increasing drug concentrations (stepwise up to 30 microg/ml) the virus retained the V106A RT mutation but acquired the novel F227L mutation in the RT genome in addition to the L100I, K1O1I, and Y181C mutations . This multiple-mutant virus proved highly resistant to virtually all nonnucleoside RT inhibitors (NNRTIs) (e.g., nevirapine, delavirdine, and loviride), but retained full sensitivity to nucleoside analogs such as AZT, ddI, (-)FTC, and 3TC . The F227 amino acid is highly conserved in HIV-1 strains and forms part of the NNRTI-binding pocket . Our model suggests a hydrophobic interaction between F227 and the chloro atom of UC-781.

Res Vet Sci, 1997 Nov-Dec, 63(3), 199 - 203
Comparison of RT-PCR assay and virus isolation in cell cultures for the detection of bovine viral diarrhoea virus (BVDV) in field samples; Laamanen UI et al.; The virus isolation-immunoperoxidase test (IPX) on cell cultures and the reverse transcription-polymerase chain reaction (RT-PCR) assay were compared for the detection of bovine viral diarrhoea virus (BVDV) directly in serum samples . Material for this study consisted of 403 sera originating from cattle in 41 BVDV-infected Finnish dairy herds and one suckler cow herd . The presence of virus was demonstrated in 48 samples by both assays . In addition, two more samples were found to be positive by the RT-PCR assay . Both methods proved to be extremely sensitive, detecting pestiviruses even in high serum dilutions, and thus to be suitable for demonstrating the occurrence of persistently infected (PI) cattle . In conclusion, the RT-PCR method used had the advantage of ascertaining BVDV nucleic acid sequences in samples in which the virus had been inactivated, eg during transport or due to the presence of neutralising antibodies.

Tsitologiia, 1997, 39(7), 566 - 70
{Intercellular contacts accelerate the growth of cell colonies in a chick embryo cell culture}; Gasparian GG et al.; In sparsely seeded (1.10(3) cells/sm2) chick embryo cell cultures no cell proliferation commonly occurs . However, such factors as increasing cell density, a conditioned medium, or addition of ethanol fixed homologous cells to the culture may accelerate the cell growth . The mitogenic action of fixed cells serves as a contact stimulation of cell proliferation (Gasparian, Grigorian, 1989, 1990) . Distant and contact cell-to cell interactions, that involve soluble and insoluble cell derived mitogens, are supposed to operate during the log phase of culture growth . The addition of an excess of cells to the previously sparse culture may mimic the cell microenvironment commonly existing in subconfluent cultures . The role of diverse cell-to cell contacts in the cell growth regulation is discussed . The addition of ethanol-fixed cells may improve the cell cloning technique.

J Neurochem, 1998 Mar, 70(3), 1054 - 60
A cell culture model for androgen effects in motor neurons; Brooks BP et al.; Androgens are known to alter the morphology, survival, and axonal regeneration of lower motor neurons in vivo . To understand better the molecular mechanisms of androgen action in neurons, we created a model system by stably expressing the human androgen receptor (AR) in motor neuron hybrid cells . Motor neuron hybrid cells express markers consistent with anterior horn cells and can be differentiated into a neuronal phenotype . When differentiated in the presence of androgen, AR-expressing cells, but not control cells, exhibit a dose-dependent change in morphology: androgen-treated cells develop larger cell bodies and broader neuritic processes while continuing to express neuronal markers . In addition, androgen promotes the survival of AR-expressing cells, but not control cells, under low-serum conditions . Our results demonstrate a direct trophic effect of androgens on lower motor neurons, mediated through the AR expressed in this population of neurons.

J Nutr, 1998 Feb, 128(2), 257 - 64
Decreased citrate improves iron availability from infant formula: application of an in vitro digestion/Caco-2 cell culture model; Glahn RP et al.; We have applied an in vitro digestion/Caco-2 cell culture model to the assessment of iron availability from human milk and a generic cow's milk-based infant formula . Experiments were designed to determine the availability of iron from human milk relative to infant formula and whether known promoters of iron absorption would increase Caco-2 cell iron uptake and availability from the infant formula . In addition, we sought to determine if decreasing the citrate concentration in the infant formula would increase the iron uptake . Although approximately twice as much iron was in solution from digests of the infant formula relative to that of human milk, smaller or equal amounts of iron were taken up from the infant formula relative to the human milk digest . These results are qualitatively similar to in vivo studies . Addition of known iron uptake promoters to infant formula did not enhance Caco-2 cell iron uptake from the infant formula digest, indicating that the iron in the infant formula existed predominantly in a tightly bound unavailable form(s) . Enzymatic pretreatment of the infant formula with citrate lyase and oxalacetate decarboxylase decreased the citrate concentration by 67% and resulted in a 64% increase of iron in solution, which corresponded to a 46% increase in the cell iron uptake . Iron uptake from the "low citrate" formula plus cysteine was 102% greater relative to the nontreated formula . The results indicate that too much citrate can reduce iron uptake, particularly if it is present at concentrations greater than promoters such as ascorbic acid and cysteine.

Am J Physiol, 1998 Feb, 274(2 Pt 1), G389 - 96
Vitamin D increases tight-junction conductance and paracellular Ca2+ transport in Caco-2 cell cultures; Chirayath MV et al.; We investigated the effects of 1 alpha,25-dihydroxyvitamin D3 {1,25(OH)2D3} on paracellular intestinal Ca2+ absorption by determination of transepithelial electric resistance (TEER), as a measure of tight-junction ion permeability and bidirectional transepithelial 45Ca2+ fluxes in confluent Caco-2 cell cultures . The rise of TEER to steady-state levels of approximately 2,000 omega.cm2 was significantly attenuated by 1,25(OH)2D3 (by up to 50%) in a dose-dependent fashion between 10(-11) and 10(-8) M . Synthetic analogs of 1,25(OH)2D3, namely, 1 alpha,25-dihydroxy-16-ene,23-yne-vitamin D3 and 1 alpha,25-dihydroxy-26,27-hexafluoro-16-ene,23-yne-vitamin D3, exhibited similar biopotency, whereas their genomically inactive 1-deoxy congeners were only marginally effective . Enhancement of transepithelial conductance of Caco-2 cell monolayers by vitamin D was accompanied by a significant increase in bidirectional transepithelial 45Ca2+ fluxes . Although 1,25(OH)2D3 also induced cellular 45Ca2+ uptake from the apical aspect of Caco-2 cell layers and upregulated the expression of calbindin-9kDa mRNA, no significant contribution of the Ca(2+)-adenosinetriphosphatase-mediated transcellular pathway to transepithelial Ca2+ transport could be detected . Therefore stimulation of Ca2+ fluxes across confluent Caco-2 cells very likely results from a genomic effect of vitamin D sterols on assembly and permeability of tight-junctional complexes.

Inflammation, 1998 Feb, 22(1), 95 - 106
Expression of CD54 (intercellular adhesion molecule-1) and the beta 1 integrin CD29 is modulated by a cyclic AMP dependent pathway in activated primary rat microglial cell cultures; Zuckerman SH et al.; Microglial cell activation plays a central role in acute and chronic inflammatory processes associated with neurodegeneration . As macrophage activation is generally associated with the up-regulation of specific surface antigens, the expression of CD54, and CD29 were evaluated on CD11b positive neonatal rat microglial cell cultures by flow cytometry . These cells when exposed to lipopolysaccharide, LPS, and gamma interferon, IFN gamma, exhibited a 2-3 fold increase in CD54 expression, an increase in CD29 and no change in CD11b . Maximal increases in CD54 and CD29 staining on CD11b positive microglial cells were apparent 20-24 h after LPS and IFN gamma while nitrite production reflecting inducible nitric oxide synthase activity, continued to increase . The increases in CD29 and CD54 staining were inhibited in a dose dependent manner by agents which increased intracellular cAMP levels including 100 microM 8-bromoadenosine 3':5'-cyclic monophosphate but not 8-bromoadenosine monophosphate, the phosphodiesterase inhibitor isobutyl methylxanthine and by direct activation of adenylate cyclase with forskolin . Concomitant with the dose dependent decreases in CD29 and CD54 staining were increases in intracellular cAMP and reduced TNF secretion . These results suggest that regulation of CD29 and CD54 expression on activated microglial cells involves a cAMP dependent pathway.

Neurochem Res, 1998 Jan, 23(1), 17 - 23
Effect of halothane in cortical cell cultures exposed to N-methyl-D-aspartate; Beirne JP et al.; In vivo studies have shown potent protection by volatile anesthetic agents against cerebral ischemic insults . Volatile agents have also been shown to antagonize glutamatergic neurotransmission at the N-methyl-D-aspartate (NMDA) receptor . This study examined the potential for halothane to reduce neuronal excitotoxic lesions caused by NMDA . Fetal rat cortical cell cultures were allowed to mature 13-16 d . Culture wells (n = 13-16) were treated with 0 mM - 3.96 mM halothane in the presence/absence of 30 microM NMDA . Additional cultures were exposed to 30 microM NMDA in the presence/absence of 10 microM MK-801 or 10 microM ACEA 1021 . Cellular lethality was assessed by measurement of lactate dehydrogenase (LDH) 24 hrs later . A maximal effect of halothane was observed at 0.70 mM (2.1 vol%) wherein a 36% reduction in NMDA-stimulated LDH release occurred relative to untreated controls . Both MK-801 and ACEA 1021 caused complete inhibition of NMDA-stimulated LDH release . These data confirm that halothane has modulatory effects at the NMDA receptor but potency of this drug is less than that of specific antagonists of either glutamate or glycine . These findings suggest that halothane protection in vivo can be partially explained by anti-excitotoxic properties although other mechanisms of action are probably also important.

Virology, 1998 Feb 1, 241(1), 1 - 13
One amino acid change on the capsid surface of poliovirus sabin 1 allows the establishment of persistent infections in HEp-2c cell cultures; Pelletier I et al.; Poliovirus mutants (PVpi) selected during the persistent infection of human neuroblastoma cells can establish secondary persistent infections in nonneural HEp-2c cells (I . Pelletier, T . Couderc, S . Borzakian, E . Wyckoff, R . Crainic, E . Ehrenfeld, and F . Colbere-Garapin, 1991, Virology, 180, 729-737) . Previous results from our laboratory have also shown that, in the genome of PVpi S11 derived from the Sabin 1 strain, the genomic region involved in this phenotype contains 11 missense mutations which map exclusively to the genes encoding the capsid proteins VP1 and VP2 . We report here the identification of precise viral determinants able to confer the capacity to establish persistent infections in HEp-2c cell cultures to the lytic Sabin 1 strain . We used a strategy based on the observation that PVpi, after a few months of persistent infection in HEp-2c cells, tend to regain a more lytic phenotype in uninfected HEp-2c cell cultures . We constructed mutant viruses carrying only a few mutations potentially involved in the phenotype of persistence . Two mutations were identified, one corresponding to the substitution His>Tyr of amino acid 142 of VP2 and another corresponding to the substitution Val>Ile of amino acid 160 of VP1 . Mutants carrying one or the other of the two determinants established persistent infections in HEp-2c cell cultures in about 20% of the infections . Higher frequencies were obtained with the mutant carrying both determinants (30%), and with PVpi S11 (63%), indicating that the effects of several determinants can be cumulative . The two determinants are localized on the capsid surface in a region known to be involved in the interactions between poliovirus and its cell receptor and in fact, we demonstrate here that in the case of the two persistent mutants, these interactions are modified .

Environ Health Perspect, 1997 Dec, 105(12), 1326 - 32
Dioxinlike components in incinerator fly ash: a comparison between chemical analysis data and results from a cell culture bioassay; Till M et al.; Potent polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxinlike polychlorinated biphenyls (PCBs) are among the most relevant toxic emissions from incinerators . Induction of cytochrome P450 1A1-catalyzed 7-ethoxyresorufin O-deethylase (EROD) activity in mammalian cell culture (EROD bioassay) is thought to be a selective and sensitive parameter used for the quantification of dioxinlike compounds . Fly ash extracts from municipal waste incinerators (MWI), a crematorium, wood combustors, and a noble metal recycling facility were analyzed in the EROD bioassay using rat hepatocytes in primary culture . Fractions containing 2,3,7,8-substituted PCDDs/PCDFs, dioxinlike PCBs, and 16 major polycyclic aromatic hydrocarbons (PAHs) were isolated from the extract and analyzed by gas chromatography-mass spectrometry (GC-MS) and by the EROD bioassay . It was found that with MWI samples the bioassay of the extract resulted in a two- to fivefold higher estimate of TCDD equivalents (TEQ) than the chemical analysis of PCDDs/PCDFs and PCBs . However, the outcome of both methods was significantly correlated, making the bioassay useful as a rough estimate for the sum of potent PCDDs/PCDFs and dioxinlike PCBs in extracts from MWI fly ash samples and in a fly ash sample from a crematorium . In noble metal recycling facility and wood combustor samples, higher amounts of PAHs were found, contributing to more pronounced differences between the results of both methods . The remaining unexplained inducing potency in fly ash samples probably results from additional dioxinlike components including certain PAHs not analyzed in this study.The hypothesis that emissions from MWI of hitherto unidentified dioxinlike compounds are higher by orders of magnitude than emissions of potent PCDDs/PCDFs and dioxinlike PCBs could not be confirmed . We found no indication for a marked synergistic interaction of dioxinlike fly ash components in the bioassay.

Brain Res Brain Res Protoc, 1998 Jan, 2(2), 160 - 4
An improved method for detection of apoptosis in tissue sections and cell culture, using the TUNEL technique combined with Hoechst stain; Whiteside G et al.; We describe a novel procedure for combining a fluorescent variant of the TUNEL technique with Hoechst 33342 stain (bis-benzimide) to identify apoptosis in tissue sections and cell culture . The biochemical hallmark of apoptosis is internucleosomal DNA cleavage, which gives rise to oligonucleosome-sized fragments (multiples of approximately 180 bp) that can be directly visualised by labelling with biotinylated or digoxygenin-conjugated nucleosides in a reaction that employs terminal deoxynucleotide transferase (TUNEL) . TUNEL and Hoechst 33342 have been used separately to identify apoptosis . TUNEL specifically labels dying cells, yet a low background makes comparison of labelled cells with surrounding normal cells difficult and causes disorientation in tissue sections . Hoechst 33342 binds all DNA therefore staining all nuclear material, allowing identification of apoptotic nuclei, but the analysis is laborious . Combining the two fluorescent labels allows the initial recognition of apoptotic cells using the TUNEL technique then, by simply changing the filter, the TUNEL positive nuclei can be compared to surrounding normal nuclei to assess changes in morphology and size . Hoechst 33342 acts as a counterstain, allowing identification of anatomical structures, and permits quantitative comparison between TUNEL positive versus normal cells . We have evaluated the technique using sections of rat embryo, post-axotomy neonatal dorsal root ganglia and adult dorsal root ganglia cell culture.

Med Pregl, 1997 Nov-Dec, 50(11-12), 565 - 8
{Economical production of rabies vaccine on cell cultures}; Lalosevic D et al.; By producing the rabies vaccine from cell culture, this vaccination has become safe, with minimal postvaccinal reactions . The first vaccine according to this technology was produced by Pavle (Paul) Fenje, former chief of department of the Pasteur Institute in Novi Sad . Many cell cultures have been introduced so far for the rabies virus multiplication: primary hamster kidney, fetal bovine kidney, chick embryo, continuous cell line monkey kidney (VERO), human diploid cell (HDC), etc . Some possibilities of an economical rabies vaccine production from a continuous BHK-21 cell line have been discussed and recommended.

Int J Cancer, 1998 Feb 9, 75(4), 635 - 42
Combination of cisplatin and radiation in cell culture: effect of duration of exposure to drug and timing of irradiation; Gorodetsky R et al.; Responses to the combination of cisplatin (CDDP) and radiation in experimental and clinical studies have been reported to vary from high radiosensitization to clear sub-additivity . We examined the combined effect of CDDP with ionizing radiation in both murine mammary adenocarcinoma (EMT-6) and human ovarian carcinoma (OV-1063) cells with special reference to the duration of CDDP exposure and timing of irradiation . Cell survival was measured with a colorimetric assay of cell density . The nature of interaction of cisplatin and radiation was evaluated using isobolograms and a combination index (CI) . Exposure of both cell lines to CDDP for 24 hr before irradiation yielded an additive or slightly sub-additive response only if the exposure was extended for a few more hours after irradiation . In EMT-6 cells, the combination of radiation with subsequent continuous as well as short-term (4 to 6 hr) CDDP treatment was found to have a clear sub-additive effect; dose escalation of each modality reduced the additional effect of the other . The sub-additive effect may be explained by a radiation-induced arrest of cells in late S phase, which was dose- and time-dependent . Post-radiation exposure to CDDP further increased the S-phase arrest . In contrast, a 2 hr post-radiation drug exposure resulted in a supra-additive combined effect . Our results stress the crucial role of the timing and the doses of both modalities as well as the duration of post-radiation drug exposure on their combined effect.

J Dent Res, 1998 Feb, 77(2), 406 - 11
"In vitro" dissolution of coral in peritoneal or fibroblast cell cultures; Fricain JC et al.; Previous studies have shown that in vivo coral resorption involves a biphasic process: First, the edges of the coral block become powdery, then extracellular fluid and phagocytosis contribute to the dissolution of the crystals . The authors examined some types of cells that could be involved in phagocytosis, particularly the ability of both dermal fibroblasts and mouse-resident peritoneal cells to phagocytose and dissolve coral powder "in vitro" . Radioactive coral was incubated for 24, 48, or 72 hrs with cells in the presence or absence of cytochalasin B (a phagocytic inhibitor) or chloroquine (a lysosomotropic agent) . Furthermore, to specify the role of crystal cell contacts in the solubilization process, they incubated radioactive coral in conditioned media (obtained from two-day human fibroblastic or macrophagic cell culture in the presence or absence of non-radioactive coral) or at a distance from the cells using culture inserts . Measurements of the radioactivity in the different supernatants were performed . Transmission electron microscopy was carried out on the cells cultivated in the presence or absence of radioactive coral . The data suggest that both fibroblasts and macrophages dissolve the coral, and that the intracellular degradation in phagolysosomes is one of the mechanisms explaining coral powder dissolution.

Ann Rheum Dis, 1997 Nov, 56(11), 693 - 5
Cytokine production in whole blood cell cultures of patients with rheumatoid arthritis; Swaak AJ et al.; OBJECTIVE: The measurement of cytokine production of activated lymphocytes and monocytes in the whole blood cell (WBC) culture system may provide a sensitive tool for evaluating the actual ongoing immune response of patients with rheumatoid arthritis (RA) . METHODS: Lipopolysaccharide (LPS) up to 250 pg/ml was used for the stimulation of monocytes for measuring the production of tumour necrosis factor alpha (TNF alpha), interleukin 6 (IL6) and IL12, while the anti-CD3 (1 microgram/ml) and anti-CD28 (5 micrograms/ml) combination was used for T cell stimulation with the measuring of IL4 and interferon gamma (INF gamma) production . Twenty seven patients with RA and 23 healthy controls were studied . RESULTS: The results showed a decreased IL6 (LPS stimulus 4-6 pg/ml) and IL-12 (LPS stimulus 16-62 pg/ml) production in the RA patients . The maximal production of both cytokines was comparable with the normal controls . T cell stimulation showed a significant decreased INF gamma production in the RA patients . CONCLUSIONS: These findings obtained in the WBC culture system are highly suggestive for a decreased TH-1 derived cytokine production by a diminished IL12 production in RA patients . Another possibility is that both IL12 and INF gamma production in WBCs are inhibited by eventual circulating serum factors.

J Gen Virol, 1998 Jan, 79 ( Pt 1), 1 - 10
Quasispecies evolution of a hypervariable region of the feline calicivirus capsid gene in cell culture and in persistently infected cats; Radford AD et al.; Feline calicivirus (FCV) is a respiratory pathogen of cats that is capable of causing persistent infections . This study examined the evolution of a hypervariable region of the FCV capsid gene both during 90 passages in cell culture and during replication in persistently infected cats . This region of the capsid protein is known to contain neutralization epitopes and may be a target for immune evasion during virus persistence in the host . Sequence analysis showed that FCV exists as a quasispecies which evolved both in cell culture and in persistently infected cats . Changes involved both loss of sequence present in the infecting isolate and a gain of both synonymous and non-synonymous nucleotide substitutions to generate sequences not detected within earlier isolates . Overall, these changes led to a reduction in population heterogeneity over time . Where virus populations were highly homogeneous allowing a consensus sequence to be determined, evolution rates for the consensus sequence ranged from 0.10-1.07 substitutions per nucleotide per year . Marked changes in virus neutralization profiles were seen in isolates obtained sequentially from a persistently infected cat . This was not the case with cell culture passaged virus, suggesting that the individual amino acid changes found only in virus from persistently infected cats may significantly alter the antigenic profile of FCV, and may be the result of immune selection.

Cancer Genet Cytogenet, 1998 Feb, 101(1), 16 - 23
A new serial transfer explant cell culture system for human prostatic cancer tissues preventing selection toward diploid cells; Zwergel T et al.; An improved explant cell culture technique to avoid selection of prostatic adenocarcinoma cells toward diploid cells is described . This method is based on 1) histologically characterized tissue explants, 2) the use of polyethylenteraphthalate (PET) membranes as growth surface, which are part of special inserts in six-well-plates to allow 3) cocultivation with heterologous fibroblasts, and 4) coating of the membranes with elements of the extracellular matrix . The main characteristic of this particular approach is the serial transfer of the tissue explant from one membrane to the other . Up to ten serial transfer steps could be performed to produce cell monolayers growing out of the same tissue specimen . Using this approach, 21 prostatic carcinoma specimens that were obtained from 13 primary prostatic adenocarcinomas after radical prostatectomy were cultivated . Ploidy of the cells was monitored by fluorescence in situ DNA hybridization using the centromere specific DNA probes pUC1.77, p alpha 7t1, and pY3.4 . Interestingly, a high aneuploidy rate of the cell cultures was found with maintainance of aneuploidy in 18 (86%) of the 21 paraffin-embedded cancer tissue specimens with proved aneuploidy . Although a slight decrease of the proportion of aneuploid cells during serial transfer was observed, significant aneuploid cell populations were retained up to a maximum of ten transfer steps . These findings indicate that selection toward diploid cells can be prevented by improved cell culture techniques that mimic the in vivo situation.

Calcif Tissue Int, 1998 Feb, 62(2), 114 - 21
Gene and protein expression during differentiation and matrix mineralization in a chondrocyte cell culture system; Kergosien N et al.; Endochondral bone formation occurs through a series of developmentally regulated cellular stages, from initial formation of cartilage tissue to calcified cartilage, resorption, and replacement by bone tissue . Nasal cartilage cells isolated by enzymatic digestion from rat fetuses were seeded at a final density of 10(5) cell/cm2 and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum in the presence of ascorbic acid and beta-glycerophosphate . First, cells lost their phenotype but in this condition they rapidly reexpressed the chondrocyte phenotype and were able to form calcified cartilaginous nodules with the morphological appearance of cartilage mineralization that occurs in vivo during endochondral ossification . In this mineralizing chondrocyte culture system, we investigated, between day 3 and day 15, the pattern expression of types II and X collagen, proteoglycan core protein, characteristic markers of chondrocyte differentiation, as well as alkaline phosphatase and osteocalcin associated with the mineralization process . Analysis of labeled collagen and immunoblotting revealed type I collagen synthesis associated with the loss of chondrocyte phenotype at the beginning of the culture . However, our culture conditions promoted extracellular matrix mineralization and cell differentiation towards the hypertrophic phenotype . This differentiation process was characterized by the induction of type X collagen mRNA, alkaline phosphatase, and diminished expression of type II collagen and core protein of large proteoglycan after an increase in their mRNA levels before the mineralizing process . These results revealed distinct switches of the specific molecular markers and indicated a similar temporal expression to that observed in vivo recapitulating all stages of the differentiation program in vitro.

FEBS Lett, 1997 Dec 29, 420(2-3), 143 - 6
Alternative pathways of xanthone biosynthesis in cell cultures of Hypericum androsaemum L; Schmidt W et al.; The biosynthesis of xanthones was studied in cell cultures of Hypericum androsaemum L . We have detected a new benzophenone synthase, for which the preferred substrate is benzoyl-CoA, itself supplied by 3-hydroxybenzoate:coenzyme A ligase . The stepwise condensation of benzoyl-CoA with three molecules of malonyl-CoA, catalyzed by benzophenone synthase, yields 2,4,6-trihydroxybenzophenone . This intermediate is subsequently converted by benzophenone 3'-hydroxylase, a cytochrome P450 monooxygenase . These biosynthetic steps, leading to the formation of 2,3',4,6-tetrahydroxybenzophenone, represent an alternative pathway to that recently proposed for cell cultures of Centaurium erythraea {Peters et al., Planta (1997) in press}.

J Steroid Biochem Mol Biol, 1997 Nov-Dec, 63(4-6), 317 - 28
Mechanisms of the actions of aromatase inhibitors 4-hydroxyandrostenedione, fadrozole, and aminoglutethimide on aromatase in JEG-3 cell culture; Yue W et al.; Selective inhibition of estrogen production with aromatase inhibitors has been found to be an effective strategy for breast cancer treatment . Most studies have focused on inhibitor screening and in vitro kinetic analysis of aromatase inhibition using placental microsomes . In order to determine the effects of different inhibitors on aromatase in the whole cell, we have utilized the human choriocarcinoma cell line, JEG-3 in culture to compare and study three classes of aromatase inhibitors, 4-hydroxyandrostenedione, fadrozole (CGS 16949A), and aminoglutethimide . Fadrozole is the most potent competitive inhibitor and aminoglutethimide is the least potent among the three . However, stimulation of aromatase activity was found to occur when JEG-3 cells were preincubated with aminoglutethimide . In contrast, 4-OHA and fadrozole caused sustained inhibition of aromatase activity in both JEG-3 cells and placental microsomes, which was not reversed even after the removal of the inhibitors . 4-OHA bound irreversibly to the active site of aromatase and caused inactivation of the enzyme which followed pseudo-first order kinetics . However, 4-OHA appears to be metabolized rapidly in JEG-3 cells . Sustained inhibition of aromatase induced by fadrozole occurs by a different mechanism . Although fadrozole bound tightly to aromatase at a site distinct from the steroid binding site, the inhibition of aromatase activity by fadrozole does not involve a reactive process . None of the inhibitors stimulated aromatase mRNA synthesis in JEG-3 cells during 8 h treatment . The stimulation of aromatase activity by AG appeared to be due to stabilization of aromatase protein . According to these results, 4-OHA and fadrozole would be expected to be more beneficial in the treatment of breast cancer patients than AG . The increase in aromatase activity by AG may counteract its therapeutic effect and might be partially responsible for relapse of breast cancer patients from this treatment.

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