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.;