Biol Chem, 1997 Aug, 378(8), 873 - 81 Matrix induced re-differentiation of cultured rat hepatocytes and changes of CCAAT/enhancer binding proteins; Runge D et al.; Rat hepatocytes de-differentiate and proliferate when cultured on collagen-coated dishes in a chemically defined Hepatocyte Growth Medium in the presence of hepatocyte growth factor and epidermal growth factor . The addition of biomatrix derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma stops this process and leads to re-differentiation of the cells . We monitored DNA binding activity and protein levels of CCAAT/Enhancer Binding Proteins (C/EBPs) during these events by electrophoretic mobility shift assays and western blot analysis . We used plasma protein gene expression as a marker for the proliferation and differentiation phases . During the initial proliferation phase the DNA binding activity of C/EBPs decreased about 5-10 fold, mainly due to reduction of C/EBP alpha protein to nearly undetectable levels . Addition of EHS-gel prevented the further loss of C/EBP alpha protein and established a new steady state level . Since C/EBP beta proteins were affected to a much lesser extent, the C/EBP alpha:C/EBP beta ratio was greater in the presence of EHS-gel . Transferrin, alpha 1-antitrypsin, and albumin mRNA expression increased substantially . Thus stabilized C/EBP alpha expression, an increased C/EBP alpha:C/EBP beta ratio, and increased expression of liver specific mRNAs all correlated with the transition of proliferative to differentiated cells. Glia, 1997 Sep, 21(1), 74 - 83 Regulation of energy metabolism by neurotransmitters in astrocytes in primary culture and in an immortalized cell line; Pellerin L et al.; Evidence suggests that astrocytes might play an important role in cerebral energy metabolism . A recently developed cell line, called DI TNC1, displays several characteristic features of astrocytes . Thus, we have investigated in these cells a number of parameters related to energy metabolism . First, glycogen, the major energy reserve in the brain, is present in these cells and its levels are influenced by the glucose content of the growth medium and the presence of serum . Second, several neurotransmitters including noradrenaline and vasoactive intestinal peptide (VIP) induce a glycogenolytic response . Their effect on glycogen is paralleled by a similar effect on the formation of cyclic AMP, which is presumably the second messenger involved . Third, noradrenaline stimulates glucose utilization (as reflected by 2-deoxyglucose uptake) in DI TNC1 cells, an effect which is mimicked by the second messenger arachidonate . Interestingly, two actions of neurotransmitters, which are well characterized in primary astrocytes, are absent in DI TNC1 cells . These are the noradrenaline- and VIP-induced resynthesis of glycogen and the glutamate-stimulated glycolysis . In summary, the observations reported here lend further support to the concept that astrocytes are important for the control of brain energy metabolism . In addition, DI TNC1 cells might represent an interesting preparation to help decipher some of the astrocytic functions related to energy metabolism. Vopr Virusol, 1997 May-Jun, 42(3), 133 - 4 {Culturing T-lymphoblastic cell lines in media containing growth proteins}; Kornilaeva GV et al.; Two optimal variants of growth medium with different content of growth proteins obtained from cattle and porcine blood sera are recommended for culturing lymphoblastoid cells . Use of growth proteins helps optimize the conditions of cell culturing by ruling out the toxic effect and Mycoplasma contamination and ensures the standardization of virological experiments due to the absence of gamma-globulin fraction. J Biol Chem, 1997 Sep 19, 272(38), 23532 - 9 Isolation of animal cell mutants defective in long-chain fatty aldehyde dehydrogenase . Sensitivity to fatty aldehydes and Schiff's base modification of phospholipids: implications for Sj-ogren-Larsson syndrome; James PF et al.; Using tritium suicide, we have isolated a variant of the Chinese hamster ovary cell line, CHO-K1, that is deficient in long-chain fatty alcohol:NAD+ oxidoreductase (FAO; EC 1.1.1.192) . Specifically, it was the fatty aldehyde dehydrogenase component that was affected . The enzymatic deficiency found in this mutant strain, designated FAA . K1A, was similar to that displayed by fibroblasts from patients with Sjogren-Larsson syndrome (SLS), an inheritable neurocutaneous disorder . Complementation analyses suggested that the deficiency in fatty alcohol oxidation in the FAA.K1A cells and the SLS fibroblasts is a result of lesions in homologous genes . The FAA.K1A cells were unable to convert long chain fatty aldehydes to the corresponding fatty acids . This resulted in a hypersensitivity of the FAA.K1A cells to the cytotoxic effects of long chain fatty aldehydes . The difference between the mutant and wild-type cells was most obvious when using fatty aldehydes between 14 and 20 carbons, with the greatest difference between wild-type and mutant cells found when using octadecanal . Fibroblasts from a patient with SLS also displayed the hypersensitivity phenotype when compared with FAldDH+ human fibroblasts . In both CHO and human FAldDH- cell lines, addition of long chain fatty aldehydes to the medium caused a dramatic increase in aldehyde-modified phosphatidylethanolamine, presumably through Schiff's base addition to the primary amine of the ethanolamine head group . When 25 microM hexadecanal was added to the growth medium, approximately 10% of the phosphatidylethanolamine was found in the fatty aldehyde-modified form in FAA.K1A, although this was not observed in wild-type cells . Modified phosphatidylethanolamine could be detected in FAldDH- cells even when exogenous fatty aldehydes were not added to the medium . We propose a possible role for fatty aldehydes, or other aldehydic species, in mediating some of the symptoms associated with Sjogren-Larsson syndrome. J Bacteriol, 1997 Sep, 179(18), 5963 - 6 Purification of the Azotobacter vinelandii nifV-encoded homocitrate synthase; Zheng L et al.; The nifV gene product (NifV) from Azotobacter vinelandii was recombinantly expressed at high levels in Escherichia coli and purified . NifV is a homodimer that catalyzes the condensation of acetyl coenzyme A (acetyl-CoA) and alpha-ketoglutarate . Although alpha-ketoglutarate supports the highest level of activity, NifV will also catalyze the condensation of acetyl-CoA and certain other keto acids . E . coli cells in which a high level of nifV expression is induced excrete homocitrate into the growth medium. Radiat Res, 1997 Sep, 148(3), 285 - 92 Tests of the double-strand break, lethal-potentially lethal and repair-misrepair models for mammalian cell survival using data for survival as a function of delayed-plating interval for log-phase Chinese hamster V79 cells; Lange CS et al.; Our data (Reddy et al., Radiat . Res . 141, 252-258, 1995) on the kinetics of the repair of potentially lethal damage in log-phase Chinese hamster V79 cells are used to test some predictions which arise from the different assumptions of the repair-misrepair (RMR) (C . A . Tobias, Radiat . Res . 104, S77-S95, 1985), lethal-potentially lethal (LPL) (S . B . Curtis, Radiat . Res . 106, 252-270, 1986) and double-strand break (DSB) (J . Y . Ostashevsky, Radiat . Res . 118, 437-466, 1989) models . The LPL model defines the time available for repair of PLD (t(rep)) as the time taken to reach maximal survival in a delayed-plating recovery experiment . Those data show that after this time has elapsed, contrary to the expectation of the LPL model, survival can be increased by changing the medium used for delayed plating from fresh growth medium to conditioned medium . According to the RMR model, all potentially lethal lesions should also be committed by that time and be unavailable for repair in the new medium . Only the DSB model correctly predicted that PLD (= DSBs) would still be available for repair after that time . Second, data for split-dose recovery are used to predict the first-order kinetics time constant for DSB repair (tau(DSBR)) using the DSB model (24 +/- 1.5 min) . This value is nearly identical to the value of 27 +/- 1 min determined from the data obtained by Cheong et al . using pulsed-field gel electrophoresis (PFGE) (Mutat . Res . 274, 111-122, 1992) . The value based on PFGE is used to calculate the value of t(rep) predicted by the DSB model (2.6 +/- 0.1 h), which agrees with the value determined experimentally as the time when changing the delayed-plating medium from growth medium to conditioned medium no longer gives the full recovery seen with delayed plating in conditioned medium (2.5 h) . However, some recovery was seen for a change in the medium (growth medium to conditioned medium) up to 5-6 h postirradiation . Reanalysis of the original data on DSB repair shows that they are consistent with two first-order repair rates (18 +/- 7 min and about 52 min) . These results are consistent with two pools of DSBs (or cells), each with their own t(rep) . The early t(rep), associated with tau(fast), is predicted to be 1.7 +/- 0.7 h, and the late t(rep), associated with tau(slow), is predicted to be about 5 h . Both values are in excellent agreement with the times at which changing from growth medium to conditioned medium no longer gives the full recovery seen in conditioned medium only (the early t(rep)), and the time when changing from growth medium to conditioned medium produces no further increase in survival (the late t(rep)), respectively . It is noted that attempts to correlate radiosensitivity with the rates of DSB repair, rather than using an explicit model such as the DSB model, are unlikely to be productive since survival depends on both tau(DSBR) and t(rep) (as defined in the DSB model) and the latter may be the more important determinant of radiosensitivity (as it appears to be for ataxia telangiectasia cells compared to normal fibroblasts and for irs compared to V79 cells). Genetics, 1997 Sep, 147(1), 43 - 55 Characterization of synthetic-lethal mutants reveals a role for the Saccharomyces cerevisiae guanine-nucleotide exchange factor Cdc24p in vacuole function and Na+ tolerance; White WH et al.; Cdc24p is the guanine-nucleotide exchange factor for the Cdc42p GTPase, which controls cell polarity in Saccharomyces cerevisiae . To identify new genes that may affect cell polarity, we characterized six UV-induced csl (CDC24 synthetic-lethal) mutants that exhibited synthetic-lethality with cdc24-4ls at 23 degrees . Five mutants were not complemented by plasmid-borne CDC42, RSR1, BUD5, BEM1, BEM2, BEM3 or CLA4 genes, which are known to play a role in cell polarity . The csl3 mutant displayed phenotypes similar to those observed with calcium-sensitive, Pet- vna mutants defective in vacuole function . CSL5 was allelic to VMA5, the vacuolar H(+)-ATPase subunit C, and one third of csl5 cdc24-4ls cells were elongated or had misshapen buds . A cdc24-4ls delta vma5::LEU2 double mutant did not exhibit synthetic lethality, suggesting that the csl5/vma5 cdc24-4ls synthetic-lethality was not simply due to altered vacuole function . The cdc24-4ls mutant, like delta vma5::LEU2 and csl3 mutants, was sensitive to high levels of Ca2+ as well as Na+ in the growth media, which did not appear to be a result of a fragile cell wall because the phenotypes were not remedied by 1 M sorbitol . Our results indicated that Cdc24p was required in one V-ATPase mutant and another mutant affecting vacuole morphology, and also implicated Cdc24p in Na+ tolerance. J Neurochem, 1997 Sep, 69(3), 1252 - 8 Oxidant injury in PC12 cells--a possible model of calcium "dysregulation" in aging: I . Selectivity of protection against oxidative stress; Joseph JA et al.; Previous research has suggested that the initial effects of cellular free radical neurotoxic insult involve large increases in intracellular Ca2+ . However, the exact role of oxidative stress on the various parameters involved in these increases has not been specified . The present experiments were performed to examine these parameters in PC12 cells exposed to 5, 25, or 300 microM H2O2 for 30 min in growth medium alone or containing either nifedipine (L-type Ca2+ antagonist), conotoxin (N-type antagonist), Trolox (vitamin E analogue), or alpha-phenyl-n-tert-butylnitrone (nitrone trapping agent; PBN) . The concentrations of H2O2 were chosen by examining the degree of cell killing induced by exposure to graded concentrations of H2O2 . The 5 and 25 microM concentrations of H2O2 produced no significant cell killing at either 30 min or 24 h after treatment, whereas the 300 microM concentration produced a moderate degree of cell killing that did not increase between the two times . Fluorescent imaging was used to visualize intracellular Ca2+ changes in fura-2-loaded cells . Baseline (pre-30 mM KCl) Ca2+ levels were increased significantly by H2O2 treatment (e.g., 300 microM, 200%), but the rise in the level of free intracellular Ca2+ after KCl stimulation (i.e., peak) was decreased (e.g., 300 microM, 50%) and the cell's ability to sequester or extrude the excess Ca2+ (i.e., Ca2+ recovery time) after depolarization was decreased significantly . All compounds prevented baseline Ca2+ increases and, with the exception of conotoxin, antagonized the peak decreases in Ca2+ . It is interesting that after 300 microM H2O2 exposure, only Trolox was partially effective in preventing these deficits in recovery . Conotoxin increased the decrement recovery in the absence of H2O2 . However, in cells exposed to 5 or 25 microM H2O2, conotoxin as well as the other agents were effective in preventing the deficits in recovery. J Virol, 1997 Sep, 71(9), 6898 - 904 The autonomous growth of human papillomavirus type 16-immortalized keratinocytes is related to the endothelin-1 autocrine loop; Venuti A et al.; Some human papillomaviruses (HPVs) such as HPV type 16 (HPV16) and HPV18 are involved in cervical carcinoma, and they can immortalize and transform keratinocytes . Endothelin-1 (ET-1) is produced in keratinocytes and has been shown to act through ETA receptors as an autocrine growth factor for keratinocytes . This study examines whether HPV16 alters the ET-1-mediated autocrine loop in human keratinocytes, providing a selective growth advantage for transformed cells . ET-1 is released in similar amounts from normal and HPV-transfected keratinocytes . All HPV-transfected cell lines express high-affinity ETA receptors . A two-fold increase in ET-1 binding sites is present in HPV16-immortalized keratinocytes, and this effect seems to be linked to the overexpression of mRNA for this receptor rather than to differences in the surface/internalized ratio of the receptors . ET-1 induces significant increases in {3H}thymidine incorporation and cell proliferation . Furthermore, HPV-transfected keratinocytes can proliferate in the absence of any growth factor added to the growth medium, and the ETA receptor antagonist BQ123 prevents this proliferation . These data suggest a new mechanism in the growth control of HPV-transformed cells mediated by the upregulation of ET-1 autocrine loop. J Biol Chem, 1997 Aug 15, 272(33), 20321 - 3 Gpa2p, a G-protein alpha-subunit, regulates growth and pseudohyphal development in Saccharomyces cerevisiae via a cAMP-dependent mechanism; Kubler E et al.; The small GTP-binding protein Ras and heterotrimeric G-proteins are key regulators of growth and development in eukaryotic cells . In mammalian cells, Ras functions to regulate the mitogen-activated protein kinase pathway in response to growth factors, whereas many heterotrimeric GTP-binding protein alpha-subunits modulate cAMP levels through adenylyl cyclase as a consequence of hormonal action . In contrast, in the yeast Saccharomyces cerevisiae, it is the Ras1 and Ras2 proteins that regulate adenylyl cyclase . Of the two yeast G-protein alpha-subunits (GPA1 and GPA2), only GPA1 has been well studied and shown to negatively regulate the mitogen-activated protein kinase pathway upon pheromone stimulation . In this report, we show that deletion of the GPA2 gene encoding the other yeast G-protein alpha-subunit leads to a defect in pseudohyphal development . Also, the GPA2 gene is indispensable for normal growth in the absence of Ras2p . Both of these phenotypes can be rescued by deletion of the PDE2 gene product, which inactivates cAMP by cleavage, suggesting that these phenotypes can be attributed to low levels of intracellular cAMP . In support of this notion, addition of exogenous cAMP to the growth media was also sufficient to rescue the phenotype of a GPA2 deletion strain . Taken together, our results directly demonstrate that a G-protein alpha-subunit can regulate the growth and pseudohyphal development of S . cerevisiae via a cAMP-dependent mechanism . Heterologous expression of mammalian G-protein alpha-subunits in these yeast GPA2 deletion strains could provide a valuable tool for the mutational analysis of mammalian G-protein function in an in vivo null setting. J Mol Cell Cardiol, 1997 Aug, 29(8), 2087 - 93 Purification of endothelin from a conditioned medium of cardiac fibroblastic cells using beating rate assay of myocytes cultured in a serum-free medium; Suzuki T et al.; A conditioned medium from cardiac fibroblastic cells stimulated the beating of quiescent cardiac myocytes cultured in a serum-free medium . The aim of this study was to isolate and characterize the myocyte beat-stimulating activity of the conditioned medium of fibroblastic cells . Cardiac myocytes and fibroblastic cells were isolated individually from neonatal rats . The fibroblastic cells were grown in a growth medium until they became confluent, then serum-free conditioned medium was obtained from them . For the beating-rate assay, the cardiac myocytes were cultured in a completely serum-free medium . The beat-stimulating factor of myocytes in the conditioned medium was purified by reverse-phase liquid chromatographies and gel filtration, and was characterized by measuring the molecular weight of the activity and a pharmacological antagonistic study . The beat-stimulating activity in the conditioned medium was purified into two active fractions . Both of the activities have a molecular weight of 2.5 kDa, and the activities were abolished similarly by FR139317, an endothelin type-A receptor antagonist . These results indicate that cardiac fibroblastic cells secrete endothelin and that this may contribute in part to the functional abnormalities of the heart in patients with myocardial fibrosis. Am J Physiol, 1997 Aug, 273(2 Pt 1), C434 - 41 Substrate-dependent expression of Na+ transport and shunt conductance in A6 epithelia; Helman SI et al.; A6 epithelia grown in tissue culture vary enormously in their baseline rates of Na+ transport due to differences in growth media, serum, and other unknown factors . To evaluate the effect(s) of substrates on expression of Na+ transport, we determined short-circuit currents, open-circuit voltages, and electrical resistances of mature confluent A6 epithelia grown on a variety of commercially available permeable supports . Because the cells, growth conditions, and all other factors were the same, differences in transport could be attributed alone to the substrate on which the cells were grown . Tissues were grown on both large- and small-diameter inserts of the same type with differing ratios of edge length to area so that the contribution of the edge and tight junction conductances to the combined shunt conductance of the inserts could be evaluated . Shunt and cellular conductances and the cellular Thevenin electromotive force were determined after aldosterone stimulation and amiloride inhibition of Na+ transport . Marked and extreme differences were observed not only for expression of Na+ transport (controls, 0.09-3.94 microA/cm2; aldosterone, 1.53-28.2 microA/cm2) due to changes of apical membrane conductance but also for the development of junctional conductances (3,250 to < infinity omega.cm2) and edge conductances (13,175 to < infinity omega.cm) among substrates. Microbiology, 1997 Aug, 143 ( Pt 8), 2657 - 64 Suppression of Escherichia coli formate hydrogenlyase activity by trimethylamine N-oxide is due to drainage of the inducer formate; Abaibou H et al.; The effect of the addition of trimethylamine N-oxide (TMAO) in the growth medium on Escherichia coli anaerobic fermentative and respiratory pathways was examined . Formate dehydrogenase H (FDH-H) activity was totally repressed by the addition of 40 mM TMAO, whereas the overall hydrogenase (HYD) activity was reduced by 25% . Accordingly, expression of lacZ operon fusions with the fdhF and hycB structural genes specifying FDH-H and HYD3 was reduced sevenfold and eightfold, respectively, leading to suppression of an active formate hydrogenlyase system . In contrast, global respiratory formate-dependent phenazine methosulphate reductase (FDH-PMS) activity, which consists of both the major anaerobic FDH-N enzyme and the aerobic FDH-Z isoenzyme, was increased approximately twofold . This was corroborated by a 2.5-fold stimulation of the sole fdoG-uidA transcriptional fusion which reflects the synthesis of the respiratory aerobic FDH-Z enzyme . In fdhD, fdhE or torA mutants lacking either FDH-PMS activity or TMAO reductase (TOR) activity, the formate hydrogenlyase pathway was no longer inhibited by TMAO . In addition, introduction of 30 mM formate in the growth medium was found to relieve the repressive effect of TMAO in the wild-type strain . When TMAO was added as terminal electron acceptor a significant enhancement of anaerobic growth was observed with the wild-type strain and the fdoG mutant . It was associated with the concomitant suppression of the formate hydrogenlyase enzymes . This was in contrast to the fdnG and torA mutants whose growth pattern and fermentative enzymes remained unaffected . Taken together, these results strongly suggest that formate-dependent reduction of TMAO via FDH-N and TOR reduces the amount of formate available for induction of the formate hydrogenlyase pathway. Protein Expr Purif, 1997 Aug, 10(3), 320 - 4 Low concentration of inducer favors production of active form of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in Escherichia coli; Yang QH et al.; Expression of chicken and rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, in Escherichia coli encountered two common problems: the chicken enzyme was liable to proteolysis and the rat enzyme was prone to form inclusion bodies . Reducing the rate of protein synthesis by lowering either growth temperature or isopropyl-beta-D-thiogalactopyranoside (IPTG) concentration alleviated these two problems . Growth at 22 degrees C was optimum for expression of both enzymes . The optimum range of IPTG concentration for expression was 0.1-1 microM for the chicken liver bifunctional enzyme and 10 microM for rat liver enzyme . The components of growth medium also influenced the production . Compared with Luria-Bertani medium, an enriched medium-tryptone-phosphate medium-tripled the production of the active enzymes . Addition of glucose (0.2%) doubled the expression level of active chicken liver enzyme, but reduced the production of active rat liver enzyme to half the maximal level, while the phosphate in tryptone-phosphate medium had no effect on the production of the two enzymes. J Cell Physiol, 1997 Aug, 172(2), 146 - 54 Decrease of Ca(2+)-ATPase activity in human keratinocytes during calcium-induced differentiation; Cho JK et al.; Ca2+ regulates keratinocyte differentiation by increasing intracellular Ca2+ levels . Ca(2+)-ATPase in the Ca(2+)-induced differentiation of human keratinocytes was investigated by measuring Ca(2+)-ATPase mRNA, protein, and activity levels . Human keratinocytes were grown in Keratinocyte Growth Medium containing 0.03, 0.1, or 1.2 mM Ca2+ and assayed on days 2, 5, 7, 14, and 21 . Ca(2+)-ATPase mRNA levels were found to be modestly increased in 5-, 7-, and 14-day cultured cells as compared with 2-day cultured cells, but levels fell below that of the 2-day cultured cells in the 21-day cultured cells . The Ca(2+)-ATPase mRNA levels were not affected by Ca2+ levels . A 135-kDa protein in human keratinocytes cross reacted with the monoclonal antibody against human erythrocyte Ca(2+)-ATPase . The level of this protein was decreased by Ca2+ and lost during differentiation, in parallel with the loss of enzymatic activity . Ca2+ influx of postconfluent 1.2 mM Ca(2+)-grown cells was higher than that of cells grown in lower Ca2+ concentrations . Ca2+ efflux from postconfluent cells grown in 0.03 mM Ca2+ was less than that from cells grown in stronger Ca2+ concentrations . These results suggest that the loss of the plasma membrane Ca(2+)-ATPase with time in culture contributes to the rise in intracellular Ca2+, thus promoting keratinocyte differentiation. J Biol Chem, 1997 Aug 1, 272(31), 19488 - 96 Genetic analysis of purine metabolism in Leishmania donovani; Hwang HY et al.; To dissect the contributions of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenine phosphoribosyltransferase (APRT), and adenosine kinase (AK) to purine salvage in Leishmania donovani, null mutants genetically deficient in HGPRT and/or APRT were generated by targeted gene replacement in wild type cells and preexisting mutant strains lacking either APRT or AK activity . These knockouts were obtained either by double targeted gene replacement or by single gene replacement followed by negative selection for loss-of-heterozygosity . Genotypes were confirmed by Southern blotting and the resultant phenotypes evaluated by enzymatic assay, resistance to cytotoxic drugs, ability to incorporate radiolabeled purine bases, and growth on various purine sources . All mutant strains could propagate in defined growth medium containing any single purine source and could metabolize exogenous {3H}hypoxanthine to the nucleotide level . The surprising ability of mutant L . donovani lacking HGPRT, APRT, and/or AK to incorporate and grow in hypoxanthine could be attributed to the ability of the parasite xanthine phosphoribosyltransferase enzyme to salvage hypoxanthine . These genetic studies indicate that HGPRT, APRT, and AK, individually or in any combination, are not essential for the survival and growth of the promastigote stage of L . donovani and intimate an important, if not crucial, role for xanthine phosphoribosyltransferase in purine salvage. Mol Cell Biol, 1997 Aug, 17(8), 4312 - 21 The replication origin decision point is a mitogen-independent, 2-aminopurine-sensitive, G1-phase event that precedes restriction point control; Wu JR et al.; At a distinct point during G1 phase (the origin decision point {ODP}), Chinese hamster ovary (CHO) cell nuclei experience a transition (origin choice) that is required for specific recognition of the dihydrofolate reductase (DHFR) origin locus by Xenopus egg extracts . We have investigated the relationship between the ODP and progression of CHO cells through G1 phase . Selection of the DHFR origin at the ODP was rapidly inhibited by treatment of early G1-phase cells with the protein kinase inhibitor 2-aminopurine (2-AP) . Inhibition of the ODP required administration of 2-AP at least 3 h prior to phosphorylation of the retinoblastoma tumor suppressor protein (Rb) and the restriction point (R point) . Cells deprived of either serum or isoleucine from metaphase throughout early G1 phase acquired the capacity to replicate in Xenopus egg extract (replication licensing) and subsequently passed through the ODP on the same schedule as cells cultured in complete growth medium . After growth arrest at the R point with hypophosphorylated Rb protein, serum- or isoleucine-deprived cells experienced a gradual loss of replication licensing . However, recognition of the DHFR origin by Xenopus egg cytosol remained stable in growth-arrested cells until the point at which all nuclei had lost the capacity to initiate replication . These results provide evidence that the ODP requires a mitogen-independent protein kinase that is activated after replication licensing and prior to R-point control. J Neurochem, 1997 Aug, 69(2), 693 - 703 Domoic acid neurotoxicity in cultured cerebellar granule neurons is mediated predominantly by NMDA receptors that are activated as a consequence of excitatory amino acid release; Berman FW et al.; The participation of NMDA and non-NMDA receptors in domoic acid-induced neurotoxicity was investigated in cultured rat cerebellar granule cells (CGCs) . Neurons were exposed to 300 microM L-glutamate or 10 microM domoate for 2 h in physiologic buffer at 22 degrees C followed by a 22-h incubation in 37 degrees C conditioned growth media . Excitotoxic injury was monitored as a function of time by measurement of lactate dehydrogenase (LDH) activity in both the exposure buffer and the conditioned media . Glutamate and domoate evoked, respectively, 50 and 65% of the total 24-h increment in LDH efflux after 2 h . Hyperosmolar conditions prevented this early response but did not significantly alter the extent of neuronal injury observed at 24 h . The competitive NMDA receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid and the non-NMDA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX) reduced glutamate-induced LDH efflux totals by 73 and 27%, respectively, whereas, together, these glutamate receptor antagonists completely prevented neuronal injury . Domoate toxicity was reduced 65-77% when CGCs were treated with competitive and noncompetitive NMDA receptor antagonists . Unlike the effect on glutamate toxicity, NBQX completely prevented domoate-mediated injury . HPLC analysis of the exposure buffer revealed that domoate stimulates the release of excitatory amino acids (EAAs) and adenosine from neurons . Domoate-stimulated EAA release occurred almost exclusively through mechanisms related to cell swelling and reversal of the glutamate transporter . Thus, whereas glutamate-induced injury is mediated primarily through NMDA receptors, the full extent of neurodegeneration is produced by the coactivation of both NMDA and non-NMDA receptors . Domoate-induced neuronal injury is also mediated primarily through NMDA receptors, which are activated secondarily as a consequence of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor-mediated stimulation of EAA efflux. J Virol, 1997 Aug, 71(8), 6191 - 3 Truncation of the C-terminal acidic transcriptional activation domain of herpes simplex virus VP16 produces a phenotype similar to that of the in1814 linker insertion mutation; Smiley JR et al.; We examined the phenotype of a herpes simplex virus (HSV) type 1 mutant (V422) in which the C-terminal acidic activation domain of the virion transactivator VP16 is truncated at residue 422 . The efficiency of plaque formation by V422 on Vero cells was boosted by approximately 100-fold by including hexamethylene bis-acetimide (HMBA) in the growth medium, as previously observed with the in1814 VP16 linker insertion mutant isolated by Preston and colleagues . V422 displayed severely reduced levels of the immediate-early transcripts encoding ICP0 and ICP4 during infection in the presence of cycloheximide, and this defect was partially overcome by the addition of HMBA . The defect in plaque formation exhibited by V422 and in 1814 was efficiently complemented in U2OS osteosarcoma cells, which had previously been shown to complement ICP0 null mutations . Taken in combination, these data confirm the key role of VP16 in triggering the onset of the HSV lytic cycle. Proc Natl Acad Sci U S A, 1997 Jul 22, 94(15), 8168 - 72 Gene expression from plasmids containing the araBAD promoter at subsaturating inducer concentrations represents mixed populations; Siegele DA et al.; Gene expression from plasmids containing the araBAD promoter can be regulated by the concentration of arabinose in the growth medium . Guzman et al . {Guzman, L.-M., Belin, D., Carson, M . J . & Beckwith, J . (1995) J . Bacteriol . 177, 4121-4130} showed that expression of a cloned gene could be modulated over several orders of magnitude in cultures grown in the presence of subsaturating concentrations of arabinose . We constructed plasmids expressing a fast-folding mutant Aequorea victoria green fluorescent protein from the araBAD promoter to examine the distribution of expressed gene products in individual cells at intermediate induction levels . Microscopic examination of cells grown at low arabinose concentrations shows mixtures of brightly fluorescent and dark cells, suggesting that intermediate expression levels in cultures reflect a population average of induced and uninduced cells . The kinetics of green fluorescent protein induction suggest that this reflects an "autocatalytic" induction mechanism due to accumulation of the inducer by active transport . This mechanism, which is analogous to the induction of the lac operon at subsaturating inducer concentrations in lacY+ cells, was described 40 years ago by Novick and Weiner {Novick, A . & Weiner, M . (1957) Proc . Natl . Acad . Sci . USA 43, 553-566}. Biochem Biophys Res Commun, 1997 Jul 18, 236(2), 347 - 50 Long-term, stable expression of green fluorescent protein in mammalian cells; Gubin AN et al.; Despite the proven utility of green fluorescent protein (GFP) as a reporter molecule for transient gene expression, the adequacy of this marker for models requiring durable, high-level gene expression has not been fully tested . To address this issue, we performed the transfection of Chinese Hamster Ovary (CHO) cells with plasmid DNA encoding both GFP and neomycin phosphotransferase (neo) cassettes . The expression of GFP was measured after the cells were cultured in the presence or absence of G418-mediated selective pressure . After removal of G418 from the growth medium, the percentage of pooled G418 resistant transfectants which co-expressed the GFP transgene increased or remained unchanged . Flow cytometric and visual isolation of GFP-expressing cells was possible without continued selection in G418 . One cloned cell line, C463, maintained high-level green fluorescence for 18 weeks in G418 and an additional 12 weeks in nonselective medium . Our data suggest expression of GFP does not confer a growth disadvantage in mammalian cells. J Biol Chem, 1997 Jul 11, 272(28), 17821 - 6 Analysis of the psbU gene encoding the 12-kDa extrinsic protein of photosystem II and studies on its role by deletion mutagenesis in Synechocystis sp . PCC 6803; Shen JR et al.; The gene encoding the 12-kDa extrinsic protein of photosystem II from Synechocystis sp . PCC 6803 was cloned based on N-terminal sequence of the mature protein . This gene, named psbU, encodes a polypeptide of 131 residues, the first 36 residues of which were absent in the mature protein and thus served as a transit peptide required for its transport into the thylakoid lumen . A psbU gene deletion mutant grew photoautotrophically in normal BG11 medium at almost the same rate as that of the wild type strain . This mutant, however, grew apparently slower than the wild type did upon depletion of Ca2+ or Cl- from the growth medium . Photosystem II oxygen evolution decreased to 81% in the mutant as compared with that in the wild type, and the thermoluminescence B- and Q-bands shifted to higher temperatures accompanied by an increase in the Q-band intensity . These results indicate that the 12-kDa protein is not essential for oxygen evolution but may play a role in optimizing the ion (Ca2+ and Cl-) environment and maintaining a functional structure of the cyanobacterial oxygen-evolving complex . In addition, a double deletion mutant lacking cytochrome c-550 and the 12-kDa protein grew photoautotrophically with a phenotype identical to that of the single deletion mutant of cytochrome c-550 . This supports our previous biochemical results that the 12-kDa protein cannot bind to photosystem II in the absence of cytochrome c-550 (Shen, J.-R., and Inoue, Y . (1993) Biochemistry 32, 1825-1832). Pediatr Dent, 1997 Jul-Aug, 19(5), 347 - 8 Milk and egg albumen are superior to human saliva in preserving human skin fibroblasts; Rozenfarb N et al.; The purpose of this in vitro study was to compare egg albumen, whole bovine milk, human saliva, and tissue culture medium (MEM) for effect on the viability of human skin fibroblasts and their osmolalities . Confluent monolayers of fibroblasts were grown . Growth medium was poured off and dishes were divided into five groups, 15 dishes each of: 1) chick egg albumen; 2) fresh whole milk; 3) human saliva; 4) tissue culture medium; and 5) bench-dried storage without any media . After 15, 45, and 90 min the average number of vital cells was measured using the trypan blue dye exclusion test . Tissue culture medium represented the best preservation media for human skin fibroblast cells (92.8% at 45 min, 87.6% at 90 min) . No significant differences were observed between milk and albumen, with a majority of the cells surviving after 90 min (67.6% and 70.2%, respectively) . Human saliva, due to its hypotonicity, markedly swelled the cells, causing decreased cell viability (27.4% at 90 min) . Bench-dried cells, as expected, showed no viable cells as early as 15 min . The osmolality of the MEM, milk and egg albumen ranged between 251-298 mOsm/kg, whereas the saliva was hypotonic, with an osmolality of 73 mOsm/kg. J Lipid Res, 1997 Jul, 38(7), 1473 - 81 Molecular cloning and functional expression of cDNA encoding the pig plasma phospholipid transfer protein; Pussinen PJ et al.; Humans and the pig show marked similarities in lipoprotein metabolism; therefore, the pig has been used as a model in numerous nutritional studies . Pig plasma displays no activity of cholesteryl ester transfer protein (CETP), which is known to be responsible for half of the phospholipid mass transfer in human plasma, the other half being accounted for by the plasma phospholipid transfer protein (PLTP) . This makes the pig an ideal subject for the study of PLTP structure and function . Here we report the molecular cloning of pig PLTP and the eukaryotic cell expression of its complementary DNA . Pig PLTP was found to share 93% amino acid sequence identity with human PLTP and 81% with mouse PLTP . Tissue expression of PLTP mRNA was examined by a method based on reverse transcription-polymerase chain reaction (RT-PCR) and solid-phase minisequencing in nine pig tissues . The highest PLTP mRNA levels were found in the pancreas, brain, lung, and liver . Medium from COS-1 cells expressing PLTP possessed phospholipid transfer activity, and the secreted recombinant PLTP was detectable by Western blotting in the culture supernatant . A mutant protein with a substitution of Cys at position 22 by Arg was found to display impaired secretion into growth medium indicating a role for cysteines in the correct folding of PLTP . This study forms the basis for future work on the structure-function relationships in pig PLTP. Shock, 1997 Jul, 8(1), 45 - 54 Establishment and characterization of a line of adipose-derived human microvascular endothelial cells (HADMEC-5) transformed by simian virus 40 large T antigen expression: application to endotoxin research; Flynn JT et al.; This paper reports the establishment and initial characterization of an immortalized line of human, adipose tissue-derived microvascular endothelial cells . Transfection of primary endothelial cell cultures was accomplished by the introduction of a plasmid, which contained simian virus 40 large T antigen DNA as well as a Rous sarcoma viral promoter region . One emergent colony, termed HADMEC-5, was isolated and has been passaged 45 times to date . The cells express simian virus 40 large T antigen protein, are immunohistochemically positive for factor VIII-related antigen, bind Ulex europaeus lectin, and accumulate Dil-labeled acetylated low density lipoprotein . The HADMEC-5 line demonstrates a highly proliferative growth rate in the absence of supplemental growth factors, when compared with primary cultures of nontransformed endothelial cells . HADMEC-5 growth remains serum dependent, but exhibits a lower serum requirement than nontransformed cells . The transformed cells grow well upon a variety of matrix compounds and in a variety of growth media . When grown upon Matrigel, the HADMEC-5 cells form three dimensional tube-like structures . The HADMEC-5 line was also tested for its ability to produce eicosanoids in response to bacterial lipopolysaccharide . The transformed cells, tested at passages 7 and 45, displayed a dose-dependent production of prostaglandin E2 in response to lipopolysaccharide in a manner similar to that seen in primary cell cultures . Threshold sensitivity to lipopolysaccharide was 10 pg/mL of media . The HADMEC-5 cell line represents a unique model in which to investigate lipopolysaccharide interactions with microvessel-derived endothelial cells and is of potential value in the study of other aspects of endothelial cell physiology. Yeast, 1997 Jul, 13(9), 837 - 48 A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae; Gari E et al.; A set of Saccharomyces cerevisiae expression vectors has been developed in which transcription is driven by a hybrid tetO-CYC1 promoter through the action of a tetR-VP16 (tTA) activator . Expression from the promoter is regulated by tetracycline or derivatives . Various modalities of promoter and activator are used in order to achieve different levels of maximal expression . In the presence of antibiotic in the growth medium at concentrations that do not affect cell growth, expression from the tetO promoter is negligible, and upon antibiotic removal induction ratios of up to 1000-fold are observed with a lacZ reporter system . With the strongest system, overexpression levels comparable with those observed with GAL1-driven promoters are reached . For each particular promoter/tTA combination, expression can be modulated by changing the tetracycline concentration in the growth medium . These vectors may be useful for the study of the function of essential genes in yeast, as well as for phenotypic analysis of genes in overexpression conditions, without restrictions imposed by growth medium composition. FEMS Microbiol Lett, 1997 Jul 1, 152(1), 75 - 81 Ornithine cycle in Nostoc PCC 73102: presence of an in vitro functional argininosuccinate lyase; Troshina O et al.; The presence of argininosuccinate lyase (ASL), an enzyme catalyzing the final step of arginine biosynthesis, was demonstrated in the heterocystous cyanobacterium Nostoc PCC 73102 by measuring in vitro enzyme activity and by visualization of ASL in native protein gels . Activity staining of a native PAGE gel revealed one ASL-dependent band with a molecular mass of about 240 kDa . A colorimetric assay for ASL based on the measurement of urea produced from arginine in the presence of an excess of arginase was further used to analyze the cyanobacterial ASL . The in vitro ASL activity was highest in cells in exponential growth phase and decreased significantly during the stationary phase of growth, ranging from 4.4 to 0.8 nmol of product formed (mg protein)-1 min-1 . Including arginine, citrulline or ornithine in the growth medium resulted in no significant change in the ASL activities, indicating that Nostoc PCC 73102 ASL is not regulated by metabolites of the ornithine cycle. Invest Ophthalmol Vis Sci, 1997 Jul, 38(8), 1598 - 609 Effect of increasing glucose concentrations and protein phosphorylation on intercellular communication in cultured rat retinal pigment epithelial cells; Stalmans P et al.; PURPOSE: The intercellular communication between cultured rat retinal pigment epithelial (RPE) cells grown in increasing glucose concentrations or after modulation of the protein kinase C-induced protein phosphorylation was investigated by studying the conduction of the {Ca2+}i wave elicited by mechanical stimulation and by analyzing the fluorescence recovery after photobleaching (FRAP) . METHODS: Subconfluent monolayers of RPE cells isolated from neonatal Long Evans rats were cultured in growth medium with various glucose levels and analyzed using the fluorescent dye fluo-3 for measurements of intracellular Ca2+ after mechanical stimulation and using 6-carboxyfluorescein diacetate to investigate the intercellular communication with FRAP . RESULTS: Mechanical stimulation in 5 or 12 mM glucose resulted in a Ca2+ wave that spread centrifugally through the neighboring cells . An inhibition of the propagation of this wave, similar to that induced by halothane, could be observed in cells grown for 72 hours in 14-mM or higher concentrations of glucose . This inhibitory effect was not caused by a hyperosmotic effect, in that results of experiments on cells cultured in growth medium supplemented with mannitol instead of glucose did not differ from those of experiments in the control medium . Activation of protein kinase C by incubation of the cells for 30 minutes with phorbol 12-myristate 13-acetate (PMA) resulted in a strong inhibition of {Ca2+}i-wave propagation . This inhibition did not depend on the oxidizing effects of PMA because the addition of glaucine, a known antioxidant, did not prevent the inhibition . Cells grown for 72 hours in glucose-rich medium (25 or 50 mM) and in which all protein kinase C activity was downregulated by a previous 72-hour exposure to 1 microM PMA, did not display the inhibitory effect on the propagation of the Ca2+ wave that is normally induced by this elevated glucose level . Stimulation or inhibition of protein kinase A activity by incubating RPE cells with Sp-cyclic adenosine monophosphate or Rp-cyclic adenosine monophosphate respectively, or inhibition of tyrosine kinase activity with herbimycin A did not alter the intercellular communication after mechanical stimulation . To determine whether the observed changes were caused by alterations in gap junction conductance (GJC), FRAP experiments were performed in control conditions, after a 30-minute incubation with PMA, and in cells cultured in 50 mM glucose in the presence and in the absence of 1 microM PMA . The measured GJC was consistent with the inhibitory effect on propagation of an intercellular Ca2+ wave in all tested conditions . CONCLUSIONS: In RPE cells, a glucose concentration of 14 mM (224 mg/dl) or higher inhibits Ca(2+)-wave propagation and intercellular GJC . This effect may be mediated by protein kinase C activity. Vet Clin North Am Food Anim Pract, 1997 Jul, 13(2), 345 - 61 Pathogenesis, diagnosis, and management of trichomoniasis in cattle; BonDurant RH; Trichomoniasis is a disease of the pregnancy, but apparently not of either the cow or the bull, except in the case of postcoital pyometra . Its self-limiting nature in the cow and chronic nature in the bull mean that a positive diagnosis for the herd can more easily be obtained from bulls than from cows . Incubation of preputial scrapings or washings (or pyometritic fluid, if available) in a selective growth medium such as the InPouch system is the diagnostic method of choice . The diagnosis is based on identification of the morphology and characteristic rolling motility of the trichomonad . "High tech" molecular approaches may eventually offer greater diagnostic sensitivity than can culture methods, but currently they are no more accurate . In addition, serologic screening of the female herd (but interestingly, not the bulls) may become possible and may allow the practitioner to at least determine whether exposure has occurred in an unvaccinated herd . Control in an infected herd involves no pharmacologic treatment but rather culling of infected bulls, retention of younger, culture-negative bulls, and segregation of the female herd by reproductive status. Arch Microbiol, 1997 Jul, 168(1), 68 - 71 Adaptive response of Haloferax mediterranei to low concentrations of NaCl (< 20%) in the growth medium; D'Souza SE et al.; Halobacteria require 20-25% NaCl for optimal growth and lyse when the salt concentration falls below 10% . The response of Haloferax mediterranei cells to low concentrations of NaCl (< 20%) in the medium was studied . The cells adapted to and grew in concentrations of NaCl as low as 10% and survived in concentrations lower than 5% . The cells synthesised a red pigment, bacterioruberin, in response to stress caused by a low concentration of NaCl (< 20%). J Bacteriol, 1997 Jul, 179(13), 4129 - 37 6-phospho-alpha-D-glucosidase from Fusobacterium mortiferum: cloning, expression, and assignment to family 4 of the glycosylhydrolases; Bouma CL et al.; The Fusobacterium mortiferum malH gene, encoding 6-phospho-alpha-glucosidase (maltose 6-phosphate hydrolase; EC 3.2.1.122), has been isolated, characterized, and expressed in Escherichia coli . The relative molecular weight of the polypeptide encoded by malH (441 residues; Mr of 49,718) was in agreement with the estimated value (approximately 49,000) obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the enzyme purified from F . mortiferum . The N-terminal sequence of the MalH protein obtained by Edman degradation corresponded to the first 32 amino acids deduced from the malH sequence . The enzyme produced by the strain carrying the cloned malH gene cleaved {U-14C}maltose 6-phosphate to glucose 6-phosphate (Glc6P) and glucose . The substrate analogs p-nitrophenyl-alpha-D-glucopyranoside 6-phosphate (pNP alphaGlc6P) and 4-methylumbelliferyl-alpha-D-glucopyranoside 6-phosphate (4MU alphaGlc6P) were hydrolyzed to yield Glc6P and the yellow p-nitrophenolate and fluorescent 4-methylumbelliferyl aglycons, respectively . The 6-phospho-alpha-glucosidase expressed in E . coli (like the enzyme purified from F . mortiferum) required Fe2+, Mn2+, Co2+, or Ni2+ for activity and was inhibited in air . Synthesis of maltose 6-phosphate hydrolase from the cloned malH gene in E . coli was modulated by addition of various sugars to the growth medium . Computer-based analyses of MalH and its homologs revealed that the phospho-alpha-glucosidase from F . mortiferum belongs to the seven-member family 4 of the glycosylhydrolase superfamily . The cloned 2.2-kb Sau3AI DNA fragment from F . mortiferum contained a second partial open reading frame of 83 residues (designated malB) that was located immediately upstream of malH . The high degree of sequence identity of MalB with IIB(Glc)-like proteins of the phosphoenol pyruvate dependent:sugar phosphotransferase system suggests participation of MalB in translocation of maltose and related alpha-glucosides in F . mortiferum. Infect Immun, 1997 Jul, 65(7), 2829 - 36 Macrophages and enriched populations of T lymphocytes interact synergistically for the induction of severe, destructive Lyme arthritis; DuChateau BK et al.; Hamsters receiving both macrophages exposed to Formalin-inactivated Borrelia burgdorferi (Mphi-FBb) and enriched populations of either immune or naive T lymphocytes developed severe swelling of the hind paws when infected with B . burgdorferi . Swelling was detected 6 days after infection, peaked on day 10, and gradually decreased . Swelling was also observed in the hind paws of hamsters infused with only Mphi-FBb or only enriched populations of either immune or naive T lymphocytes after infection with B . burgdorferi . However, the swelling detected in these hamsters was less severe and of shorter duration . In addition, hamsters receiving both macrophages not exposed to Formalin-inactivated B . burgdorferi (Mphi-NFBb) and enriched populations of either immune or naive T lymphocytes failed to develop severe swelling after infection with B . burgdorferi . No swelling was also observed in hamsters infused with both Mphi-FBb and enriched populations of immune T lymphocytes and then inoculated with spirochetal growth medium . We further showed that macrophages and enriched populations of T lymphocytes did not interact synergistically for controlling B . burgdorferi infection, as spirochetes were readily recovered from the tissues of all cell transfer recipients infected with B . burgdorferi . These findings demonstrate that hamsters infused with both Mphi-FBb and enriched populations of either immune or naive T lymphocytes develop a more fulminate arthritis after infection with B . burgdorferi than recipients infused with either cell type alone . These findings suggest that macrophages and T lymphocytes interact synergistically for the induction of severe, destructive Lyme arthritis. Mol Cell Biol, 1997 Jul, 17(7), 3966 - 76 Genetic analysis of regulatory mutants affecting synthesis of extracellular proteinases in the yeast Yarrowia lipolytica: identification of a RIM101/pacC homolog; Lambert M et al.; Depending on the pH of the growth medium, the yeast Yarrowia lipolytica secretes both an acidic proteinase and an alkaline proteinase, the synthesis of which is also controlled by carbon, nitrogen, and sulfur availability, as well as by the presence of extracellular proteins . Recessive mutations at four unlinked loci, named PAL1 to PAL4, were isolated which prevent alkaline proteinase derepression under conditions of carbon and nitrogen limitation at pH 6.8 . These mutations markedly affect mating and sporulation . A dominant suppressor of all four PAL mutations was isolated from a wild-type genomic library, which turned out to be a C-terminally truncated form of a 585-residue transcriptional factor of the His2Cys2 zinc finger family, which we propose to call YlRim101p . Another C-terminally truncated version of YlRim101p (419 residues) is encoded by the dominant RPH2 mutation previously isolated as expressing alkaline protease independently of the pH . YlRim101p is homologous to the transcriptional activators Rim101p of Saccharomyces cerevisiae, required for entry into meiosis, and PacC of Aspergillus nidulans and Penicillium chrysogenum, which were recently shown to mediate regulation by ambient pH . YlRim101p appears essential for mating and sporulation and for alkaline proteinase derepression . YlRIM101 expression is autoregulated, maximal at alkaline pH, and strongly impaired by PAL mutations. Yeast, 1997 Jun 30, 13(8), 727 - 34 Identification and preliminary characterization of p31, a new PSTAIRE-related protein in fission yeast; Tournier S et al.; One of the defining characteristics of the catalytic subunit of the cyclin-dependent protein kinases (cdks) is the so-called PSTAIRE motif . Western blots of fission yeast cytosolic extracts using a monoclonal antibody against the PSTAIRE peptide revealed two bands at 34 kDa (p34cdc2) and 31 kDa (p31) . Polyclonal antibodies to the C-terminus of p34cdc2 or to the full-length protein recognized the 34 kDa band but not p31 . Overexpression of the cdc2+ gene resulted in the increase of the 34 kDa band but not p31 . Like p34cdc2, the level of p31 revealed no obvious cell cycle regulation but the protein was present in spores where p34cdc2 was barely detectable . p31 expression was unaffected by removal of either phosphate or ammonium from the growth medium, although the level of p34cdc2 was reduced in the absence of phosphate . p31 was not associated with cyclin B, nor was it adsorbed to p13suc1 Sepharose beads, two characteristics of p34cdc2 . p31 did, however, interact with p15, the starfish homologue of p13suc1 . p31 was present in cells in which cdc2+ was replaced by its budding yeast homologue CDC28 . When fission yeast cytosolic extracts were subjected to gel filtration chromatography, p31 eluted in two peaks, one at approximately 100 kDa, the other at approximately 30 kDa . We conclude that p31 is a novel fission yeast PSTAIRE protein and therefore, potentially, a new cdk. Biochim Biophys Acta, 1997 Jun 23, 1346(3), 253 - 60 Biosynthesis of furan fatty acids (F-acids) by a marine bacterium, Shewanella putrefaciens; Shirasaka N et al.; A mutant derived from Shewanella putrefaciens 8CS7-4 treated with N-methyl-N'-nitro-N-nitrosoguanidine was found to produce 15-20 mg of a furan fatty acid (F-acid), 10,13-epoxy-11-methyloctadeca-10,12-dienoic acid (F18), per liter of growth medium (10-15% of total fatty acids) . Capillary gas chromatography-mass spectrometry and proton nuclear magnetic resonance analysis of the fatty acid methyl esters of the mutant revealed the presence of other F-acids, 8,11-epoxy-9-methylhexadeca-8,10-dienoic acid (F16), 6,9-epoxy-7-methyltetradeca-6,8-dienoic acid (F14), and methyl branched unsaturated fatty acids, 11-methyl-12E-octadecenoic acid (11-me-18:1) and 11-methyl-10E,12E-octadecadienoic acid (1-me-18:2) . About 90% of F-acids were present in phospholipids, in which the F-acids were found to be exclusively linked at the sn-1 position . 11-me-18:1 and 11-me-18:2 were also detected in the sn-1 position . Firstly, 11-me-18:1 increased and reached a maximum at 12 h, and then decreased rapidly . Secondly, the 11-me-18:2 content reached a maximum at 24 h, when 11-me-18:1 was little detected, and then decreased . Finally, the amount of F18 began to increase after 20 h and reached a plateau at 36 h . These results suggest that 11-me-18:1 and 11-me-18:2 are precursors of F18. J Biol Chem, 1997 Jun 20, 272(25), 15841 - 8 Differential extraction and protein sequencing reveals major differences in patterns of primary cell wall proteins from plants; Robertson D et al.; The proteins of the primary cell walls of suspension cultured cells of five plant species, Arabidopsis, carrot, French bean, tomato, and tobacco, have been compared . The approach that has been adopted is differential extraction followed by SDS-polyacrylamide gel electrophoresis (PAGE), rather than two-dimensional gel analysis, to facilitate protein sequencing . Whole cells were washed sequentially with the following aqueous solutions, CaCl2, CDTA (cyclohexane diaminotetraacetic acid, DTT (dithiothreitol), NaCl, and borate . SDS-PAGE analysis showed consistent differences between species . From the 233 proteins that were selected for sequencing, 63% gave N-terminal data . This analysis shows that (i) patterns of proteins revealed by SDS-PAGE are strikingly different for all five species, (ii) a large number of these proteins cannot be identified by data base searches indicating that a significant proportion of wall proteins have not been previously described, (iii) the major proteins that can be identified belong to very different classes of proteins, (iv) the majority of proteins found in the extracellular growth media are absent from their respective cell wall extracts, and (v) the results of the extraction process are indicative of higher order structure . It appears that aspects of speciation reside in the complement of extracellular wall proteins . The data represent a protein resource for cell wall studies complementary to EST (expressed sequence tag) and DNA sequencing strategies. Arch Biochem Biophys, 1997 Jun 15, 342(2), 373 - 82 Viral infection . II . Hemin induces overexpression of p67 as it partially prevents appearance of an active p67-deglycosylase in baculovirus-infected insect cells; Saha D et al.; The roles of p67-deglycosylase (p67-DG) in the regulation of protein synthesis in baculovirus-infected insect cells were studied . Like vaccinia viral infection, baculovirus infection of insect cells also induced the appearance of a p67-DG . However, p67-DG activity could not be detected because these cells do not contain a detectable level of p67 . The baculovirus expression vector system (BEVS), however, promotes significant expression of cloned p67-cDNA . The expression of p67 was significantly enhanced by the addition of hemin to the growth medium . Maximum enhancement was observed at 5 microM hemin . Data suggest that hemin prevents the activation of latent p67-DG inside the cell and does not have any effect on p67 gene transcription . To gain a better understanding of the mechanism of p67-DG activation and hemin stimulation of p67 synthesis, we have now purified p67-DG from baculovirus-infected insect cells . We prepared antibodies against this protein . These antibodies reacted with a 105-kDa protein in cell extracts from the uninfected insect cells (Sf9), KRC-7, and L929 (animal cells) . In addition, these antibodies reacted with an additional 60-kDa protein in the cell extracts of baculovirus-infected Sf9 cells and vaccinia virus-infected KRC-7 and L929 cells . Data are also presented to show that the antibodies against p67-DG reacted more efficiently (40%) with the 60-kDa protein in both hemin-deficient reticulocyte lysate and hemin-deficient baculovirus-infected cells . We suggest that hemin prevents the conversion of an inactive p67-DG into an active form possibly by covalent modification such as protein phosphorylation or protein glycosylation . The active form is more efficiently recognized by the p67-DG antibodies since these antibodies were prepared against the active form of p67-DG. J Cell Biochem, 1997 Jun 15, 65(4), 574 - 90 Heparan sulfate-binding peptide promotes the deposition of proteoglycans in the extracellular matrix; Colburn P et al.; A synthetic peptide, which was shown to bind extracellular matrix heparan sulfate chains with a high degree of affinity and specificity {Colburn et al . (1996): Arch Biochem Biophys 325:129-138}, has now been found to promote the transfer and the deposition of endothelial cell surface proteoglycans in the extracellular matrix . The peptide also induces preferential binding of extracellular matrix heparan sulfate proteoglycans, which have been added to the supernatant growth medium, and the requirement for its presence is stringent in that only a negligible amount of proteoglycans are bound to the cell layer in the absence of the peptide . In addition, antibodies directed against the peptide detect the accumulation of the peptide in the matrix compartment where the peptide is found associated with the proteoglycans transferred from the cell surface . The sequence of events induced by the peptide appears to be an extension of a naturally occurring process since proteoglycans with properties similar to those of the species ordinarily present in the extracellular matrix have been observed to transfer from the cell surface to the matrix during a pulse-chase experiment . We suggest that formation of the complex peptide-proteoglycan with consequent displacement of the proteoglycan from its anchorage on the cell initiates the process of transfer of the heparan sulfate-bound peptide from the cell surface to the extracellular matrix. Nucleic Acids Res, 1997 Jun 15, 25(12), 2389 - 95 Expression of herpes virus thymidine kinase in Neurospora crassa; Sachs MS et al.; The expression of thymidine kinase in fungi, which normally lack this enzyme, will greatly aid the study of DNA metabolism and provide useful drug-sensitive phenotypes . The herpes simplex virus type-1 thymidine kinase gene ( tk ) was expressed in Neurospora crassa . tk was expressed as a fusion to N.crassa arg-2 regulatory sequences and as a hygromycin phosphotransferase-thymidine kinase fusion gene under the control of cytomegalovirus and SV40 sequences . Only strains containing tk showed thymidine kinase enzyme activity . In strains containing the arg-2 - tk gene, both the level of enzyme activity and the level of mRNA were reduced by growth in arginine medium, consistent with control through arg-2 regulatory sequences . Expression of thymidine kinase in N.crassa facilitated radioactive labeling of replicating DNA following addition of {3H}thymidine or {14C}thymidine to the growth medium . Thymidine labeling of DNA enabled demonstration that hydroxyurea can be used to block replication and synchronize the N.crassa mitotic cycle . Strains expressing thymidine kinase were also more sensitive than strains lacking thymidine kinase to anticancer and antiviral nucleoside drugs that are activated by thymidine kinase, including 5-fluoro-2'-deoxyuridine, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouridine and trifluorothymidine . Finally, expression of thymidine kinase in N . crassa enabled incorporation of bromodeoxyuridine into DNA at levels sufficient to separate newly replicated DNA from old DNA using equilibrium centrifugation. Biochim Biophys Acta, 1997 Jun 2, 1346(2), 185 - 92 Bacterial expression and characterization of human pancreatic phospholipase A2; Han SK et al.; Mammalian pancreatic phospholipases A2 (PLA2) have recently been implicated in cell surface receptor-mediated inflammation . As a first step toward understanding how human pancreatic PLA2 (hp-PLA2) interacts with membranes and other biological targets including cell-surface receptors, we constructed its bacterial expression vector which can be used for the mutagenesis and protein over-expression . The expression vector (pSH-hp) was constructed using a synthetic hp-PLA2 gene whose transcription is controlled by T7 promoter . hp-PLA2 was expressed as a mature protein in high concentration in Escherichia coli cells and formed inclusion body . The solubilization of inclusion body protein followed by the refolding and purification produced ca . 5 mg of pure protein from one liter of growth medium . Kinetic studies of recombinant human, bovine and porcine pancreatic PLA2s using polymerized mixed liposomes and micelles as substrates showed that despite their highly homologous structures these mammalian pancreatic PLA2s have distinct phospholipid head group specificity and different activity toward various lipid substrates. J Wound Care, 1997 Jun, 6(6), 272 - 4 The cytocompatibility of hydrocolloid dressings; Agren M; The purpose of this study was to evaluate the effect on fibroblast proliferation of hydrophilic particles isolated from six commercial hydrocolloid dressings . The hydrophobic adhesive matrix of six hydrocolloid dressings was removed using a reflux extraction method with an organic solvent (xylene) . The remaining hydrophilic particles were dissolved in complete cell growth medium containing 10% (v/v) foetal calf serum and added to confluent human dermal fibroblasts grown in monolayer in final concentrations of 0.1 and 0.01% (w/v) . Control cells received growth medium alone . The fibroblasts were incubated with the hydrophilic particles and the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) for 24 hours . The incorporation of BrdU into DNA was used as a measure of cell proliferation and determined using an ELISA kit . The results were expressed in percentage of control-treated wells and analysed using analysis of variance . Apart from Comfeel Plus, the hydrophilic particles of hydrocolloid dressings significantly inhibited fibroblast proliferation at 0.1% compared to control-treated fibroblasts (p < 0.05). J Biomol NMR, 1997 Jun, 9(4), 437 - 40 Improved labeling strategy for 13C relaxation measurements of methyl groups in proteins; Lee AL et al.; Selective incorporation of 13C into the methyl groups of protein side chains is described as a means for simplifying the measurement and interpretation of 13C relaxation parameters . High incorporation (> 90%) is accomplished by using pyruvate (3-13C, 99%) as the sole carbon source in the growth media for protein overexpression in E . coli . This improved labeling scheme increases the sensitivity of the relaxation experiments by approximately fivefold when compared to randomly fractionally 13C-labeled protein, allowing high-quality measurements on relatively dilute (< 1 mM) protein samples at a relatively low cost. Can J Microbiol, 1997 Jun, 43(6), 517 - 25 Use of Tn5-gusA5 to investigate environmental and nutritional effects on gene expression in the coronatine biosynthetic gene cluster of Pseudomonas syringae pv . glycinea; Palmer DA et al.; Pseudomonas syringae pv . glycinea PG4180 produces coronatine (COR), a chlorosis-inducing phytotoxin that consists of the polyketide coronafacic acid (CFA) coupled via an amide bond to the ethylcyclopropyl amino acid coronamic acid (CMA) . Both CFA and CMA function as intermediates in the pathway to coronatine, and genes encoding their synthesis have been localized: however, the precise factors that regulate the production of COR and its precursors remain unclear . In the present study, a lambda delivery system for Tn5-gusA5 was developed and used to obtain transcriptional fusions in the COR gene cluster . Selected carbon (fructose and xylose) and amino acid (isoleucine and valine) sources significantly decreased COR biosynthesis at the transcriptional level . Transcriptional activity in the COR gene cluster was temperature dependent with maximal expression at 18-24 degrees C and significantly less expression at 14 and 30 degrees C . Interestingly, changes in osmolarity and the addition of complex carbon and nitrogen sources to the growth medium did not significantly affect COR gene expression, although both factors significantly impacted the quantity of COR produced . These results indicate that multiple factors impact COR production and only some of these directly affect transcription in the COR gene cluster. Clin Otolaryngol, 1997 Jun, 22(3), 275 - 83 Otomycosis: the detection of fungi in ears by immunofluorescence microscopy; Gurr PA et al.; The procedure currently used to diagnose infection in otitis externa has several limitations: it is slow to culture organisms on growth media, fungal infections are often missed, and extensive laboratory facilities and mycological expertise are required . A rapid, accurate and sensitive assay would greatly improve patient care by initiating appropriate antifungal treatment at the onset of disease . We report the development of a rapid detection assay for otomycosis using fungal-specific monoclonal antibodies to detect fungi in ear swabs by immunofluorescence microscopy . This assay could form the basis of a detection assay for fungal infections of the head and neck. Carcinogenesis, 1997 Jun, 18(6), 1265 - 70 Butyrate can act as a stimulator of growth or inducer of apoptosis in human colonic epithelial cell lines depending on the presence of alternative energy sources; Singh B et al.; In vivo, butyrate is a major energy source for the colonic epithelium and is thought to stimulate proliferation . In contrast, butyrate in vitro has been shown to inhibit proliferation and induce differentiation and apoptosis in colonic epithelial cells . Most colon cell cultures are grown in medium containing high concentrations of glucose, whereas in vivo, the main energy source used by the colon cells is butyrate . The aim of this study was to determine whether the apparent contrasting roles of butyrate in vivo and in vitro could be as a consequence of differences in glucose availability . The sensitivity of two human colorectal tumour cell lines, one adenoma (S/RG/C2) and one carcinoma (HT29) to butyrate-induced growth inhibition and apoptosis was investigated to determine whether these cellular effects were altered under glucose depleted culture conditions . Glucose depletion resulted in increased apoptosis in both cell lines in the absence of butyrate . Butyrate in standard culture conditions (containing 25 mM glucose and 1 mM pyruvate) inhibited growth and induced apoptosis in both cell lines . However, low concentrations of butyrate in glucose depleted culture conditions (i.e . standard growth medium without glucose and pyruvate supplements) were found to reduce apoptosis induced by glucose deprivation and increase cell yield in both cell lines . The results show that in glucose depleted culture conditions, butyrate at low concentrations (0.5 mM for S/RG/C2, and 0.5 and 2 mM for HT29 cells) was found to be growth stimulatory whereas in the presence of glucose, these same concentrations of butyrate induced apoptosis . Thus, whether butyrate is growth stimulatory or growth inhibitory may depend on the availability of other energy sources . These observations may, in part, provide an explanation for the apparent opposite effects of butyrate on proliferation reported in vivo and in vitro. Radiat Res, 1997 Jun, 147(6), 674 - 9 Caffeine-induced apoptosis reveals a persistent lesion after treatment with bromodeoxyuridine and ultraviolet-B light; Hagan MP et al.; Ultraviolet-B (UVB) light treatment of synchronized V79 Chinese hamster cells after pulse-labeling with bromodeoxyuridine (BrdUrd) reveals a marked age response for cell killing . Incubation after treatment in growth medium containing caffeine increases cell killing during the resistant portions of the cell cycle, resulting in a much less marked age response to UVB irradiation . Examination of the split-dose survival curves for BrdUrd and UVB light in the presence or absence of caffeine indicates that sensitization by caffeine is completely independent of the sparing effect of dose fractionation . Further, sensitization by caffeine is nearly complete after the first mitosis after the UVB exposure . With delayed addition of caffeine, however, nearly full sensitization can be elicited as late as two cell cycles after the treatment with BrdUrd and UVB light . Also, synchronized cells exposed to caffeine after treatment with BrdUrd and UVB in the S phase cease to incorporate radiolabeled thymidine and undergo apoptosis after the second mitosis . Thus exposure to caffeine reveals a persistent sensitivity lasting for at least two cell cycles, after the injury induced by treatment with BrdUrd and UVB. Nat Biotechnol, 1997 Jun, 15(6), 574 - 80 Bipotent progenitor cell lines from the human CNS; Sah DW et al.; Human central nervous system (CNS) cell lines would substantially facilitate drug discovery and basic research by providing a readily renewable source of human neurons . We isolated clonal human CNS cell lines that had been immortalized with a tetracycline (Tc)-responsive v-myc oncogene; addition of Tc to the growth medium suppressed the oncoprotein rapidly and virtually completely, allowing differentiation to proceed . Two classes of bipotent precursor cells were immortalized: the first class had a default differentiation pathway of neurons only, and the second class had a default differentiation pathway of neurons and astrocytes . We found that after exposure to different external signals in vitro, the environment is capable of redirecting the fate of a particular cell, even in the case of the bipotent precursor cell whose default differentiation pathway was neurons only . These data suggest that extrinsic cues can prevail over intrinsic determinants in directing cell fate in the human CNS. Appl Environ Microbiol, 1997 Jun, 63(6), 2206 - 12 Variation of microcystins, cyanobacterial hepatotoxins, in Anabaena spp . as a function of growth stimuli; Rapala J et al.; Cyanobacterial hepatotoxins, microcystins, are specific inhibitors of serine/threonine protein phosphatases and potent tumor promoters . They have caused several poisonings of animals and also pose a health hazard for humans through the use of water for drinking and recreation . Different strains of the same cyanobacterial species may variously be nontoxic, be neurotoxic, or produce several microcystin variants . It is poorly understood how the amount of toxins varies in a single strain . This laboratory study shows the importance of external growth stimuli in regulating the levels and relative proportions of different microcystin variants in two strains of filamentous, nitrogen-fixing Anabaena spp . The concentration of the toxins in the cells increased with phosphorus . High temperatures (25 to 30 degrees C), together with the highest levels of light studied (test range, 2 to 100 mumol m-2 s-1), decreased their amount . Different structural variants of microcystins responded differently to growth stimuli . Variants of microcystin (MCYST)-LR correlated with temperatures below 25 degrees C, and those of MCYST-RR correlated with higher temperatures . Nitrogen added into the growth medium and increasing temperatures increased the proportion of microcystin variants demethylated in amino acid 3 . All variants remained mostly intracellular . Time was the most important factor causing the release of the toxins into the growth medium . Time, nitrogen added into the growth medium, and light fluxes above 25 mumol m-2 s-1 significantly increased the concentrations of the dissolved toxins . According to the results, it thus seems that the reduction of phosphorus loads in bodies of water might play a role in preventing the health hazards that toxic cyanobacterial water blooms pose, not only by decreasing the cyanobacteria but also by decreasing their toxin content. J Bacteriol, 1997 Jun, 179(11), 3761 - 6 Role of GATA factor Nil2p in nitrogen regulation of gene expression in Saccharomyces cerevisiae; Rowen DW et al.; We have identified the product of the NIL2 gene of Saccharomyces cerevisiae which contains a zinc finger region highly homologous to those of the GATA factors Gln3p and Nil1p as an antagonist of Nil1p and to a lesser extent of Gln3p . The expression of many nitrogen-regulated genes of Saccharomyces cerevisiae requires activation by GATA factor Gln3p or Nil1p and is prevented by the presence of glutamine in the growth medium . Disruption of NIL2 results in a great increase in the expression of NIL1 and of GAP1, the structural gene for the general amino acid permease, in glutamine-grown cells in response to activation by Nil1p . The primary effect of the elimination of Nil2p appears to be an increase in the intracellular level of Nil1p, which in turn is responsible for increased expression of GAP1 . Experiments using an artificial UAS (upstream activating site) consisting of three GATAAGATAAG sites revealed that Nil2p exerts its effect by competing primarily with Nil1p and less effectively with Gln3p for these sites . Apparently, the principal role of Nil2p is to prevent activation of transcription by Nil1p unless Nil1p has been converted to a more active state by the absence of glutamine and glutamate. J Inorg Biochem, 1997 Jun, 66(4), 231 - 40 Incorporation of copper into the yeast Saccharomyces cerevisiae . Identification of Cu(I)--metallothionein in intact yeast cells; Presta A et al.; Copper is an essential metal ion to many living organisms, including mammals, as it mediates a wide variety of important biochemical processes . At elevated concentrations, copper is extremely toxic to host cells . This paradoxical nature of copper has necessitated a highly regulated procedure for its cellular accumulation, transport, and excretion . One important group of proteins involved in eukaryotic copper speciation is the protein metallothionein . Luminescence microscopy data, emission, and circular dichroism spectral data are reported as copper is incorporated into metallothionein by the yeast Saccharomyces cerevisiae . These techniques provide information on the mechanism of copper uptake by S . cerevisiae . A two-stage kinetic mechanism for the uptake of copper from the growth medium by the yeast cells is observed . The first stage displays an uptake rate that is dependent on the initial copper concentration of the growth medium, and lasts for approximately 6 h . The second stage has a slower rate of copper uptake than the first, but the kinetics are independent of the initial copper concentration . Emission spectra recorded directly from the intact yeast cells (at 77 K) show that the cellular incorporation of copper proceeds via several species, eventually leading to storage of the copper in the form of Cu-metallothionein . The photomicrographs of yeast cells grown in a copper-containing medium clearly show an orange luminescence, indicating the formation of a Cu(I)-thiolate species . The identification of this species as copper-metallothionein was confirmed by measurement of the circular dichroism and emission properties following excretion and isolation of the copper-containing protein from the yeast cells . Analysis of the emission spectrum from S . cerevisiae Cu-metallothionein at 77 K reveals two emission bands, centered at 570 and 700 nm . The high-energy emission band exhibits a two-component decay, with excited state lifetimes of 4.70 and 48.5 microseconds . The low-energy emission exhibits one major decay component with a lifetime of 1.13 microseconds . A high-molecular-weight, copper-containing species is also isolated from the yeast cells and is characterized spectroscopically. J Biol Chem, 1997 May 9, 272(19), 12430 - 6 Identification of framework residues in a secreted recombinant antibody fragment that control production level and localization in Escherichia coli; Forsberg G et al.; The monoclonal antibody 5T4, directed against a human tumor-associated antigen, was expressed as a secreted Fab superantigen fusion protein in Escherichia coli . The product is a putative agent for immunotherapy of non-small cell lung cancer . During fermentation, most of the fusion protein leaked out from the periplasm to the growth medium at a level of approximately 40 mg/liter . This level was notably low compared with similar products containing identical CH1, CL, and superantigen moieties, and the Fv framework was therefore engineered . Using hybrid molecules, the light chain was found to limit high expression levels . Substituting five residues in VL increased the level almost 15 times, exceeding 500 mg/liter in the growth medium . Here, the substitutions Phe-10 --> Ser, Thr-45 --> Lys, Thr-77 --> Ser, and Leu-78 --> Val were most powerful . In addition, replacing four VH residues diminished cell lysis during fermentation . Thereby the product was preferentially located in the periplasm instead of the growth medium, and the total yield was more than 700 mg/liter . All engineered products retained a high affinity for the tumor-associated antigen . It is suggested that at least some of the identified framework residues generally have to be replaced to obtain high level production of recombinant Fab products in E . coli. Biochim Biophys Acta, 1997 May 2, 1352(1), 73 - 84 The regulation of the vanadium chloroperoxidase from Curvularia inaequalis; Barnett P et al.; The effects of carbon and nitrogen source on the regulation of the vanadium chloroperoxidase secreted by the fungus Curnularia inaequalis were investigated . The addition of glucose showed a repressing effect on both the observed messenger RNA level and the measured enzyme activities, whereas the addition of glutamate as nitrogen source and the addition of both glutamate and glycerol had no effect . Addition of vanadate had no effect on the level of mRNA . Eight hundred base pairs of the upstream promoter region of vCPO were sequenced and various features of interest are highlighted . Closer inspection of the mycelium revealed that once secreted, vCPO probably remains tightly associated with the hyphae in two forms, one of which may be a proform of the enzyme . A possible cleavage event at the C-terminus may lower its potential for hyphal association and permit its disassociation into the growth medium . A putative role for the vanadium chloroperoxidase is put forward. J Biol Chem, 1997 May 2, 272(18), 11763 - 9 Negative control of heavy metal uptake by the Saccharomyces cerevisiae BSD2 gene; Liu XF et al.; We have previously shown that mutations in the Saccharomyces cerevisiae BSD2 gene suppress oxidative damage in cells lacking superoxide dismutase and also lead to hyperaccumulation of copper ions . We demonstrate here that bsd2 mutant cells additionally accumulate high levels of cadmium and cobalt . By biochemical fractionation and immunofluorescence microscopy, BSD2 exhibited localization to the endoplasmic reticulum, suggesting that BSD2 acts at a distance to inhibit metal uptake from the growth medium . This BSD2 control of ion transport occurs independently of the CTR1 and FET4 metal transport systems . Genetic suppressor analysis revealed that hyperaccumulation of copper and cadmium in bsd2 mutants is mediated through SMF1, previously shown to encode a plasma membrane transporter for manganese . A nonsense mutation removing the carboxyl-terminal hydrophobic domain of SMF1 was found to mimic a smf1 gene deletion by eliminating the copper and cadmium toxicity of bsd2 mutants and also by precluding the bsd2 suppression of superoxide dismutase deficiency . However, inactivation of SMF1 did not eliminate the elevated cobalt levels in bsd2 mutants . Instead, this cobalt accumulation was found to be specifically mediated through the SMF1 homologue, SMF2 . Hence, BSD2 prevents metal hyperaccumulation by exerting negative control over the SMF1 and SMF2 metal transport systems. Can J Microbiol, 1997 May, 43(5), 447 - 55 Denitration of 2,4,6-trinitrotoluene by Pseudomonas savastanoi; Martin JL et al.; Past disposal of wastewaters containing 2,4,6-trinitrotoluene (TNT) at the former Nebraska Ordnance Plant has resulted in numerous acres of TNT-contaminated soil . Examining the microbial population of these soils revealed several TNT-tolerant Pseudomonas spp . We selected one species, P . savastanoi, to determine its ability to transform TNT . Pure culture experiments were performed in pseudomonas minimal medium containing 0.31 mM TNT (70 mg TNT . L(-1)) under varied nutrient and cell density regimes . Experiments with TNT as a sole C or N source showed that P . savastanoi has the ability to denitrate TNT, as evidenced by production of 2,4-dinitrotoluene (2,4-DNT) and NO2- with time . TNT denitration and formation of 2,4-DNT were enhanced by removing NH4+ and adding NO2- to the growth medium . In all experiments, 2-amino-4,6-dinitrotoluene (2-ADNT) and 4-amino-2,6-dinitrotoluene (4-ADNT) appeared as incidental reduction products . Glucose addition to the medium enhanced 2-ADNT and 4-ADNT production and decreased denitration of TNT . Mid-log phase cells rapidly transformed {ring-14C(U)}TNT but were unable to mineralize significant quantities of TNT, as evidenced by conversion of less than 1% of the label to 14CO2 . These results indicate that P . savastanoi is a TNT-tolerant pseudomonad that can promote TNT degradation through reductive denitration and nitro moiety reduction. In Vitro Cell Dev Biol Anim, 1997 May, 33(5), 344 - 51 GTSF-2: a new, versatile cell culture medium for diverse normal and transformed mammalian cells; Lelkes PI et al.; The aim of this study was to test the versatility of a new basal cell culture medium, GTSF-2 . In addition to traditional growth-factors, GTSF-2 contains a blend of three sugars (glucose, galactose, and fructose) at their physiological levels . For these studies, we isolated normal endothelial cells from human, bovine, and rat large blood vessels and microvessels . In addition, GTSF-2 was also tested as a replacement for high-glucose-containing medium for PC12 pheochromocytoma cells and for other, transformed cell lines . The cell growth characteristics were assessed with a novel cell viability and proliferation assay, which is based on the bioreduction of the fluorescent dye, Alamar Blue . After appropriate calibration, the Alamar Blue assay was found to be equivalent to established cell proliferation assays . Alamar Blue offers the advantage that cell proliferation can be measured in the same wells over an extended period of time . For some of the cell types (e.g., endothelial cells isolated from the bovine aorta, the rat adrenal medulla, or the transformed cells), proliferation in unmodified GTSF-2 was equivalent to that in the original culture media . For others cell types (e.g., human umbilical vein endothelial cells and PC12 cells), GTSF-2 proved to be a superior growth medium, when supplemented with simple additives, such as endothelial cell growth supplement (bFGF) or horse serum . Our results suggest that GTSF-2 is a versatile basal medium that will be useful for studying organ-specific differentiation in heterotypic coculture studies. Antonie Van Leeuwenhoek, 1997 May, 71(4), 353 - 61 Regulation of ammonia assimilation in an obligate methylotroph Methylobacillus flagellatum under steady-state and transient growth conditions; Baev MV et al.; The obligate methylotroph Methylobacillus flagellatum was grown in the presence of different ammonium concentrations and the regulation of the enzymes associated with ammonium assimilation was investigated in steady-state and transient growth regimes . As the medium changed from C-limitation to dual C/N- and finally to N-limitation, the culture passed through three definite growth phases . The NADP(+)-dependent glutamate dehydrogenase (GDH) was present under ammonium limitation of the culture growth (at 2 mmol 1(-1) of ammonium in the growth medium) and increased in response to an increase in nitrogen availability . Glutamine synthetase (GS) and glutamate synthase (GOGAT) activities were negligible during C- and C/N-limitation . In N-limited cells the GOGAT activity increased as the dilution rate increased up to 0.35 h-1, and then sharply dropped . In the N-sufficient cultures both NAD(+)-and NADP(+)-dependent isocitrate dehydrogenase (NAD-ICDH and NADP-ICDH) activities were up-regulated as dilution rate increased, but in the N-limited culture the NAD-ICDH activity was up-regulated whereas NADP-ICDH one was down-regulated . Pulse additions of ammonium and methanol demonstrated the coordinate regulation of the GDH and ICDHs activities . When pulses were added to the C/N-limited cultures, there was an immediate utilization of the nutrients, resulting in an increase in biomass; at the same time the GDH and ICDH activities increased and the GS and GOGAT activities decreased . When the same ammonium/methanol pulse was added into the N-limited culture, there was a 3 h delay in the culture response, after which the substrates were utilized at rates close to the ones shown by the C/N-limited culture after the analogous pulse. Biotechnol Prog, 1997 May-Jun, 13(3), 301 - 10 Effect of oxygen limitations on monoclonal antibody production by immobilized hybridoma cells; Riley MR et al.; The productivity of an immobilized cell biocatalyst is often limited by the amount of oxygen that reaches cells located at interior regions of the biocatalyst . These diffusive limitations depend on a multitude of factors including the oxygen supply, the cellular uptake kinetics, and the cell density of the material . Large cell densities, which are desired for high productivity, are also likely to reduce the percentage of cells that receive an adequate supply of oxygen . To develop a better understanding of how different conditions affect biocatalyst behavior, a computational model of immobilized hybridoma cells was developed . The model accounts for oxygen diffusion and consumption, cell proliferation and death, and monoclonal antibody production . This model assumes that cellular productivity is limited only by the supply of oxygen and that the growth media is continually replenished so that nutrient levels remain high and wastes are eliminated . Biocatalyst performance is evaluated by monitoring the amount of monoclonal antibody produced by the cells . Model predictions agree with experimental measurements reported in the literature and indicate that for long operation time the supply of oxygen, biocatalyst size, and cell kinetics have a significant effect on biocatalyst performance, whereas the initial cell loading has only a relatively small effect . Under typical culture conditions, we find that oxygen penetrates to a maximum depth of about 0.4 mm . Accordingly, cells immobilized farther than this threshold distance receive an insufficient supply of oxygen. Yeast, 1997 May, 13(6), 529 - 39 The osmotic hypersensitivity of the yeast Saccharomyces cerevisiae is strain and growth media dependent: quantitative aspects of the phenomenon; Blomberg A; Osmotic hypersensitivity is manifested as cellular death at magnitudes of osmotic stress that can support growth . Cellular capacity for survival when plated onto high NaCl media was examined for a number of laboratory and industrial strains of Saccharomyces cerevisiae . During respiro-fermentative growth in rich medium with glucose as energy and carbon source, the hypersensitivity phenomenon was fairly strain invariant with a threshold value of about 1 M-NaCl; most strains fell within a 300 mM range in LD10 values (lethal dose yielding 10% survival) . Furthermore, all but one of the strains displayed similar differential death responses above the threshold value, i.e . ten-fold decreased viability for every 250 mM increase in salinity . Addition of small amounts of salt to the growth medium drastically improved tolerance and shifted the hypersensitivity threshold to higher NaCl concentrations . This salt-instigated tolerance could partly be reversed by washing in water . The washing procedure depleted cells of the glycerol that they had accumulated under saline growth, and the contribution from glycerol to the improved tolerance was about 50% in the two strains examined . Growth on derepressing carbon sources like galactose, ethanol or glycerol gave strain-dependent responses . The laboratory strain X2180-1A drastically improved tolerance while the bakers' yeast strain Y41 did so only marginally . It was concluded that all strains of S . cerevisiae display the osmotic hypersensitivity phenomenon in qualitative terms while the quantitative values differ . It was also proposed that growth rate does not dictate the level of osmotic hypersensitivity of S . cerevisiae. Antimicrob Agents Chemother, 1997 May, 41(5), 1064 - 8 Pentostam induces resistance to antimony and the preservative chlorocresol in Leishmania donovani promastigotes and axenically grown amastigotes; Ephros M et al.; An axenic amastigote culture system was utilized to directly assess the stage-specific antileishmanial effects of antimony on amastigotes of Leishmania donovani devoid of the macrophage host cell . Pentostam, which contains antimony in the form of sodium stibogluconate and the preservative chlorocresol, was used . Cell density was quantified by measuring the activity of the stable enzyme ornithine decarboxylase . Dose-response curve analyses show that Leishmania promastigotes are susceptible to Pentostam, with the 50% inhibitory concentration (IC50) being 104 microg/ml, while amastigotes are more susceptible, with the IC50 being 24 microg/ml . Promastigotes and amastigotes are also susceptible to chlorocresol, with IC50s being 1.27 and 1.82 microg/ml, respectively . Given that promastigotes are insensitive to antimony, these results suggest that the increased susceptibility of amastigotes to Pentostam is due to the stage-specific activity of sodium stibogluconate . To further study this phenomenon, spontaneous resistance to Pentostam was induced in L . donovani promastigotes by increasing the concentration of Pentostam in the growth medium in a stepwise fashion . Two mutants, Ld1S.04 and Ld1S.20, grew at 0.4 and 2.0 mg of Pentostam per ml, respectively . Promastigotes of these mutants were 11 and 21 times, respectively, more resistant to Pentostam than the wild type . Amastigotes were 40 and 148 times, respectively, more resistant than the wild type . The mutants were also chlorocresol resistant; promastigotes were 6 and 9 times, respectively, more resistant than the wild type, and amastigotes were 14 and 35 times, respectively, more resistant than the wild type . These data show that resistance to Pentostam induced in antimony-insensitive promastigotes is manifested in amastigotes as resistance both to pentavalent antimony and to chlorocresol . The axenic amastigote system is a unique tool which enables direct evaluation of the activity of antileishmanial compounds on the amastigote devoid of its host cell. Appl Environ Microbiol, 1997 May, 63(5), 1712 - 4 Biodegradation of glyceryl trinitrate by Penicillium corylophilum Dierckx; Zhang YZ et al.; Penicillium corylophilum Dierckx, isolated from a contaminated water wet, double-base propellant, was able to completely degrade glyceryl trinitrate (GTN) in a buffered medium (pH 7.0) containing glucose and ammonium nitrate . In the presence of 12 mg of initial fungal inoculum, GTN (48.5 to 61.6 mumol) was quantitatively transformed in a stepwise process to glyceryl dinitrate (GDN) and glyceryl mononitrate (GMN) within 48 h followed by a decrease in the GDN content with a concomitant increase in the GMN level . GDN was totally transformed to GMN within 168 h, and the complete degradation of GMN was achieved within 336 h . The presence of glucose and ammonium nitrate in the growth medium was essential for completion of the degradation of GTN and its metabolites . Complete degradation of GTN by a fungal culture has not been previously reported in the literature. Biochim Biophys Acta, 1997 Apr 10, 1351(3), 333 - 40 Modulation of type VII collagen (anchoring fibril) expression by retinoids in human skin cells; Chen M et al.; We examined the effects of retinoids on the expression of type VII collagen, a major component of anchoring fibrils, in human keratinocytes and amnion cells (WISH) . All-trans retinoic acid (RA) (5 X l0(-6) M) decreased the steady-state levels of type VII collagen mRNA by at least 80% after 18 h . The inhibition was evident within 6 h after the addition of RA, maximal at 18 h, and was dose-dependent . Reduction of type VII mRNA expression also occurred when cell cultures were incubated with retinol, retinal, and 13-cis RA . Retinoid-mediated inhibition of type VII collagen mRNA expression was observed in keratinocytes growing in either serum-free keratinocyte growth medium (KGM) or KGM supplemented with 1.4 mM Ca2+ . Cycloheximide blocked RA-mediated inhibition of type VII collagen mRNA, demonstrating the need for de novo protein synthesis . The mRNA levels for fibronectin and glyceraldehyde phosphate dehydrogenase were not affected by the retinoids, suggesting selective inhibition on type VII collagen expression . In addition, the decrease in type VII collagen mRNA was accompanied by a parallel decrease in secretion of the 290 kDa, type VII collagen alpha chains. Hum Gene Ther, 1997 Apr 10, 8(6), 709 - 17 The bystander effect of the nitroreductase/CB1954 enzyme/prodrug system is due to a cell-permeable metabolite; Bridgewater JA et al.; The bystander effect is an important part of tumor kill using gene-directed enzyme prodrug therapy (GDEPT) . Recently, we have described a novel enzyme prodrug system using bacterial nitroreductase and the prodrug CB1954 (NTR/CB1954) . We demonstrate here the presence of a cell-permeable cytotoxic activity in the conditioned growth medium of nitroreductase (NTR)-transduced cells treated with CB1954 and show that its appearance corresponds to the appearance of two metabolites of CB1954 previously identified (Friedlos et al., 1992) . The degree of bystander effect and the degree of transferred cytotoxicity correlates with the level of NTR enzyme expression . Two other prodrugs for NTR show little bystander killing and do not produce detectable cell permeable metabolites . The elucidation of the mechanism of the bystander effect may allow the more effective use of NTR/CB1954. FEBS Lett, 1997 Apr 7, 406(1-2), 175 - 8 In vitro cytotoxic effects of tumor necrosis factor-alpha in human breast cancer cells may be associated with increased glucose consumption; Kaplan O et al.; Tumor necrosis factor-alpha inhibited growth of cultured MCF-7 human breast cancer cells in a dose dependent manner . Tumor necrosis factor-alpha also markedly increased glucose consumption, and its cytotoxicity was modified by glucose concentrations in the growth medium; higher glucose levels were associated with increased cell survival . However, when the cells were perfused in physiological conditions, very high levels of tumor necrosis factor-alpha (200 ng/ml) in the perfusion solution had no inhibitory effects . Moreover, tumor necrosis factor-alpha had no effects on 31P nuclear magnetic resonance spectra of the perfused cells . In the traditional growth inhibition assays, cells are incubated for several days with a drug, a situation where their metabolism is altered due to the depletion of nutrients, the accumulation of toxic waste materials and pH changes . Perfusion experiments are more relevant to in vivo conditions, and may be used for studying metabolic processes and the mechanisms of action of therapeutic agents. J Biol Chem, 1997 Apr 4, 272(14), 9332 - 43 The PPS1 gene of Saccharomyces cerevisiae codes for a dual specificity protein phosphatase with a role in the DNA synthesis phase of the cell cycle; Ernsting BR et al.; We report the identification of the PPS1 gene of Saccharomyces cerevisiae . The deduced amino acid sequence of PPS1p shows similarity with protein-tyrosine phosphatases (PTPases) and is most closely related to a subfamily of PTPases that are capable of dephosphorylating phosphoseryl and phosphothreonyl residues as well as phosphotyrosyl residues . Analysis of the predicted amino acid sequence suggests that the protein consists of an active phosphatase domain, an inactive phosphatase-like domain, and an NH2-terminal extension . Mutation of the catalytic cysteinyl residue in the active phosphatase domain reduced the in vitro activity of the mutant protein to less than 0.5% of wild type activity, while mutation of the corresponding cysteinyl residue of the inactive phosphatase-like domain had no effect on in vitro activity . The PPS1 protein was expressed in Escherichia coli, and the protein was shown to catalyze the hydrolysis of p-nitrophenyl phosphate, dephosphorylate phosphotyrosyl, and phosphothreonyl residues in synthetic diphosphorylated peptides and to inactivate the human ERK1 protein . PPS1 transcript abundance is coregulated with that of the divergently transcribed DPB3 gene, which codes for a subunit of DNA polymerase II, with both transcripts showing peak abundance in S phase . pps1Delta mutant strains did not differ from PPS1 strains under any of the conditions tested, but overexpression of the PPS1 protein in S . cerevisiae led to synchronous growth arrest and to aberrant DNA synthesis . A screen for suppressors of this growth arrest identified the RAS2 gene as a multicopy suppressor of the PPS1p overexpression arrest . The arrest was not suppressed by the presence of multicopy RAS1, TPK2, or TPK3 genes or by the presence of 5 mM cAMP in the growth medium, suggesting that PPS1 functions in a pathway involving RAS2, but not TPK kinases or adenylate cyclase. Biochemistry (Mosc), 1997 Apr, 62(4), 401 - 11 Expression of functionally active cytochrome b5 in Escherichia coli: isolation, purification, and use of the immobilized recombinant heme protein for affinity chromatography of electron-transfer proteins; Chudaev MV et al.; Cytochrome b5 is an integral membrane protein which is localized in endoplasmic reticulum membranes . In this paper we present the results on expression in E . coli, purification, and characterization of recombinant rat cytochrome b5 . The full-length cDNA for rat liver microsomal cytochrome b5 has been modified using polymerase chain reaction (PCR) to introduce corresponding restriction sites as well as to insert silent mutations in the N-terminal sequence to increase the content of A and T nucleotides that prevents formation of elements of secondary structure of the mRNA transcripts and facilitates high expression . The expression plasmid was constructed by cloning of amplified cDNA to pCWori+ plasmid and used for transformation of E . coli DH5 alpha . The optimization of recombinant cytochrome b5 expression procedure induces expression level up to 3000 nmoles per liter of growth medium; this confers in the cells a deep pink color . The most interesting fact is that cytochrome b5 is expressed in this system in the reduced state . Recombinant cytochrome b5 was purified from solubilized cell membranes by a combination of ion-exchange chromatography and gel filtration . During purification, part of the cytochrome b5 is subjected to limited proteolysis with formation of truncated form . Sequencing of the N-terminal part of the recombinant cytochrome b5 indicates that it coincides with the sequence of rat cytochrome b5 . Recombinant cytochrome b5 was found to have physicochemical, catalytic, and immunochemical properties to that of the native protein and was used as an efficient affinity matrix for purification of the various electron-transfer proteins. Arch Physiol Biochem, 1997 Apr, 105(2), 158 - 66 Isolation of cells from ovine fetal long bone and characterization of their osteoblastic activities during in vitro mineralization; Collignon H et al.; Studies about bone formation and regulation are complex due to a close relationship between bone cells . Primary cell cultures allow to understand osteoblastic function . We isolated cells from the cortical metacarpal bone of 85 or 120 day-old ovine fetuses by an enzymatic method . After first passage and cell amplification, the growth medium (DMEM, ascorbic acid and fetal calf serum 10%) was replaced at confluence by a mineralization medium (MM: DMEM, ascorbic acid, beta-glycerophosphate, insulin) . Alkaline phosphatase (ALP) activity in cell-matrix layer increased after 4 days of cultures in MM and maximized at day 6 . We also measured osteocalcin, ALP and IGF-I secretion simultaneously during mineralization . PTH, PTHrP and 1.25(OH)2D3 decreased ALP activity in cell-matrix layer after 4 days of treatment in MM without fetal calf serum (FCS) . Cells from 120 day-old fetuses were cultivated in MM with 10% FCS during 32 days to induce mineralization . Inorganic phosphorus concentration increased in medium between days 5 and 12, Ca concentration decreased in medium after 12 days of culture . Mineralization started at day 12, in the same time ALP activity appeared in medium . Osteocalcin secretion increased between days 6 and 12, decreased at day 14 and increased from day 16 until day 32 . Ovine fetal bone cells produced IGF-I until first days of culture in MM . Such ovine osteoblast phenotype cells having the capacity to differentiate and mineralize in vitro would be a model to study the endocrine regulation of osteoblastic function in large mammals. J Clin Microbiol, 1997 Apr, 35(4), 823 - 9 Western immunoblot analysis of Haemobartonella muris and comparison of 16S rRNA gene sequences of H . muris, H . felis, and Eperythrozoon suis; Rikihisa Y et al.; Infectious agents were isolated from the spleens of three wild mice (Apodemus argenteus) by intraperitoneal inoculation of the spleen homogenate into laboratory mice . The laboratory mice developed clinical signs and splenomegaly, and three isolates were maintained by passage in mice . Tetracyclines were effective in preventing infection of mice with these agents, but streptomycin and penicillin were ineffective . The agents did not grow in bacterial growth media or chicken embryos . In smears of blood from infected mice stained by the Giemsa or the indirect immunofluorescence method, numerous organisms were found on the surfaces of erythrocytes . Electron microscopy revealed cell wall-less pleomorphic cocci of 350 to 700 nm in diameter . On the basis of these results, the isolates were identified as Haemobartonella muris . There was no antigenic cross-reactivity with Rickettsia or Ehrlichia spp . or other related organisms . Western immunoblot analysis of three strains of H . muris with mouse antisera to H . muris revealed identical major antigens of 118, 65, 53, 45, and 40 kDa . By heteroduplex analysis of the three PCR-amplified segments of the 16S rRNA genes, the three strains of H . muris were found to be identical . The 16S rRNA genes of one of the H . muris strains, four strains of H . felis, and two strains of Eperythrozoon suis were sequenced and compared . The sequences of two strains of H . felis from cats in California were identical, as were the sequences of a strain from a cat in Ohio and a strain from a cat in Florida, but the similarity of sequences between the California and the Ohio-Florida strains was only 85% . The sequence of an H . muris strain was unique and was more closely related to that of the Ohio-Florida strain of H . felis (89%) than to that of the California strain of H . felis (84%) . The sequence of E . suis from a pig in Illinois was identical to that from another pig from Taiwan . The similarity of the 16S rRNA gene sequence of E . suis with those of three Haemobartonella strains was 84 to 92%, with that of E . suis being most similar to that of the H . felis strain from California . In the phylogenetic analysis based on 16S rRNA gene sequences, the Haemobartonella spp . and E . suis formed a distinct clade more closely related to Mycoplasma spp . (79 to 83% similarity) than to Anaplasma marginale (72 to 75% similarity) . Our results suggest that the Haemobartonella spp . and E . suis may be reclassified in the same genus in the family Mycoplasmataceae. Tissue Cell, 1997 Apr, 29(2), 207 - 15 Conditions for the culture of bovine embryonic myogenic cells; Woods TL et al.; The objective of this experiment was to determine the growth characteristics of bovine embryonic muscle cells and to optimize the growth conditions for these cells using commercially-prepared media and sera . In the first study, the growth of muscle cells isolated from the hindlimb was determined by measuring DNA content . The DNA concentration was lowest (P < 0.001) at 24 h post-plating and increased to a maximum at approximately 60 h . The slopes of creatine kinase activity and fusion index curves were similar to the DNA; however, the creatine kinase activity achieved a maximum at 140 h post-plating, while the fusion index reached maximum at 120 h . In the second study, cells were cultured on different substrata, either plastic, gelatin, or collagen . There were no differences (P > 0.05) in the cell growth rates for any of the three substrata . In the third study, cells were grown in 10% fetal bovine serum (FBS) and either a balanced salt solution (BSS; 30 mM Hepes, 10 mM glucose, 120 mM NaCl, 2.5 mM Na2HPO4, and 3 mM KCl), McCoy's 5A, Dulbecco's Minimal Essential Medium/Ham's F12 (DMEM/F12), or 70% DMEM/20% M-199 . Cell numbers adhering to the plate at 26 h post-plating were different (P > 0.001) between each medium (DMEM/M-199 > McCoy's 5A > DMEM/F12 > BSS) . Cell proliferation rates for each treatment medium were greatest for DMEM/M-199, followed by McCoy's 5A, DMEM/F12, and BSS . Cell differentiation was highest (P < 0.05) in the DMEM/F12, followed by McCoy's 5A, DMEM/M-199, and BSS . In the final study, the cells were treated with different sources of serum added at 10% to DMEM/M-199 . The sera consisted of FBS, newborn calf serum (NCS), horse serum (HS) and iron-supplemented calf serum (Fe(2+)-CS) . The cells were added to each well at 10(4) cells . At 24 h post-plating, the serum-free, NCS, and FBS-treated cell numbers were greater (P < 0.05) than the cells treated with HS or Fe(2+)-CS, which may reflect the efficient adherence to the surface or faster adaptation to the serum by the cells . The proliferation rate was greatest (P < 0.001) for the cells treated with Fe(2+)-CS, followed by FBS = NCS, HS, and no serum . Therefore, the muscle cells obtained from bovine embryos grow and differentiate similar to muscle cells from other species . The optimal growth medium for growing these cells in vitro is DMEM/M-199 plus 10% Fe(2+)-CS, while the optimal differentiation medium is McCoy's 5A. Metabolism, 1997 Apr, 46(4), 425 - 30 Inhibitory effects of galanin on growth hormone (GH) release in cultured GH-secreting adenoma cells: comparative study with octreotide, GH-releasing hormone, and thyrotropin-releasing hormone; Giustina A et al.; The aim of the present study was to characterize in a large series (N = 12) of cultured somatotrope adenomas the in vitro effects of the neuropeptide galanin on growth hormone (GH) secretion . This was contrasted with two peptides known to be GH secretagogues (GH-releasing hormone {GHRH} and thyrotropin-releasing hormone {TRH}) and a peptide with a known GH-inhibitory effect (the somatostatin analog octreotide) . Groups of three wells were incubated for 4 hours with growth medium alone (control incubation), galanin, GHRH(1-29)NH2, TRH, or octreotide . Galanin and octreotide were applied at concentrations of 0.1, 1, and 10 mumol/L, and GHRH and TRH at concentrations of 0.01, 0.1, and 1 mumol/L . Galanin was able to inhibit GH release in nine of 12 cultured somatotrope adenoma cells . This inhibitory effect was clearly dose-dependent in five adenomas . Overall, the mean GH nadir after galanin was -36.1% in nine responder adenoma cultures versus control wells . Octreotide inhibited GH release in five of eight cultured somatotrope adenoma cells . The mean GH nadir after octreotide was -32.7% in five responder adenoma cultures compared with control wells . GHRH and TRH were able to stimulate GH release, respectively, in seven of 11 and in six of seven cultured somatotrope adenoma cells . The mean GH peaks after either GHRH or TRH in responder adenoma cultures were, respectively, +71.5% and +143.7% compared with levels in the control wells . In conclusion, the consistency and potency of the in vitro GH-inhibitory eff