J In Vitro Fert Embryo Transf, 1988 Oct, 5(5), 286 - 9 Effects of charcoal-extracted serum as a growth medium supplement on in vitro development of mouse embryos; Padilla SL et al.; Two-cell mouse embryos (N = 952) were cultured in modified Ham's F-10 medium supplemented with 15% charcoal-extracted serum or 15% nonextracted serum from 17 patients . Each sample was assayed independently and all experiments run in duplicate . Ten nonextracted samples inhibited development to the blastocyst stage compared to controls in F-10 alone . Charcoal extraction significantly improved (P less than 0.05) development compared to nonextracted serum in eight of these samples . Seven samples supported development and no difference was noted between F-10 alone and extracted and nonextracted serum-supplemented media in this group . In conclusion, charcoal extraction significantly reversed the "embryotoxic" effects of some sera in the two-cell mouse embryo model. Carcinogenesis, 1988 Oct, 9(10), 1801 - 10 Intracellular 58-kd selenoprotein levels correlate with inhibition of DNA synthesis in mammary epithelial cells; Morrison DG et al.; Five mouse mammary epithelial cell lines (MMEC) with different growth rates were used to examine the relationship between selenoprotein levels and selenite-mediated inhibition of DNA synthesis . The inhibition of DNA synthesis preceded and was significantly greater than inhibition of protein synthesis . Cycloheximide, a protein synthesis inhibitor, caused a coordinate inhibition of both DNA and protein synthesis over a 50-fold dose range . Of the selenoproteins detected by one-dimensional SDS-PAGE, the 58-, 26- and 23-kd proteins were the only major selenoproteins observed in common among the five MMEC lines before and during inhibition of DNA synthesis . Other selenoproteins were present in some cell lines or after inhibition of DNA synthesis . The level of the 58-kd selenoprotein was most closely correlated with the degree of inhibition of DNA synthesis (r2 = 0.85), whereas the 26- and 23-kd proteins most closely correlated with selenite retention (r2 = 0.78) . Upon selenite withdrawal from the growth medium, the decrease in 58-kd, but not in the 26- and 23-kd proteins correlated with resumption of DNA synthesis . Similarly, dose-response studies indicated that the 58-kd protein increased greater than 20-fold, whereas the 26- and 23-kd proteins increased only 5-fold . At high doses of selenite, other selenoproteins of mol . wts 30-101 kd were present but these proteins seemed to appear after inhibition of DNA synthesis . The possibility exists that these proteins may be product/precursors of the major selenoproteins . Since these experiments attempted to quantitate selenoproteins by densitometry of one-dimensional SDS-PAGE autoradiographs, the quality controls used for the experiments are discussed . The results are discussed in terms of the hypothesis that the 58-kd selenoproteins may mediate the effects of selenite on DNA synthesis. Biomed Environ Mass Spectrom, 1988 Oct, 16(1-12), 457 - 9 Monitoring of bacterial drug response by mass spectrometry of single cells; Seydel U et al.; The application of the laser microprobe mass analyser LAMMA 500 to the solution of problems in the field of microbiology is reported . The special features of this instrument allow the analysis of single bacterial cells, and questions can be answered which are not accessible to the normally applied integral methods . Thus it is possible to establish distributions of, for instance, elemental concentrations within a bacterial population and of correlations between measured characteristics of a bacterium with its morphology . The mass spectrum of a single bacterial cell comprises information on its intracellular cation contents as well as on the organic matrix . The relation between the sodium and potassium contents can serve as a criterion of the physiological state of a cell and of its viability . The information from the organic matrix can be extracted from the complex spectra of fragment ions produced by the interaction of the laser with the cell by applying multivariate data analysis, thus rendering additional information . Examples will be given for in vitro drug screening and in vivo therapy control in leprosy, an infection which is caused by a mycobacterial species, Mycobacterium leprae; this organism does not multiply in artificial growth media so that only limited numbers of organisms are available for microbiological investigations. J Bacteriol, 1988 Oct, 170(10), 4816 - 22 Activation of protease-constitutive recA proteins of Escherichia coli by all of the common nucleoside triphosphates; Wang WB et al.; To understand why the RecA proteins of the protease-constitutive recA1202 and recA1211 mutants show very high protease activities in vivo without the usual need for DNA damage (E . S . Tessman and P . Peterson, J . Bacteriol . 163:677-687, 1985), we examined the activation of the mutant proteins by nucleoside triphosphates (NTPs) in vitro . In vivo, the mutant protease activities are resistant to inhibition by cytidine plus guanosine (C + G) in the growth medium, in contrast to the activities of weaker mutants, such as recA441, which are sensitive to C + G inhibition . We found that RecA1202 and RecA1211 proteins, in contrast to RecA+, can use natural NTPs other than ATP and dATP as cofactors in the cleavage of LexA repressor . The effectiveness of NTPs in promoting LexA cleavage by RecA1202 and RecA1211 proteins decreased in roughly the following order: dATP greater than ATP greater than UTP greater than ATP-gamma S greater than dCTP greater than CTP greater than dGTP greater than GTP greater than TTP . These mutant proteins showed higher affinities for ATP and single-stranded DNA and higher repressor cleavage activities than RecA+ protein . With the various effectors (single-stranded DNA or NTPs), the RecA1202 protein always showed more activity than RecA1211 in the cleavage of LexA repressor in vitro, which is consistent with the greater activity of the recA1202 mutant in vivo . The results explain, in part, why some recA mutants have unusually high constitutive RecA protease activity and why that activity is more or less resistant to C + G inhibition. Biochim Biophys Acta, 1988 Sep 15, 944(1), 79 - 84 Effect of exogenous gangliosides on the lipid composition of chick neurons in culture; Leray C et al.; When exogenous gangliosides are added to the growth medium of neuronal cell cultures they are inserted into their plasma membranes and are afterwards metabolized in the cytoplasmic interior . The action of exogenous gangliosides brings important morphological and biochemical changes to neurons in culture . The present report shows that the treatment with exogenous gangliosides of a primary culture of chick neurons modified the distribution of fatty acids in phosphatidylinositol (PI), mainly that of arachidonic acid and the fatty acids of the (n - 3) series without affecting the other phospholipids . The composition of neutral lipids did not change but their content was increased up to 2-3-fold depending upon the concentration of gangliosides . The change of the growth medium from one containing fetal calf serum to a chemically defined one reduced dramatically the content of free fatty acids while the addition of gangliosides raised this content to normal levels . The increase in the amount of diacylglycerol (DG) confirmed the finding that gangliosides stimulate phosphoinositide degradation . Finally the fatty acid composition of DG suggests indirectly that this compound might be produced also by degradation of phosphatidylcholine and not only of PI. Mutat Res, 1988 Sep, 194(2), 101 - 8 Detection of DNA damage and repair by alkaline elution using human lymphocytes; Munzer JS et al.; The repair of DNA alkylation damage in human cells is poorly understood . We have adapted the alkaline elution technique for use with human peripheral blood lymphocytes in culture . We have also established conditions necessary for short-term culture of human lymphocytes . Lymphocyte growth which can be maintained for up to 30 days is dependent upon irradiated TK6 feeder cells and T-cell growth factor (crude TCGF) . The amount of damage induced by a given concentration of methyl methane-sulfonate (MMS) is dependent upon cell number per ml of growth medium . The DNA damage measured, in lymphocytes, by alkaline elution is a composite of single strand breaks and alkali-labile lesions . Repair of this damage after appropriate recovery periods is also detectable . The irradiated feeder TK6 cells do not contribute to the number of strand breaks detected or the amount of recovery after treatment . This method offers a quick and reproducible means of detecting DNA damage and repair in human T-lymphocytes. Anal Biochem, 1988 Sep, 173(2), 278 - 84 Determination of nitrilotriacetate in biological matrices using ion exclusion chromatography; Schneider RP et al.; A sensitive method for the determination of nitrilotriacetate in biological growth media and cell-free extracts by ion exclusion chromatography is described using HCl as an eluant . The eluant conductivity was chemically suppressed with a membrane suppressor and a conductivity detector was used for subsequent detection . The membrane was continuously regenerated with a tetrabutylammoniumhydroxide solution . The detection limit for nitrilotriacetate in cell-free extract was 11 mg/liter, while for nitrilotriacetate in growth media it was 1 mg/liter . Interference by compounds present in biological matrices with the determination is discussed. In Vitro Cell Dev Biol, 1988 Sep, 24(9), 900 - 4 A factor produced by human cells in vitro that changes HeLa cell colonial morphology; Foley JF et al.; Colonies of HeLa cells cultured in media supplemented with human or bovine serum or both can be morphologically described as three types: diffuse, intermediate, and compact, with their modal distribution depending on the serum or sera added to the growth medium . We have observed that for a particular medium or serum system, the percentage of compact colonies remains fairly constant under normal culture conditions, 0.2%, whereas the diffuse and intermediate colonies vary over a much wider range . The presence of certain substances as trypsin, heparin and Darvan in the medium favor the increase of compact colonies at the expense of other types . Furthermore, we have discovered that colonial morphology is influenced by cocultivation of the HeLa cells with human fibroblastlike cells, the compact colonies increasing as the density of the fibroblast element introduced into the mixed cultures is increased . Subsequent investigation revealed that conditioned medium from confluent fibroblast and HeLa cell cultures contained a factor(s), that significantly increased the percentage of compact colonies . The factor is nondialyzable, heat-stable and can be neutralized by serum . Recorded in this presentation are preliminary observations on the kinetics of colony formation and the interaction among the three HeLa cell colony types, the diffuse, the intermediate, and the compact . The factor's effect on HeLa cell colonial morphology is time dependent and rapidly reversed if the factor(s) is removed and fresh medium added. Biochem Cell Biol, 1988 Sep, 66(9), 951 - 7 Regulation of uptake of inositol by glucose in cultured retinal pigment epithelial cells; Khatami M et al.; Long term and acute effects of glucose on myo-inositol (MI) uptake were studied in primary cultures of bovine retinal pigment epithelial (RPE) cells . RPE cells were grown under low (5 mM) or high (20, 40, or 50 mM) glucose levels in the growth medium for up to 18 days . The concentrative capacity of confluent RPE cells to accululate {3H}MI (10 microM) was reduced up to 41% as the glucose concentration in the growth medium increased . When the growth medium glucose was switched from 5 to 40 mM, or vice versa, the capacity of cells to accumulate MI was reversed . Treatment of cells grown in 40 or 50 mM glucose with 0.1 mM Sorbinil (an aldose reductase inhibitor) minimally reversed the ability of cells to accumulate MI . RPE cells, grown in 5 mM glucose, were incubated with 10-60 mM D-glucose or its nonmetabolizable analogues (acute effect) . Kinetics of MI uptake inhibition by alpha-methyl glucose according to Dixon plots were characteristic of competitive inhibition (Ki = 28 mM) . MI uptake was strongly inhibited by phlorizin . The ability of RPE cells to bind 5 microM {3H}phlorizin also was reduced by increased glucose levels in the growth medium . These studies indicated that MI and glucose shared the same transporter system . Glucose in the incubation medium directly interfered with MI binding to the transporter . High glucose feeding of the cells also down-regulated the transporter density . The uptake and function of solutes such as MI in tissues that operate on the glucose carrier system may be severely impaired in diabetes. J Bacteriol, 1988 Sep, 170(9), 4286 - 92 Transcriptional regulation of katE in Escherichia coli K-12; Schellhorn HE et al.; Escherichia coli produces two distinct species of catalase, hydroperoxidases I and II, which differ in kinetic properties and regulation . To further examine catalase regulation, a lacZ fusion was placed into one of the genes that is involved in catalase synthesis . Transductional mapping revealed the fusion to be either allelic with or very close to katE, a locus which together with katF controls the synthesis of the aerobically inducible hydroperoxidase (hydroperoxidase II) . katE was expressed under anaerobic conditions at levels that were approximately one-fourth of those found in aerobically grown cells and was found to be induced to higher levels in early-stationary-phase cells relative to levels of exponentially growing cells under both anaerobic and aerobic conditions . katE was fully expressed in air and was not further induced when the growth medium was sparged with 100% oxygen . Expression of katE was unaffected by the addition of hydrogen peroxide or by the presence of additional lesions in oxyR or sodA, indicating that it is not part of the oxyR regulon . When katF::Tn10 was introduced into a katE::lacZ strain, beta-galactosidase synthesis was largely eliminated and was no longer inducible, suggesting that katF is a positive regulator of katE expression. Mol Pharmacol, 1988 Sep, 34(3), 354 - 62 Mechanism of gamma-aminobutyric acid/benzodiazepine receptor turnover in neuronal cells: evidence for nonlysosomal degradation; Borden LA et al.; In a previous report we described the use of flunitrazepam as a photoaffinity label to monitor the turnover of the gamma-aminobutyric acid/benzodiazepine receptor complex in primary brain and spinal cord cell cultures {Science (Wash . D . C.) 226:857-860 (1984)} . In the present communication we have extended our studies on the kinetics of receptor turnover and have examined the mechanism of receptor degradation . There are approximately 60,000 irreversible binding sites per neuron, and photolabeling is stereospecific . Photolabeling does not demonstrably alter the kinetics of degradation, and a complete rate equation relating the kinetic constants for degradation to receptor number is described . The rapid phase of degradation is slowed at low temperature (Q10 = 5, which corresponds to an apparent energy of activation of 25 kcal/mol) and by inhibitors of ATP production (sodium azide, 2-deoxyglucose, and 2,4-dinitrophenol) . The fast phase for the degradation of photolabeled receptor is not affected by lysosomotropic agents (methylamine, ammonium chloride, and chloroquine) or by elimination of horse serum and chick embryo extract from the growth medium . In contrast, overall protein degradation is inhibited by methylamine and enhanced in serum-free medium . The results suggest that the gamma-aminobutyric acid receptor complex is degraded through an energy-dependent nonlysosomal pathway. J Neurochem, 1988 Sep, 51(3), 950 - 9 Neurite extension and increased expression of RI cyclic AMP-binding protein in ouabain-resistant neuroblastoma mutants; Meyer SA et al.; Morphological and biochemical parameters of neuroblastoma differentiation were assessed in 12 clonal derivatives of the N-18 mouse neuroblastoma cell line selected for their ouabain-resistant (ouar) property . When cultured in a normal growth medium, nine of the 12 ouar cell lines exhibited a more complex pattern of neurite outgrowth than the parental N-18 cells . The morphological pattern most frequently observed with the ouar cells was the extension of several branched processes per cell . This pattern of spontaneous neurite outgrowth in the ouar cell lines can be correlated with an increase in expression of the 47,000-dalton RI cyclic AMP (cAMP)-binding protein . The growth rate, intracellular level of cAMP, and acetylcholinesterase activity of the ouar cell lines were not significantly different from those of the parental N-18 neuroblastoma cells . Treatment of the parental and ouar neuroblastoma cell lines with 1 mM N6, O2-dibutyryl cAMP promoted an elaborate pattern of neurite outgrowth and marked increases in acetylcholinesterase and RI cAMP-binding activities . The distinctive pattern of differentiation phenotype exhibited by the ouar cells and the dibutyryl cAMP-induced differentiated neuroblastoma cell suggests that these two protocols yielded different degrees of differentiation . Furthermore, our results suggest a linkage of the biochemical events underlying ouabain resistance and expression of differentiation phenotypes in the mouse neuroblastoma cells. Isr J Med Sci, 1988 Sep-Oct, 24(9-10), 505 - 11 Variable efficacy of interferon-alpha treatment on growth of human hepatoma cell lines in vitro; Ilan Y et al.; Three human hepatoma cell lines, PLC/PRF/5, Mahlavu and Sk-Hep 1, two of which contain integrated HBV DNA, were grown in culture and treated with human alpha-IFN for up to 14 days . IFN treatment caused a varying suppression of cell growth of the three hepatoma cell lines . While doubling time and cloning efficiency were significantly reduced for all three hepatoma cell lines tested, 3{H}thymidine incorporation was markedly suppressed, in a dose-dependent fashion, only in treated PLC/PRF/5 cells but not in Sk-Hep 1 and Mahlavu cells . The inhibiting effect of interferon treatment on growth of PLC/PRF/5 cells in vitro was neutralized by antibodies to human IFN . IFN treatment caused a significant suppression of HBsAg and alpha FP secretion by PLC/PRF/5 hepatoma cells . This effect, while constant throughout the observation period for HBsAg, was cumulative for alpha FP secretion . Following discontinuation of treatment, suppression of PLC/PRF/5 hepatoma cell growth was rapidly reversed, and HBsAg and alpha FP secretion returned to their pretreatment levels . These experiments suggest that human alpha-IFN suppresses the growth of some human hepatoma cells in culture but that this effect is dependent on the continuous presence of IFN in the growth medium . Finally, the inhibitory effects of IFN on cell growth differed for the various hepatoma cell lines tested. Clin Chem, 1988 Sep, 34(9), 1693 - 6 Production of murine hybrid-hybridomas secreting bispecific monoclonal antibodies for use in urease-based immunoassays; Takahashi M et al.; To produce bispecific antibodies (BiAbs) for enzyme immunoassay (EIA) to replace antibody-enzyme conjugates, we developed a panel of 8-azaguanine/ouabain-resistant anti-urease variant hybridoma cell lines for use in hybridoma-hybridoma fusions . These variants represent mouse immunoglobulin subclasses IgM, IgG1, IgG2a, and IgG2b and have growth rates equal to those of the parental hybridomas . We fused an anti-urease-secreting variant hybridoma with an anti-human choriogonadotropin (hCG)-secreting hybridoma (both of IgG1 subclass) and selected the desired product with growth media containing hypoxanthine-aminopterin-thymidine (HAT) and ouabain . Over 95% of the resulting hybrids secreted anti-urease, and 60% of these secreted anti-hCG . The bispecific nature of secreted antibodies was demonstrated in a simultaneous EIA where BiAbs, hCG, and urease (EC 3.5.1.5) were incubated together in anti-hCG-coated microwells . As little as 25 int . units of hCG per liter could be reliably detected, which is equivalent to that for antibody-enzyme conjugates in EIA. Thromb Haemost, 1988 Aug 30, 60(1), 63 - 7 Regulation of plasminogen activator inhibitor-1 mRNA in human endothelial cells; van den Berg EA et al.; The plasminogen activator inhibitor (PAI-1) from endothelial cells is a potentially important regulator of plasminogen activator activity . Cultured human endothelial cells increase their PAI-1 production upon stimulation with LPS and TNF, agents that are known to cause an increase in PAI-1 levels in vivo . We isolated a PAI-1 cDNA probe, and by RNA hybridization analysis studied the regulation of PAI-1 mRNA synthesis in human endothelial artery cells . Freshly isolated endothelial cells do not contain detectable amounts of PAI-1 mRNA, but after adherence and incubation for 18 h in growth medium produce considerable amounts of PAI-1 activity and contain PAI-1 mRNA levels comparable to those found in subcultured cells . When subcultured endothelial cells are incubated for 6 h with LPS or TNF, both species of PAI-1 mRNA increase 10 to 20 fold, while PAI-1 activity in the growth medium increases only 1.5 to 2 fold . Stimulation of endothelial cells in the presence of cycloheximide (CHX) results in superinduction of mainly the 3.0 kb PAI-1 mRNA . The 3' end of this mRNA contains a 60 bp AT-rich sequence, that resembles 3' sequences present in a number of other genes superinducible with CHX. Cancer Lett, 1988 Aug 15, 41(2), 225 - 33 Sensitivity of a human oral carcinoma cell line to retinoic acid; Sarkar R et al.; We have examined the effect of retinoic acid (RA) on proliferation of KB cells in both anchorage dependent and independent growth conditions . Our study shows that RA can cause a reduction in cell proliferation rate both in monolayer and in agar cultures . After 48 h of exposure to RA, the cells started to assume a flattened appearance and no longer formed multilayers . RA treatment also caused increase in generation time, reduction in saturation density and induced cell-to-substratum adhesiveness . These changes were reversed within 48 h after removal of RA from growth medium . The results show that this cell line is sensitive to RA-induced growth inhibition and morphologic alterations which are generally associated with reduced expression of the malignant phenotype. Biochem J, 1988 Aug 15, 254(1), 301 - 2 Use of 3-aminopropanol as an ethanolamine analogue in the study of phospholipid metabolism in Tetrahymena; Smith JD et al.; About 50% of the ethanolamine in phosphatidylethanolamine in Tetrahymena is replaced by 3-aminopropan-1-ol when the compound is added to the growth medium . The phosphatidylpropanolamine which is formed is not converted into the corresponding phosphatidylcholine analogue by methylation . There is an increase in phosphatidylcholine formed by the phosphotransferase pathway from free {3H}choline and a decrease in the phosphatidylcholine formed by the methylation pathway from {14C}methionine . The nature of the observed phospholipid alterations suggests that the regulation of phosphatidylcholine biosynthesis in Tetrahymena may be different from that found in higher eukaryotes. J Med Chem, 1988 Aug, 31(8), 1656 - 9 Effects of sulfur-containing analogues of stearic acid on growth and fatty acid biosynthesis in the protozoan Crithidia fasciculata; Rahman MD et al.; A variety of analogues of stearic acid in which one of the methylene groups was replaced by a sulfur atom were examined as inhibitors of growth and fatty acid biosynthesis in the trypanosomatid protozoan Crithidia fasciculata . The 8-, 9-, 10-, and 11-thiastearic acids were found to suppress the synthesis of the cyclopropane-containing fatty acid dihydrosterculic acid (9,10-methyleneoctadecanoic acid) at micromolar concentrations in the growth medium, and all but the 9-thiastearate were found to inhibit the growth of the protozoa at concentrations . The most potent inhibitor, 8-thiastearic acid (I50 for growth = 0.8 microM; I50 dihydrosterculate synthesis = 0.4 microM), was also observed to inhibit the synthesis of gamma-linolenic acid at a similar concentration . The sulfoxide derivatives of the 9- and 10-thiastearates were found to have little effect on growth or fatty acid synthesis, and several long-chain amides of 3-amino-1,2-propanediol were found to have effects similar to those of the fatty acids from which they were derived. J Gen Microbiol, 1988 Aug, 134 ( Pt 8), 2385 - 92 Characterization of membrane components of the flask-shaped mycoplasma Mycoplasma mobile; Fischer M et al.; The cell membrane of Mycoplasma mobile was isolated by either ultrasonic or French press treatment of intact cells . The membrane fraction contained all of the cellular lipids, but only one-third of cellular proteins and had a density of 1.14 g ml-1 . The soluble fraction contained the NADH dehydrogenase activity of the cells, as well as a protein with an apparent molecular mass of 55 kDa that was phosphorylated in the presence of ATP . Lipid analyses of M . mobile membranes revealed that membrane lipid could be labelled by radioactive glycerol, oleate and to a much higher extent by palmitate but not by acetic acid . The membrane lipid fraction was composed of 54% neutral and 46% polar lipid . The major constituents of the neutral lipid fraction were free fatty acid, free cholesterol and cholesterol esters (45, 25 and 20%, respectively, of total neutral lipid fraction) . The free cholesterol count was 13% (w/w) of total membrane lipids with a cholesterol:phospholipid molar ratio of about 0.9 . Among the polar lipids, both phospho- and glycolipids were detected . The phospholipid fraction consisted of a major de novo-synthesized phosphatidylglycerol (approximately 63% of total phospholipids), plus exogenous phosphatidylcholine and sphingomyelin incorporated in an unchanged form from the growth medium . The glycolipid fraction was dominated by a single glycolipid (approximately 90% of total glycolipids) that was preferentially labelled by palmitic acid and showed a very high saturated:unsaturated fatty acids ratio. Eur J Cell Biol, 1988 Aug, 46(3), 453 - 7 Effects of medium amino acids on ouabain-sensitive 86Rb+ -uptake and membrane-potential dependent {3H}tetraphenylphosphonium accumulation in Friend erythroleukemia cells; Schaefer A et al.; The effects of amino acids present in minimal essential medium were investigated on 86Rb+ -fluxes and on the membrane-potential dependent accumulation of the lipophilic cation {3H}tetraphenylphosphonium (TTP+) in logarithmically growing Friend erythroleukemia cells . The ouabain-sensitive 86Rb+ -uptake measured as well in complete growth medium as in Earle's balanced salt solution (EBSS) with amino acid composition present in growth medium, was 3 to 4-fold increased in comparison to the 86Rb+-uptake measured in pure EBSS only . The Na+,K+,2Cl- -cotransport measured as piretanide-sensitive 86Rb+-uptake was reduced in the presence of amino acids . Stimulation of the ouabain-sensitive 86Rb+ -uptake could be brought about by the addition of alanine alone or of the sodium ionophore monensin . In spite of the activation of the Na+,K+ -pump the membrane-potential dependent accumulation of {3H}TPP+ was about 40 per cent reduced in the presence of medium amino acids indicating a decreased membrane potential under these conditions . On the other hand, monensin which induces an electrically silent Na+ -influx via Na+/H+ -exchange was shown to hyperpolarize the membrane on the basis of {3H}TPP+-accumulation . These results suggest that the intensive uptake of neutral amino acids by Na+-cotransport in rapidly growing cells may be responsible for both stimulation of the Na+,K+ -pump and decrease in the transmembrane potential. J Gen Microbiol, 1988 Aug, 134 ( Pt 8), 2111 - 21 Use of carbon sources for lipid biosynthesis in Mycobacterium leprae: a comparison with other pathogenic mycobacteria; Wheeler PR et al.; Carbon from glycerol and palmitate, but not significantly from five other carbon sources tested, was incorporated into lipids by suspensions of non-growing Mycobacterium leprae organisms . However, of the five other substrates three-citrate, glucose and pyruvate-were taken up . Nongrowing Mycobacterium microti and Mycobacterium avium incorporated carbon into lipids from most simple carbon sources tested unless they were obtained from growth media including palmitate or from experimentally infected animals, when incorporation of carbon into lipids from carbon sources except palmitate occurred up to 20 times more slowly . Thus, utilization of simple carbon appeared to be repressible while utilization of the one fatty acid tested, palmitate, appeared constitutive . In M . leprae, carbon from glycerol was incorporated into the glycerol moiety of acylglycerols but not into the fatty acid moieties or into free fatty acids . M . microti and M . avium incorporated carbon from simple carbon sources into fatty acids, even (though very slowly) when these organisms were obtained from host tissue . Isocitrate lyase, malate synthase and acetate kinase were detected in M . leprae . However acetyl-CoA synthetase was not detectable and phosphoacetylase was deficient; thus, M . leprae may be incapable of making acetyl-CoA from acetate . Phosphotransacetylase was readily detected in both host-grown M . avium and M . microti. J Bacteriol, 1988 Aug, 170(8), 3601 - 10 Accumulation of trehalose by Escherichia coli K-12 at high osmotic pressure depends on the presence of amber suppressors; Rod ML et al.; When grown at high osmotic pressure, some strains of Escherichia coli K-12 synthesized substantial levels of free sugar and accumulated proline if it was present in the growth medium . The sugar was identified as trehalose by chemical reactivity, gas-liquid chromatography, and nuclear magnetic resonance spectroscopy . Strains of E . coli K-12 could be divided into two major classes with respect to osmoregulation . Those of class A showed a large increase in trehalose levels with increasing medium osmolarity and also accumulated proline from the medium, whereas those in class B showed no accumulation of trehalose or proline . Most class A strains carried suppressor mutations which arose during their derivation from the wild type, whereas the osmodefective strains of class B were suppressor free . When amber suppressor mutations at the supD, supE, or supF loci were introduced into such sup0 osmodefective strains, they became osmotolerant and gained the ability to accumulate trehalose in response to elevated medium osmolarity . It appears that the original K-12 strain of E . coli carries an amber mutation in a gene affecting osmoregulation . Mutants lacking ADP-glucose synthetase (glgC) accumulated trehalose normally, whereas mutants lacking UDP-glucose synthetase (galU) did not make trehalose and grew poorly in medium of high osmolarity . Trehalose synthesis was repressed by exogenous glycine betaine but not by proline. In Vitro Cell Dev Biol, 1988 Aug, 24(8), 811 - 6 Regulation of glucocorticoid receptors and Na-K ATPase activity by hydrocortisone in proximal tubular epithelial cells; Ellis D et al.; The effect of hydrocortisone (HC) in modulating glucocorticoid receptors (GR) and sodium-potassium adenosine triphosphatase (Na-K ATPase) activity was studied in primary cultures of immunoisolated murine proximal tubular epithelial cells (PTEC) . Utilizing monoclonal antibody against stage-specific embryonic antigen-1, a homogeneous population of PTEC was obtained in high yield . The cells were cultured to confluence and further treated for 48 h in serum-free growth medium containing no HC (control); 50 nM HC; or 50 nM HC plus 20 nM of the antiglucocorticoid, RU 38486 . PTEC treated with 50 nM HC had 56% of GR binding and 160% Na-K ATPase activity as compared to controls (P less than 0.01) . GR binding was abolished by incubation in RU 38486 whereas Na-K ATPase fell below control values (P less than 0.05) . Brief incubations of HC-treated PTEC with 0.5 mM ouabain resulted in a fall in GR binding without a change in Na-K ATPase activity . These data indicate that in PTEC, HC regulates GR binding and they suggest that stimulation of Na-K ATPase activity is a direct biological response to this receptor-hormone interaction . Thus, primary cultures of immunoaffinity-isolated PTEC offer a good model system for investigating the molecular basis underlying the regulation of GR binding and postreceptor events influenced by glucocorticoids. J Med Microbiol, 1988 Aug, 26(4), 301 - 6 Differentiation of phase I and variant strains of Bordetella pertussis on Congo red media; Parton R; The addition of Congo red, Trypan blue or haemin to the growth medium allowed the differentiation of phase-I and variant strains of Bordetella pertussis . Phase-I strains produced red (CR+), blue or dark brown colonies on a modified cyclodextrin solid medium containing Congo red, Trypan blue or haemin, respectively, whereas variant (Vir- and phase IV) strains grew as pale (CR-) colonies . Spontaneous CR- variants were isolated and characterised and had a phenotype like that of Vir- or phenotypically modulated, C-mode strains in that they did not produce the haemolysin, haemagglutinin(s), histamine-sensitising factor (pertussis toxin), heat-labile toxin and two major envelope polypeptides associated with phase-I strains . Two such variants had reduced virulence for mice . CR+ strains, when grown on a high nicotinic acid medium to induce modulation, gave CR- colonies . Thus the CR+ phenotype is a characteristic of phase-I B . pertussis and its expression appears to be controlled in a manner similar to that of other phase I-related factors . CR- variants of B . parapertussis and B . bronchiseptica were also deficient in these factors . Four isolates of B . avium were CR-. J Bacteriol, 1988 Aug, 170(8), 3561 - 6 Regulation of phosphatidate phosphatase activity by inositol in Saccharomyces cerevisiae; Morlock KR et al.; Regulation of phosphatidate phosphatase (EC 3.1.34) activity was examined in Saccharomyces cerevisiae cells supplemented with phospholipid precursors . Addition of inositol to the growth medium of wild-type cells resulted in a twofold increase in phosphatidate phosphatase activity . The increase in phosphatidate phosphatase activity was not due to soluble effector molecules, and inositol did not have a direct effect on enzyme activity . The phosphatidate phosphatase activity associated with the mitochondrial, microsomal, and cytosolic fractions of the cell was regulated by inositol in the same manner . Cells supplemented with inositol had elevated phospholipid levels and reduced triacylglycerol levels compared with unsupplemented cells . Serine, ethanolamine, and choline did not significantly affect the phosphatidate phosphatase activity of cells grown in the absence or presence of inositol . Enzyme activity was not regulated in inositol biosynthesis regulatory mutants, suggesting that regulation by inositol is coupled to regulation of inositol biosynthesis . Phosphatidate phosphatase activity was pleiotropically expressed in structural gene mutants defective in phospholipid biosynthesis . These results suggested that phosphatidate phosphatase was regulated by inositol at a genetic level. Proc Natl Acad Sci U S A, 1988 Aug, 85(15), 5571 - 5 Glucocorticoids facilitate the stable transformation of embryonal rat fibroblasts by a polyomavirus large tumor antigen-deficient mutant; Martens I et al.; The addition of glucocorticoids to the growth medium could substitute for the expression of the polyomavirus large tumor antigen in the transformation of rat fibroblasts in vitro . After transfection with a large tumor antigen-deficient mutant of polyomavirus, pbc1051, high-frequency permanent transformation was observed, if the cells were grown in medium containing dexamethasone . Growth of pbc1051-transfected rat fibroblasts was strictly dependent on the presence of glucocorticoids during the initial phase of transformation . In the second phase, the growth of pbc1051-transfected cells was stimulated by dexamethasone, but the hormone was not essential for growth . After approximately 10 weeks in culture, pbc1051-transfected cells had progressed to hormone independent growth . Rat embryo cells transfected with wild-type polyomavirus DNA had the second phase in which growth was stimulated by glucocorticoid, and after this phase growth was steroid independent . Addition of glucocorticoids to rat fibroblasts transfected with a plasmid encoding only the middle-sized tumor antigen resulted in only a weak stimulation of growth . In contrast, embryo cells transfected with a plasmid containing the human homologue of the cellular T24 Ha-ras gene linked to murine sarcoma virus and simian virus 40 enhancers could be efficiently established as cell lines in medium supplemented with glucocorticoids . The data suggest that, in the transformation of primary rodent cells by polyomavirus, the activity of large tumor antigen can be substituted for by stimulating normal cellular functions with dexamethasone. J Bacteriol, 1988 Aug, 170(8), 3384 - 9 Effect of molybdenum and tungsten on synthesis and composition of formate dehydrogenase in Methanobacterium formicicum; May HD et al.; The influence of sodium molybdate and sodium tungstate on formate dehydrogenase activity was studied in H2-CO2-grown cultures of Methanobacterium formicicum . Depletion of molybdate from the growth medium resulted in a 75-fold decrease of intracellular molybdenum and a 35-fold decrease in enzyme activity; however, growth rate and cell yields were not influenced . By using an indirect enzyme-linked immunoassay, the amount of formate dehydrogenase approximated 3% of the total protein in cells grown in the presence of molybdate . Molybdenum-starved cells contained approximately 15-fold less formate dehydrogenase protein; Western blot (immunoblot) analysis revealed that both subunits of the enzyme were synthesized . Molybdenum starvation resulted in an increase in the amount of mRNA that hybridized to fdh-specific DNA . The results indicated an inverse relationship between the amount of transcript and the amount of formate dehydrogenase protein detected in response to molybdenum starvation . The addition of 1 mM tungstate to molybdate-containing media resulted in nearly complete loss of enzyme activity and decreased the intracellular concentration of molybdenum 10-fold . Cells grown in the presence of tungstate synthesized high amounts of inactive formate dehydrogenase and contained mRNA that hybridized to fdh-specific DNA in amounts similar to that in cells grown with sufficient molybdate . Inactive formate dehydrogenase, purified from cells grown in the presence of tungstate, had the same subunit composition and contained amounts of molybdopterin cofactor, albeit metal-free, comparable to those in the active enzyme. Biochemistry, 1988 Jul 26, 27(15), 5700 - 7 Effects of chronic exposure to ethanol on the physical and functional properties of the plasma membrane of S49 lymphoma cells; Bode DC et al.; The effects of chronic exposure to ethanol on the physical and functional properties of the plasma membrane were examined with cultured S49 lymphoma cells . The beta-adrenergic receptor-coupled adenylate cyclase system was used as a probe of the functional properties of the plasma membrane . Steady-state fluorescence anisotropy of diphenylhexatriene and the lipid composition of the plasma membrane were used as probes of the physical properties of the membrane . Cells were grown under conditions such that the concentration of ethanol in the growth medium remained stable and oxidation of ethanol to acetaldehyde was not detected . Chronic exposure of S49 cells to 50 mM ethanol or growth of cells at elevated temperature resulted in a decrease in adenylate cyclase activity . There were no changes in the density of receptors or in the affinity of beta-adrenergic receptors for agonists or antagonists following chronic exposure to ethanol . The fluorescence anisotropy of diphenylhexatriene was lower in plasma membranes prepared from cells that had been treated with 50 mM ethanol than in membranes prepared from control cells . However, this change was not associated with changes in the fatty acid composition or the cholesterol to phospholipid ratio of the plasma membrane . There was a small but statistically significant decrease in the amount of phosphatidylserine and an increase in the amount of phosphatidylethanolamine . These changes cannot account for the decrease in anisotropy . In contrast to the effect of ethanol, a decrease in adenylate cyclase activity following growth of S49 cells at 40 degrees C was not associated with a change in anisotropy. J Biol Chem, 1988 Jul 25, 263(21), 10229 - 35 Purification and properties of the NusB protein of Escherichia coli; Swindle J et al.; Mutations in the nusB gene of Escherichia coli block transcriptional antitermination mediated by the N gene protein of bacteriophage lambda . We describe here two methods of overproducing the NusB protein in E . coli and a method of purifying NusB to apparent homogeneity on a large scale . Purified NusB directly stimulates transcriptional antitermination by the lambda N protein in vitro . It behaves as a monomer (Mr = 15,689) during gel permeation chromatography and gradient sedimentation . The number of NusB molecules in a wild type E . coli K12 cell ranges from about 3,000 to about 6,000 molecules/cell, depending on the growth medium, and is about 50-80% of the number of molecules of the core component of RNA polymerase in the cell . This implies that NusB has a major role in regulating chain elongation during the transcription of E . coli genes . Many E . coli strains with nusB mutations cannot grow at low temperature . However, a sup+ strain with the suppressible amber mutation nusBam115 can grow at 42 degrees C . Since such a strain does not produce NusB protein detectable by immunoprecipitation with anti-NusB, normal amounts of NusB are not essential for the survival of E . coli at 42 degrees C. Carbohydr Res, 1988 Jul 15, 178, 271 - 92 Blood group Tn-active macromolecules from human carcinomas and erythrocytes: characterization of and specific reactivity with mono- and poly-clonal anti-Tn antibodies induced by various immunogens; Springer GF et al.; In contrast to healthy and noncarcinoma-diseased tissues, greater than 80% of all carcinomas (CAs) tested express immunoreactive O-(2-acetamido-2-deoxy-alpha-D-galacto-pyranosyl)-(1----3)-serine/threon ine {alpha-D-GalpNAc-(1----3)-Ser/Thr} in their glycoproteins . CA cells shed, into the tumor's environment, Tn, which is involved in cancer pathogenesis as adhesion molecule and as autoimmunogen . An increase in density of Tn on primary CA frequently parallels augmented CA aggressiveness . Tn-Active glycoproteins of culture-grown human breast CA DU 4475 cells were isolated from cytoplasm and from spent growth medium, and erythrocyte (RBC) Tn antigen was prepared by (1----3)-beta-D-galactosidase treatment of isolated human O RBC MN glycoprotein-derived Thomsen-Friedenreich (T) antigen . Immunochemical, serological, physical, and chemical analyses showed close resemblance of CA- and RBC-derived Tn antigens . The preponderant carbohydrate in both Tn glycoproteins is the alpha-D-GalpNAc residue, and the antigens have a qualitatively and quantitatively similar amino acid composition . Highly specific rodent monoclonal (Mo) anti-Tn antibodies (Abs) were elicited with Tn RBC and normal O RBC-derived Tn antigen, and compared with CA-anti-Tn MoAbs unwittingly evoked by others . A sensitive enzyme immunoassay (EIA) with Tn antigen as solid phase was developed . In this system, highly purified, "naturally occurring" anti-Tn antibodies, which all humans possess, were more sensitive in quantitating breast CA Tn structures than the anti-Tn MoAbs induced by Tn RBCs, and by RBC- and CA-derived Tn-active antigens . The sensitivity of anti-Tn MoAbs was higher in detecting RBC-Tn. J Biol Chem, 1988 Jul 5, 263(19), 9402 - 8 Insulin-like growth factor-I is an essential regulator of the differentiation of 3T3-L1 adipocytes; Smith PJ et al.; Murine 3T3-L1 preadipocytes proliferate normally in medium containing fetal calf serum depleted of insulin, growth hormone, and insulin-like growth factor-I (IGF-I) . However, the cells do not differentiate into adipocytes in the presence of the hormone-depleted serum . Supplementation of the growth medium with 10-20 nM IGF-I or 2 microM insulin restores the ability of 3T3-L1 cells to develop into adipocytes . The cells acquire an adipocyte morphology, accumulate triglycerides, and express a 450-fold increase in the activity of the lipogenic enzyme glycerol-3-phosphate dehydrogenase . The increase in glycerol-3-phosphate dehydrogenase activity is paralleled by the accumulation of glycerol-3-phosphate dehydrogenase mRNA and mRNA for the myelin P2-like protein aP2, another marker for fat cell development . IGF-I or insulin-stimulated adipogenesis in 3T3-L1 cells is not dependent on growth hormone . Occupancy of preadipocyte IGF-I receptors by IGF-I (or insulin) is implicated as a central step in the differentiation process . The IGF-I receptor binds insulin with a 70-fold lower affinity than IGF-I, and 30-70-fold higher levels of insulin are required to duplicate the effects of an optimal amount of IGF-I . The effects of 10-20 nM IGF-I are likely to be mediated by high affinity (KD = 5 nM) IGF-I receptors that are expressed at a density of 13,000 sites/preadipocyte . In undifferentiated cells the IGF-I receptor concentration is twice that of the insulin receptor . After adipocyte differentiation is triggered, the number and affinity of IGF-I receptors remain constant while insulin receptor number increases approximately 25-fold as developing adipocytes become responsive to insulin at the level of metabolic regulation . Thus, preadipocytes have the potential for a maximal response to IGF-I, whereas the accumulation of more than 95% of adipocyte insulin receptors and the appearance of responsiveness to insulin are consequences of differentiation . IGF-I or insulin is essential for the induction of a variety of abundant and nonabundant mRNAs characteristic of 3T3-L1 adipocytes. J Wildl Dis, 1988 Jul, 24(3), 528 - 32 Experimental Mycoplasma gallisepticum infections in captive-reared wild turkeys; Rocke TE et al.; The effects of Mycoplasma gallisepticum (MG) infections on egg production, fertility, and hatchability were studied in captive-reared wild turkeys (Meleagris gallopavo) . Three groups of adult birds, each consisting of four hens and two toms, were exposed to MG by the respiratory route at the beginning of their breeding season . Fourteen control birds received sterile growth medium . Although no mortality of infected or control birds occurred, egg production during the first breeding season after infection was reduced . The mean number of eggs/hen/day produced by infected groups the first breeding season postexposure (PE) was significantly lower than the control value . The mean number of eggs produced daily by the same hens 1 yr later was unaffected by MG infection . The percentage of fertile eggs produced by infected groups was slightly reduced in both the first and second breeding seasons PE . Hatchability of fertile eggs from infected hens was significantly lower than eggs from control hens . Productivity may be impaired if MG infections occur in free-ranging wild turkey populations. Mutat Res, 1988 Jul-Aug, 200(1-2), 201 - 6 Modulation of DNA precursor pools, DNA synthesis, and ultraviolet sensitivity of a repair-deficient CHO cell line by deoxycytidine; Newman CN et al.; The presence of 2 mM deoxycytidine (CdR) in growth medium substantially increased the deoxycytidine triphosphate (dCTP) and deoxythymidine triphosphate (dTTP) pools in a Chinese hamster ovary cell line, CHO-K1, and in a radiation-sensitive mutant, xrs-5, derived from it (Jeggo et al., 1982) . We observed significant differences in alkaline-sucrose gradient profiles of pulse-labeled DNA from unirradiated CHO-K1 and xrs-5 cells . For the latter cell line, a sizable fraction of the DNA synthesized during 5 or 10 min of growth subsequent to a 5-min radiolabeling period was found to co-sediment with large-chromosome DNA . This characteristic of xrs-5 was dramatically reduced by the presence of 2 mM CdR in the culture medium, and the UV resistance of the mutant increased to nearly that of the parent cell line under these culture conditions . These results show that the locus conferring UV-radiation sensitivity to xrs-5 affects DNA replication and that replicative activity and UV-radiation sensitivity are jointly modulated by CdR, possibly through its impact on the size of deoxynucleoside triphosphate pools. J Gen Virol, 1988 Jul, 69 ( Pt 7), 1735 - 9 Immune serum increases arenavirus replication in monocytes; Lewis RM et al.; The U937 monocytic cell line was used to determine whether antibodies could facilitate infection and replication of the arenaviruses, Pichinde virus (PV) and Lassa fever virus (LFV) . When high dilutions of PV-immune serum were added to cultures simultaneously with PV inoculum, virus replication was dramatically (1000-fold) increased . Low dilutions of this antiserum neutralized the virus . LFV also replicated in U937 cells . The presence of LFV-specific immune serum in the growth medium increased the viral titre as much as 10,000-fold . Addition of heat-aggregated IgG partially inhibited antibody-mediated enhancement, probably by inhibiting the binding of immune complexes to the monocytic cells. J Infect Dis, 1988 Jul, 158(1), 44 - 51 Effect of magnesium on production of toxic-shock-syndrome toxin-1: a collaborative study; Kass EH et al.; We performed a series of collaborative experiments to clarify the effect of Mg++ on production of toxic-shock-syndrome toxin-1 (TSST-1) in various culture media . TSST-1 production was enhanced by adding ethylenediamine-tetraacetic acid (EDTA) at appropriate concentrations to brain-heart infusion and beef-heart medium . The magnitude of this effect depended both on the number of bacteria used to inoculate the media and on the sampling time . Large inocula prepared in media containing high levels of Mg++ introduced sufficient Mg++ to the growth medium to influence subsequent bacterial multiplication and toxin production . Small inocula of bacteria washed in Mg++-deficient medium before inoculation did not, however, multiply or produce toxin in Mg++-deficient medium . Maximal toxin expression occurred during late logarithmic phase, regardless of Mg++ concentration, and Mg++ appeared to control when late logarithmic stage of growth would be achieved . The toxin-enhancing effect of EDTA was reversed by adding excess Mg++ to treated medium. Mol Microbiol, 1988 Jul, 2(4), 527 - 30 The nirB promoter of Escherichia coli: location of nucleotide sequences essential for regulation by oxygen, the FNR protein and nitrite; Jayaraman PS et al.; Using recombinant DNA techniques, nested deletions have been made upstream of the Escherichia coli nirB transcription start site and their effects on the regulation of nirB promoter activity have been measured . Nucleotide sequences downstream of -73 are sufficient for FNR-dependent induction of activity by anaerobic growth conditions . However, nucleotide sequences between -87 and -149 are essential for further induction by nitrite in the growth medium . The nucleotide sequence at the galP1 CRP binding site located from -31 to -52 displays some similarities with the same region at the nirB promoter . When the galP1 sequence from -30 to -54 was replaced by the corresponding nirB sequence, expression from galP1 became inducible by FNR under anaerobic growth conditions. In Vitro Cell Dev Biol, 1988 Jul, 24(7), 683 - 95 Initiation and characterization of primary mouse kidney epithelial cultures; Bell CL et al.; Primary cultures of murine renal epithelial cells were established from a preparation of proximal tubule fragments . Confluent cultures exhibited multiple dome formation, indicating the presence of tight junctions and an intact transcellular transport process . Ultrastructural analysis revealed a monolayer of polarized cells, with a sparse but clearly defined microvillar surface facing the growth medium and a basolateral surface attached to the substratum . Cultures grown on collagen gels did not show domes . The epithelial monolayer exhibited several differentiated functions of the proximal tubule: a) parathyroid hormone (PTH)-stimulated cAMP synthesis; b) production of 24,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3; c) high alkaline phosphatase activity; and d) Na+-dependent transport of phosphate (Pi) and alpha-methylglucoside (alpha-MG) . The sugar uptake was selectively inhibited by phlorizin, a competitive inhibitor of glucose uptake at the luminal membrane . Kinetic analysis revealed independent transport systems for Pi and alpha-MG, with Km values corresponding to the high affinity systems identified in brush border membrane vesicles derived from the proximal tubule . Pi uptake by the epithelial monolayers was regulated by the concentration of Pi in the growth medium . Phorbol esters and PTH did not exert an effect on Pi and alpha-MG transport in mouse primary cultures . The present study demonstrates that primary cultures provide a useful in vitro preparation to investigate renal proximal tubular function. Arch Biochem Biophys, 1988 Jul, 264(1), 288 - 94 Regulation of ornithine decarboxylase from Mycobacterium smegmatis; Balasundaram D et al.; The activity of ornithine decarboxylase in Mycobacterium smegmatis is regulated by a novel macromolecular inhibitor--a ribonucleic acid . Addition of polyamines to the growth medium enhances the level of this inhibitor, suggesting that the level of this negative modulator changes in response to the intracellular concentration of polyamines . Thus, while other modes of regulation may be operational, the control by polyamines at the transcriptional level leading to the generation of a specific RNA inhibitor seems to be a key element in the regulation of ornithine decarboxylase in mycobacteria. Biochim Biophys Acta, 1988 Jun 22, 941(2), 257 - 63 External ATP-induced passive permeability change and cell lysis of cultured transformed cells: action in serum-containing growth media; Kitagawa T et al.; External ATP causes a marked increase in the passive permeability to phosphorylated metabolites in several types of transformed cells in alkaline medium containing low concentrations of Ca2+, but not in untransformed cells . Such increased membrane permeability with external ATP was also observed in B16 melanoma cells at pH 7.4-7.5 in both Tris-buffered saline and a growth medium containing 10% calf serum and divalent ions at normal concentrations, although a higher concentration of ATP was required . The permeability change in the growth medium was significantly enhanced by calmodulin-interacting drugs, such as trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) and chlorpromazine (CPZ) . As expected, prolonged exposure of the cells to ATP in the serum-containing medium led to cell lysis . This ATP-dependent cell lysis was observed only in several transformed cell lines, and not in untransformed mouse fibroblasts . These results indicate that the effect of ATP on the membrane permeability in transformed cells is elicited under the physiological conditions and this would be useful in some limited way for cancer chemotherapy management. Biochem Pharmacol, 1988 Jun 15, 37(12), 2389 - 93 Increase of daunorubicin and vincristine accumulation in multidrug resistant human ovarian carcinoma cells by a monoclonal antibody reacting with P-glycoprotein; Broxterman HJ et al.; An overexpression of plasma membrane 170-180 kDa P-glycoproteins is consistently found in multidrug-resistant (MDR) cell lines . In this study MRK-16, a monoclonal antibody (mAb) reacting with P-glycoprotein is used to study the putative functional role of this protein in vincristine (VCR) and daunorubicin (DNR) cellular accumulation in the MDR human ovarian carcinoma cell line 2780AD . We established that this cell line is highly cross-resistant to vincristine and daunomycin, related to a greatly reduced drug accumulation . Verapamil (Vp) (8 microM) caused a 3.6-fold increase in DNR as well as VCR accumulation . Exposition of 2780AD cells to MRK-16 led to an increase of 30% in cellular accumulation of VCR, both in normal growth medium as well as in medium without added glucose and with sodium azide, which largely depleted cellular ATP levels . No increase in DNR accumulation was found under these conditions . However, in the presence of 8 microM Vp, MRK-16 increased not only VCR but also DNR accumulation with about 30% . The relative increase of DNR accumulation was constant in a concentration range of 0.2-4 microM DNR . These data indicate that mAbs against P-glycoprotein might potentiate the action of calcium antagonists like Vp to increase cellular anthracycline accumulation. Appl Environ Microbiol, 1988 Jun, 54(6), 1504 - 10 Growth of and fumitremorgin production by Neosartorya fischeri as affected by temperature, light, and water activity; Nielsen PV et al.; The effects of temperature, light, and water activity (aw) on the growth and fumitremorgin production of a heat-resistant mold, Neosartorya fischeri, cultured on Czapek Yeast Autolysate agar (CYA) were studied for incubation periods of up to 74 days . Colonies were examined visually, and extracts of mycelia and CYA on which the mold was cultured were analyzed for mycotoxin content by high-performance liquid chromatography . Growth always resulted in the production of the tremorgenic mycotoxins verruculogen and fumitremorgins A and C . The optimum temperatures for the production of verruculogen and fumitremorgins A and C on CYA at pH 7.0 were 25, 30, and 37 degrees C, respectively . The production of fumitremorgin C by N . fischeri has not been previously reported . Fumitremorgin production was retarded at 15 degrees C, but an extension of the incubation period resulted in concentrations approaching those observed at 25 degrees C . Light clearly enhanced fumitremorgin production on CYA (pH 7.0, 25 degrees C), but not as dramatically as did the addition of glucose, fructose, or sucrose to CYA growth medium (pH 3.5, 25 degrees C) . Growth and fumitremorgin production was greatest at aw of 0.980 on CYA supplemented with glucose or fructose and at aw of 0.990 on CYA supplemented with sucrose . Growth and fumitremorgin production were observed at aw as low as 0.925 on glucose-supplemented CYA but not at aw lower than 0.970 on CYA supplemented with sucrose . Verruculogen was produced in the highest amount on all test media, followed by fumitremorgin A and fumitremorgin C. Br J Haematol, 1988 Jun, 69(2), 197 - 203 A simple method for culturing myeloma cells from human bone marrow aspirates and peripheral blood in vitro; Millar BC et al.; A double layer agar technique has been developed to grow myeloma colonies (MY-CFUc) from human bone marrow aspirates and peripheral blood . Heavily irradiated HL60 cells (5 x 10(5)/plate) are added to an agar underlay in growth medium containing 0.5% agar . Mononuclear cells from the test bone marrow or blood are overlayered in either 0.2 ml HL60-conditioned medium (HL60-CM) or in 0.5 ml growth medium containing 0.23% agar, and the cultures are incubated at 37 degrees C in an atmosphere of 5% CO2, 10% O2 and 85% N2 . Colonies (greater than 50 cells) form between 2 and 3 weeks . Using this method 60/68 samples of bone marrow and 7/12 samples of blood from 54 patients have produced colonies in soft agar and in liquid on an agar underlay . The cells which form these colonies are of two distinct sizes, the larger cells being plasmacytoid and the smaller lymphoid . The two cell types are usually, but not always, present in separate colonies . Both plasmacytoid and lymphoid cells carry the isotype of the respective patient's myeloma protein and the plasma cell marker (HAN PC1) . This technique has enabled us to culture myeloma cells from patients with as few as 2% plasma cells in the bone marrow but it does not permit the growth of normal B, T or granulocyte-macrophage colonies (GM-CFUc) . The drug sensitivity of myeloma cells (MY-CFUc) compared with normal haemopoietic cells (GM-CFUc) can be measured using dose-response curves in individual patients . Furthermore, this method can detect resistant subpopulations within a given myeloma sample. Am J Physiol, 1988 Jun, 254(6 Pt 1), G829 - 36 Primary culture of PYY cells from canine colon; Aponte GW et al.; In the present study we report methods for the isolation and culture of colonic peptide YY (PYY) cells and have tested the effects of sodium oleate and other chemotransmitters on PYY release . Enzyme-dispersed canine colonic mucosa cells were separated by a Beckman elutriation rotor, and the enriched PYY fraction was cultured on type I collagen in full growth medium . After 42-48 h of culture PYY cells had selectively adhered to the collagen . Forty to 45% of the adherent cells contained PYY (5.5 +/- 0.5 pmol/24-mm well), and 10-15% of the cells contained glucagon-like immunoactivity (GL29-LI; 0.95 pmol/24-mm well) . The effect of various secretagogues on PYY release from these cultures was monitored . Sodium oleate stimulated PYY release in a time-dependent fashion over a dose range from 10 microM to 10 mM . However, sodium oleate at a dose of 100 mM produced disproportionately large PYY release . During a 2-h incubation 5.1% of the total cell content of PYY was released in response to 0.1 mM sodium oleate compared with basal release of 1.1% . At doses less than 10 mM sodium oleate did not release GL29-LI, whereas concentrations of 10 and 100 mM resulted in marked GL29-LI release . These findings suggest that lower concentrations of sodium oleate selectively release PYY, whereas higher concentrations nonselectively induce peptide release probably by effects on membrane stability . We also found that bombesin, epinephrine, and forskolin, but not carbachol, produced time- and dose-dependent release of PYY from these cultures. Radiat Res, 1988 Jun, 114(3), 415 - 24 Radiation response characteristics of human cells in vitro; Hall EJ et al.; Improvements in tissue culture techniques and growth media have made it possible to culture a range of cells of human origin, both normal and malignant . The most recent addition to the list are endothelial cells from umbilical cord veins . Interesting results in radiosensitivity studies of these human cells have been obtained, some of which may have implications in radiation therapy . (i) Repair of potentially lethal damage (PLDR) has been observed in all cell lines investigated; cells of normal origin repair PLD at least as well as malignant cells, which makes clinical trials of PLDR inhibitors of doubtful usefulness . (ii) No apparent correlation can be made between the extent of PLDR and the traditional radioresponsiveness of a particular tumor type . Indeed, if anything, it could appear to have an inverse correlation since the most resistant tumor cells show the smallest amount of PLD repair . (iii) Dose-rate effects appear to be better predictors of radiosensitivity than PLDR capacity . (iv) Sublethal damage repair, manifest by a dose-rate effect, has also been observed in all human cell lines tested . Cells of normal tissue origin, including fibroblasts and endothelial cells, exhibit a dose-rate effect that is intermediate between that for cells from traditionally resistant tumors (melanoma and osteosarcoma) and cells from more sensitive tumors (neuroblastoma and breast). Bone Miner, 1988 Jun, 4(2), 157 - 65 Establishment of primary cell cultures from human and canine parathyroid gland explants; Hornicek FJ et al.; Primary parathyroid cell cultures were established from 22 canine and 20 human parathyroid glands explanted into Connaught Medical Research Laboratory-1415 (CMRL-1415) medium containing fetal calf serum (FCS) . Initiation of cellular outgrowth and the rate of cellular propagation through the first subcultures were evaluated in various media . These included Roswell Park Memorial Institute-1640 (RPMI-1640), minimal essential medium (MEM), medium-199 (M-199) and Coon's modified Ham's F-12 (F12) and CMRL-1415 media . Only CMRL-1415 medium supplemented with FCS invariably supported the vigorous rate of cellular growth as determined by the number of cells in cultures, their viability, parathyroid hormone (PTH) levels in the growth media and the number of proliferating cells . Propagation of parathyroid cells in vitro was limited to five passages . Cessation of cell division was always coincidental with the emergence of numerous, non-dividing binucleated cells . These experiments demonstrate that primary parathyroid cell cultures from canine and human parathyroid gland explants can be readily established in CMRL-1415 medium containing FCS. Lipids, 1988 Jun, 23(6), 615 - 8 Modification of the fatty acid composition of L1210 leukemia subcellular organelles; Burns CP et al.; We have examined the extent to which it is possible to modify the fatty acid composition of subcellular organelles of L1210 leukemia cells . A polyunsaturated fatty acid, docosahexaenoic acid, or a monounsaturated fatty acid, oleic acid, were added to the culture media . After 48 hr, the cells were ruptured and the subcellular fractions isolated . Fatty acid analysis revealed that nuclei, mitochondria, plasma membranes and microsomes of the cells grown in media supplemented with docosahexaenoic acid contained increased amounts of polyenoic fatty acids compared with cells grown in oleic acid . We conclude that it is possible to experimentally modify the lipids of multiple intracellular structures of L1210 cells by the addition of fatty acids to the growth media. J Bacteriol, 1988 Jun, 170(6), 2692 - 7 Characteristics of Ureaplasma urealyticum urease; Blanchard A et al.; Sonication of Ureaplasma urealyticum cells grown in a dialysate growth medium effectively separated the cytoplasmic fraction from the membrane fraction, with both fractions relatively free from exogenous contaminating proteins . The urease activity was associated with the cytoplasmic fraction, and the ureaplasmal urease exhibited a specific activity higher than that of crystalline jack bean urease . The enzymatic activity of the ureaplasmal enzyme was optimum at pH 7.5 and was resistant to the chelating agents EDTA and sodium citrate . Sulfhydryl-blocking agents such as HgCl2 and Pb(NO3)2 inhibited the ureaplasmal urease, which was also shown to be particularly sensitive to flurofamide and, to a much lesser extent, to acetohydroxamic acid . Electrophoretic analysis of the proteins of the ureaplasmal cell fractions combined with Western immunoblot with an antiserum to the ureaplasmal urease indicated that the urease constitutes a major component of the cytoplasm and is composed of several 70-kilodalton polypeptides. In Vitro Cell Dev Biol, 1988 Jun, 24(6), 593 - 600 Ultrastructural and immunohistochemical characterization of submandibular duct cells in culture and modification of outgrowth differentiation by manipulation of calcium ion concentration; Kurth BE et al.; The recognized need for epithelial cell culture models for cystic fibrosis (CF) research has resulted in ongoing efforts to improve normal and CF submandibular duct cell culture capabilities . The duct is most likely the site of the CF defect in this and other exocrine glands . In a previous report conditions required for the successful primary explant culture of normal and CF submandibular glands were outlined; however, terminal keratinization and involution of these cultures were recognized as severe limiting factors to their utilization in CF research . This report explores the effects of calcium concentrations in the medium, growth factor supplements, and matrix components on growth and differentiation of these cultures . Results of the study further confirm the ductal origin of cells in the outgrowth and demonstrate that progressive keratinization is initiated only after cells proliferate beyond the environment of the explant fragment . Keratinization with subsequent multilayering, desmosome formation, and involution in the cell outgrowth are governed in degree by the calcium concentration of the growth medium . Upon reduction of medium calcium to 0.1 mM concentration, the cells proliferate as a monolayer and subculture through 8 to 9 passages and retain the capacity to undergo ductlike differentiation. Gene, 1988 May 30, 65(2), 269 - 75 Transcription of the Rhodobacter capsulatus nifHDK operon is modulated by the nitrogen source . Construction of plasmid expression vectors based on the nifHDK promoter; Pollock D et al.; We characterized the Rhodobacter capsulatus nifHDK promoter by nucleotide sequencing and nuclease S1 analysis of mRNA-protected DNA probes . Comparison of this promoter to nifP and ntrP promoters from other species reveals extensive homology to the canonical nifP consensus sequence . Using lac fusions we have demonstrated that transcription of the nifHDK operon is totally repressed when the growth medium is supplemented with ammonia, becomes fully derepressed in ammonia-free medium, and proceeds at intermediate levels when other nitrogen sources are used . Based on this information, we constructed plasmid expression vectors in which the rates of transcription from cloned DNA fragments are determined by the nitrogen source used in the growth medium. FEBS Lett, 1988 May 23, 232(2), 354 - 8 Effect of 7 beta-hydroxycholesterol on growth and membrane composition of Mycoplasma capricolum; Lelong I et al.; 7 beta-OH cholesterol in a cholesterol rich growth medium (5-10 micrograms/ml) extended the lag period and slowed down the growth rate of Mycoplasma capricolum cells . In a cholesterol poor medium (0.5 micrograms/ml) inadequate to support growth, 7 beta-OH cholesterol exerts a synergistic effect on growth . The 7 beta-OH cholesterol was incorporated unchanged from the growth medium and could be recovered exclusively in the membrane fraction . The incorporation of the 7 beta-OH cholesterol has no effect on the total phospholipid content but the DPG to PG ratio was markedly decreased . Exchange studies with lipid vesicles revealed that whereas most of the cholesterol underwent exchange, only about 20% of the 7 beta-OH cholesterol was exchanged. Cancer Res, 1988 May 15, 48(10), 2898 - 903 Cellular proliferation and ras oncogene of p21 21,000 expression in relation to the intracellular cyclic adenosine 3':5'-monophosphate levels of a human salivary gland adenocarcinoma cell line in culture; Azuma M et al.; We have found that reversible differentiation into the myoepithelial cells of a human salivary gland adenocarcinoma cell line (HSG) occurs in growth medium containing dibutyryl cAMP (dB-cAMP) . In the current study, the relationship between intracellular cAMP levels and anchorage-dependent and -independent growth or ras oncogene of p21 levels was analyzed in the differentiation process toward myoepithelial cells of HSG cells cultured in the presence of dB-cAMP . Correlation between the concentrations of dB-cAMP and intracellular cAMP in the HSG cells was statistically significant . There was a significant inverse correlation between the concentrations of dB-cAMP and colony-forming ability of the cells in semisolid agar or on a plastic surface . We have found the expression of ras p21 protein in HSG cells . When HSG cells were cultured in the presence of dB-cAMP and were committed to differentiate into myoepithelial cells, it was shown by double-antibody labeling technique and/or immunoblotting that the committed cells expressed myosin with a concomitant decrease of ras p21 protein . Moreover, intracellular cAMP levels were found to be inversely associated with ras p21 content of the cells . These findings indicate that the intracellular cAMP levels regulate significantly cell proliferation and ras p21 expression in HSG cells. Curr Eye Res, 1988 May, 7(5), 465 - 70 Effects of vitreous from photocoagulated eyes on retinal microvascular cells in culture: a preliminary report; Boulton M et al.; Panretinal photocoagulation was carried out in mini pigs in an attempt to elucidate the mechanisms involved in laser therapy . The effect of the vitreous from these eyes on the proliferation of retinal microvascular endothelial cells was then studied . Vitreous removed 4 days after panretinal photocoagulation had no effect on the proliferation of bovine retinal microvascular endothelial cells in basal medium but inhibited proliferation in growth medium . Control vitreous was mitogenic for the microvascular cells in basal medium but this effect was not observed in growth medium. Int J Hyperthermia, 1988 May-Jun, 4(3), 333 - 44 Uncoupling of oxidative phosphorylation does not induce thermotolerance in cultured Chinese hamster cells; Rastogi D et al.; Two uncouplers of oxidative phosphorylation, 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), were tested for their ability to modify the survival of cultured Chinese hamster ovary (CHO) and Chinese hamster V79 cells treated with hyperthermia . The uncouplers were used under conditions that inhibit oxidative ATP synthesis, as judged from measurements of cellular ATP levels . Incubation of CHO cells in glucose-free Hanks' balanced salt solution (HBSS) containing 1 mM DNP for 1 h at 37 degrees C followed by reincubation at 37 degrees C in complete growth medium for 3 or 16 h, showed no substantial changes in the 45 degrees C heat survival curve as compared to heated cells not exposed to DNP . Thus, DNP treatment of CHO cells did not induce thermotolerance . Carbonyl cyanide m-chlorophenylhydrazone (CCCP), tested under similar experimental conditions, did alter cellular heat resistance . The major change in the 45 degrees C survival curve of CHO cells pretreated with CCCP was an increase in the width of the shoulder: the Dq value increased from 14 min to 24 min, for the control and CCCP-treated cells respectively . The D0 value did not change appreciably . In contrast, heat-induced thermotolerance (10 min, 45 degrees C + 16 h, 37 degrees C) was characterized primarily by an increase in the D0 parameter from 4 min (unheated cells) to 17 min . Similar results were observed with CCCP-treated V79 cells . The data demonstrate that heat resistance induced by 1.2 microM CCCP was manifest as an increased cellular capacity to accumulate and/or repair hyperthermia damage, rather than an induction of thermotolerance, and that this effect probably was not related to the action of CCCP as an uncoupler of oxidative phosphorylation. J Steroid Biochem, 1988 May, 29(5), 457 - 63 Oncogenes modulate cellular gene expression and repress glucocorticoid regulated gene transcription; Jaggi R et al.; The v-mos oncogene was subjected to the transcriptional control of the MMTV LTR and introduced by transfection into NIH 3T3 cells . The LTR v-mos gene was induced by the addition of glucocorticoid hormone to the growth medium of cells synchronized by culturing in 1.5% FCS for 36 h . The effects of p37 v-mos expression were monitored . The endogenous c-myc gene is induced as a consequence of p37 v-mos expression in a transient fashion, reaching a maximum of expression after 8 h . Induction of the c-myc gene was observed at the level of its transcriptional rate and at the level of mRNA concentration . Ornithine decarboxylase (ODC) mRNA was induced constitutively and high levels were found 8 and 25 h after v-mos induction . H4 histone mRNA is elevated at 25 h after hormone addition at a time when the mitogenic stimulus of v-mos causes DNA synthesis . The expression of actin mRNA is not affected by the v-mos oncogene . We have previously described a modulation of glucocorticoid dependent gene expression by oncogenes . In an extension of these observations the consequences of expression of the v-mos and the v-ras oncogenes were also studied in retrovirally infected NIH 3T3 cells . MMTV LTR constructs transfected into the infected cells could only be transiently induced by glucocorticoid hormone . The presence of the p37 v-mos and the p21 v-ras oncoproteins causes a repression of glucocorticoid hormone dependent gene transcription. Exp Cell Res, 1988 May, 176(1), 162 - 73 Variation in the relative synthesis of some proteins in mammalian cells exposed to hypertonic medium; Battistini A et al.; Exposure of a number of quiescent murine and human cell lines to low-graded doses of cycloheximide (CXM) results in a pattern of protein synthesis consisting of enhanced and induced species . This pattern is reminiscent of but not identical to that observed after several stress treatments {V . Sorrentino et al . (1985) J . Cell . Physiol . 125, 313} . A pattern identical to that seen after exposure to CXM is synthesized when cells are exposed to an hypertonic growth medium resulting in a full and reversible block of the initiation of polypeptide chain . This suggests that this kind of response is triggered by a reduction of overall protein synthesis rather than by a slow-down of the elongation step . Analysis of the synthesis of histones and ribosomal proteins during these two nonphysiological treatments (CXM or high salt) shows that these classes of proteins are neither stimulated nor preferentially retained . In contrast, greatly enhanced levels of steady-state histone mRNAs have been observed which have been translated in a reticulocyte lysate system, but are not apparently translated in vivo. Int J Radiat Oncol Biol Phys, 1988 May, 14(5), 947 - 55 Lactate-induced inhibition of tumor cell proliferation; Marx E et al.; Culture medium that was recovered from tumor cell or fibroblast cultures during the plateau phase, and that was replenished by addition of glucose, glutamine, and serum and readjustment of pH had a distinct growth-inhibiting effect on monolayer cell cultures . The effect, which was not specific for a given cell strain, may be partially responsible for the "density inhibition" commonly observed in malignant cells grown in monolayer cultures . By modifying fresh growth media, it was shown that the growth inhibition observed can be partly attributed to the accumulation of lactate in the culture medium of plateau phase cells . This substance reduced the plating efficiency and the number of cells per petri dish in the plateau phase . It is concluded that this effect may be used for inducing growth inhibition in tumors in vivo by manipulating the cellular production of lactate and/or by impeding its removal from the cellular microenvironment. Int J Hyperthermia, 1988 May-Jun, 4(3), 323 - 31 Protection against heat-induced cell killing by alanine; Henle KJ et al.; When L-alanine was added either to full growth medium or to Hanks' balanced salt solution (HBSS) prior to hyperthermia, survival of heated cells was significantly increased in a concentration-dependent manner . Maximal heat protection was not immediate, but required at least 1 h at 37 degrees C incubation prior to heating . Heat protection was principally reflected in an increased Dq on the 45 degrees C survival curve; for example, with 100 mM L-alanine, the Dq increased from approximately equal to 20 (control) to 30 min at 45 degrees C . Hyperthermia of 1 h at temperatures between 42 degrees C and 45 degrees C indicated that 100 mM alanine had shifted the isotoxic temperature by 0.5 degrees C . Comparable heat protection was also observed with D-alanine and amino acid dimers, such as alanyl-alanine or alanyl-leucine . Leucine at similar concentrations by itself, without alanine, did not protect cells against heat killing, but increased cellular heat sensitivity . The data suggest that heat protection by alanine does not require incorporation of alanine into cellular protein, but is mediated by the free amino acid. J Bacteriol, 1988 May, 170(5), 2031 - 9 Incorporation of LL-diaminopimelic acid into peptidoglycan of Escherichia coli mutants lacking diaminopimelate epimerase encoded by dapF; Mengin-Lecreulx D et al.; Recently a dapF mutant of Escherichia coli lacking the diaminopimelate epimerase was found to have an unusual large LL-diaminopimelic acid (LL-DAP) pool as compared with that of meso-DAP (C . Richaud, W . Higgins, D . Mengin-Lecreulx, and P . Stragier, J . Bacteriol . 169:1454-1459, 1987) . In this report, the consequences of high cellular LL-DAP/meso-DAP ratios on the structure and metabolism of peptidoglycan were investigated . For this purpose new efficient high-pressure liquid chromatography techniques for the separation of the DAP isomers were developed . Sacculi from dapF mutants contained a high proportion of LL-DAP that varied greatly with growth conditions . The same was observed with the two DAP-containing precursors, UDP-N-acetylmuramyl-tripeptide and UDP-N-acetylmuramyl-pentapeptide . The limiting steps for the incorporation of LL-DAP into peptidoglycan were found to be its addition to UDP-N-acetylmuramyl-L-alanyl-D-glutamate and the formation of the D-alanyl-DAP cross-bridges . The Km value of the DAP-adding enzyme for LL-DAP was 3.6 x 10(-2) M as compared with 1.1 x 10(-5) M for meso-DAP . When isolated sacculi were treated with Chalaropsis N-acetylmuramidase and the resulting soluble products were analyzed by high-pressure liquid chromatography, the proportion of the main peptidoglycan dimer was lower in the dapF mutant than in the parental strain . Moreover, the proportion of LL-DAP was higher in the main monomer than in the main dimer, where it was almost exclusively located in the donor unit . There are thus very few D-alanyl-LL-DAP cross-bridges, if any . We also observed that large amounts of LL-DAP and N-succinyl-LL-DAP were excreted in the growth medium by the dapF mutant. Acta Physiol Scand, 1988 May, 133(1), 41 - 7 Human granulopoiesis in vitro--medium-dependent growth regulation by a granulocyte-derived inhibitor; Helgestad J et al.; A number of reports have indicated that mature blood granulocytes produce regulators that inhibit proliferation of progenitor cells in the bone marrow . However, this concept of negative feedback of granulopoiesis is still controversial . To examine whether conflicting results may depend upon the experimental set-up, we have compared colony formation by human bone marrow cells in different growth media . Unmodified McCoy's medium, which in feeder layer cultures supports the formation of large numbers of colonies, was a poor growth medium in cultures supplied with crude or recombinant colony stimulating factor (CSF) . The colony formation improved when the medium was supplemented with defined additives . In CMRL 1066 cultures, granulocyte extract (GRE) consistently caused a strong inhibition of colony formation . In contrast, with unmodified McCoy's medium, granulocyte extract enhanced colony formation in a dose-dependent manner . The enhancing effect of granulocyte extract coincided with low colony numbers in the control cultures . The stimulatory effect of granulocyte extract in McCoy's medium, switched to strong inhibition when thymidine, a component of CMRL 1066 medium, was added . The inhibitory and stimulatory activities were found in the same molecular weight fractions (30-60 kD) after gel filtration . Both modulators in granulocyte extract appeared to be independent of monocytes and T lymphocytes in the bone marrow, as shown by removal of these cells with magnetic microspheres coated with specific monoclonal antibodies . The present work shows that regulation of cell proliferation in vitro depends strongly on culture conditions, such as choice of medium . It appears that thymidine acts as co-factor for the inhibitor in granulocyte extract. J Clin Microbiol, 1988 May, 26(5), 965 - 70 Biochemical and ultrastructural study of Blastocystis hominis; Zierdt CH et al.; This study was prompted by the paradox of strong presence of mitochondria in an anaerobic protozoan, recently reclassified from the yeasts . Stemming from publication in 1911 to 1912, Blastocystis hominis has been generally accepted as a harmless intestinal yeast of humans, with short standardized textbook (parasitology) descriptions, even to the present day . Reports since 1967 have changed the classification of B . hominis from yeast to protozoan (Sarcodina), and this has been followed by interest in B . hominis-caused disease, resulting in documentation of disease in humans and other primates . In this study of B . hominis, the basic ultrastructure of the mitochondria was shown by thin-section electron microscopy to be identical to that of an archetypical mitochondrion . There were hundreds of them in large B . hominis cells (100 to 200 microns in diameter) . Mitochondria were confined to a peripheral ring of cytoplasm bounded by the outer cell membrane (there is no cell wall) and the membrane of the large, spherical, organelle-free central body that constitutes 75% of the cell's volume . Mitochondria tended to surround the cell's usual two to four nuclei . Rhodamine 123 stained the mitochondria selectively, visualized by fluorescence microscopy . The cell was devoid of cytochromes . Addition of 0.1% cytochrome c to the growth medium increased utilization of glucose by 34% and that of lactate by 17% . Furthermore, it markedly increased the number of mitochondrion-filled cells . At higher concentrations, cytochrome c inhibited the growth of the cells . Despite the presence of large numbers of mitochondria, activities of the mitochondrial enzymes pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, isocitrate dehydrogenase, glutamate dehydrogenase, and cytochrome c oxidase were absent . Thus, the function of the mitochondria in B . hominis remains unknown . Considerable activities of aspartate aminotransferase and alanine aminotransferase were found . Aldolase activity was prominent . Pyruvate decarboxylase was present . Diaphorase and lactate dehydrogenase were detectable but in suspect quantities . Other missing enzymes were gamma glutamyl transpeptidase, alkaline phosphatase (a lysosomal marker), and creatine kinase isoenzymes. J Cell Physiol, 1988 May, 135(2), 224 - 34 Prostacyclin and prostaglandin E2 secretions by bovine pulmonary microvessel endothelial cells are altered by changes in culture conditions; Chung-Welch N et al.; The isolation and culture of pulmonary microvascular endothelial (MVE) cells from bovine lungs were established . Primary and early passaged cultures grew best in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% equine plasma-derived serum, bovine retinal growth extract (1%), and heparin (90 micrograms/ml) on gelatin coated plates . A second tissue culture procedure was prepared in which the isolation technique was the same except the culture medium consisted of DMEM supplemented with 10% plasma-derived serum . Either growth medium produced homogeneous, long term, serial cultures for up to 16 passages . MVE cells were characterized in part based on their morphology by light and electron microscopy and positive reaction to Factor VIII-related antigen and uptake of 1,1'-dioctacecyl-1,3,3,3'3-tetramethyl-indocarbocyanine perchlorate acetylated low density lipoprotein (Dil-Ac-LDL) . MVE cells were also positive for angiotensin-converting enzyme (ACE) activity and the presence of ACE was localized on the cells by indirect immunofluorescence . MVE cells maintained in the presence of heparin and growth factor principally synthesized prostaglandin (PG) E2 (1512 +/- 159 pg/mg protein at 15 min) and smaller amounts of prostacyclin (PGI2) and thromboxane (Tx) A2 (316 +/- 43 and 588 +/- 105 pg/mg protein/15 min respectively) as measured by radioimmunoassay . However, prostanoid release was not elevated from basal levels upon incubation with arachidonic acid, bradykinin, or ionophore A23187 . In contrast, MVE cells cultured without heparin and growth factor secreted more PGI2 than PGE2 (862 +/- 84 and 89 +/- 12 respectively) . Incubation with arachidonic acid, bradykinin, or ionophore A23187 induced significant increases in PGI2 and PGE2 production (P less than 0.01) . Pulmonary artery endothelial (PAE) cell cultures used as a control for comparison predominantly synthesized PGI2 . These findings suggest that in vitro the vessel source and culture conditions may qualitatively and quantitatively affect the pattern and levels of prostanoid synthesized and secreted. Mikrobiologiia, 1988 May-Jun, 57(3), 494 - 8 {Mechanisms of the protective action of cryoprotectors on Escherichia coli cells}; Smirnova LF et al.; Two classes of cryoprotectors, namely, glycerol (penetrating the cell) and dextran with a mol . mass of 15-20,000 Da (non-penetrating the cell), were tested for their ability to exert a protective effect on the permeability and viability of Escherichia coli M 17 cells subjected to different freezing conditions . Cell permeability assayed in terms of the release of low-molecular-weight compounds into the surrounding medium was shown to be disordered to a far less extent when dextran was used as a cryoprotective agent than in the case of glycerol . The cells were found to be resistant to lysis stimulated by the detergent sodium lauryl sulfate (0.02%) in the presence of dextran . The cells were still capable of forming colonies after their freezing to -10 degrees C in the presence of the cryoprotectors in media containing the detergent (2%) . Once the cells had been frozen to -196 degrees C, only those protected by glycerol were resistant to the detergent in the growth medium . Different mechanisms of the cryoprotective action on E . coli cells are discussed. J Protozool, 1988 May, 35(2), 291 - 5 Secretory hydrolases of Entamoeba histolytica; Muller FW et al.; Cells of Entamoeba histolytica grown over a period of four days contained NADP+-dependent alcohol dehydrogenase exclusively inside the cells . No activity of this enzyme could be found in the growth medium after harvesting the cells . Under the same conditions, acid phosphatase, beta-N-acetylglucosaminidase, esterase, alpha-glucosidase, and different amylases of the parasite were found both inside the cells and in the medium . The activities present in the cell homogenate and in the medium before and after growth of the amoebas were partially separated by gel filtration on Sephadex G150 and G75, respectively . The comparison of the elution diagrams revealed that NADP+-dependent alcohol dehydrogenase, acid phosphatase, esterase, and amylases occurred as multiple forms inside the cells . These activities, as well as beta-N-acetylglucosaminidase and alpha-glucosidase, were released into the extracellular environment to a different degree . The enzymes originating from the parasite were identified and distinguished from those of the ingredients of the growth medium according to their molecular mass and pH optimum . Furthermore, the amoebic origin of the secreted enzymes was shown on the basis of their inhibition by antibodies prepared against the supernatant fraction of the homogenate. J Biol Chem, 1988 Apr 25, 263(12), 5775 - 9 Rapid changes in polyphosphoinositide metabolism associated with the response of Dunaliella salina to hypoosmotic shock; Einspahr KJ et al.; The inositol phospholipids phosphatidylinositol, phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2) comprise 14.8, 1.2, and 0.3 mol %, respectively, of Dunaliella salina phospholipids . In isolated plasma membrane fractions, PIP and PIP2 are highly concentrated, together comprising 9.5 mol % of plasmalemma phospholipids . The metabolism of these inositol phospholipids and phosphatidic acid (PA) is very rapid under normal growth conditions . Within 5 min after introduction of 32Pi into the growth medium, over 75% of lipid-bound label was found in these quantitatively minor phospholipids . Within 2 min after a sudden hypoosmotic shock, the levels of PIP2 and PIP dropped to 65 and 79%, respectively, of controls . Within the same time frame, PA rose to 141% of control values . These data suggest that a rapid breakdown of the polyphosphoinositides may mediate the profound morphological and physiological changes which allow this organism to survive drastic hypoosmotic stress . In contrast to hypoosmotic shock, hyperosmotic shock induced a rise in PIP2 levels to 131% of control values, whereas the level of PA dropped to 56% of controls after 4 min . These two different types of osmotic stress affect inositol phospholipid metabolism in a fundamentally opposite manner, with only hypoosmotic shock inducing a net decrease in polyphosphoinositides. Mol Biochem Parasitol, 1988 Apr, 28(3), 189 - 95 Purification and characterization of the Giardia lamblia double-stranded RNA virus; Miller RL et al.; The dsRNA virus which infects some strains of Giardia lamblia has been purified and characterized with respect to its effect on growth of the parasite . Extensive purification of the virus from G . lamblia growth medium was accomplished by Millipore filtration and two successive CsCl gradient centrifugations . The purified virus possessed a single major protein species of 100,000 molecular weight . Effects of the extensively purified virus on growth of the virus-free parasite were studied . A cloned WB strain, sensitive to the viral infection, and a cloned E-9/M strain, resistant to the infection, were studied . With the WB strain, infection can occur at a ratio as low as 10 viral particles per organism . As the virus to parasite ratio increased, the rate of growth of the parasite decreased and the percentage of parasites not adhering to the culture tube wall also increased . These nonadhering cells, which differed from the nonadhering cells under normal growth conditions, were unable to divide . They contained an average number of 500,000 viral particles per cell which may be the threshold intracellular density of viral particles arresting the growth of G . lamblia . The results also suggest that the specific consequence of viral infection, even at extremely high multiplicity of infection, is not lysis of G . lamblia trophozoites but cessation of growth. In Vitro Cell Dev Biol, 1988 Apr, 24(4), 289 - 93 A cloned rat thymic epithelial cell line established from serum-free selective culture; Piltch A et al.; A serum-free system has been developed for selective growth and long-term culture of rat thymic epithelial cells . The growth media is a modification of McKeehan's WAJC 404, plus insulin, cholera toxin, dexamethasone, and epidermal growth factor . Cultures have been continuously passaged and maintained for over 6 mo., and a cloned cell line, TEA3A1, has been established . These cells are epithelial, judging by morphology and ultrastructure, and are positive for A2B5 and thymosin alpha 1 markers for thymic endocrine cells. Radiat Res, 1988 Apr, 114(1), 114 - 24 Stable H2O2-resistant variants of Chinese hamster fibroblasts demonstrate increases in catalase activity; Spitz DR et al.; Hydrogen peroxide (H2O2)-resistant variants of the Chinese hamster ovary HA-1 line have been derived by culturing cells in progressively higher concentrations of H2O2 (greater than 200 days, in 50-800 microM H2O2) . The H2O2-resistant phenotype has been stable for over 60 passages (240 days) following removal from the H2O2 stress . The resistant cells demonstrate both increased capacity to deplete exogenously added H2O2 from the growth medium and increased catalase activity . H2O2 resistance correlates well with catalase activity . An increase in chromosome number occurred in the cells adapted to 200-800 microM H2O2, but increases in aneuploidy and tetraploidy were not necessary for resistance . These results suggest that adaptation to chronic oxidative stress mediated by H2O2 in mammalian cells is accompanied by a stable heritable change in expression of catalase activity. Carcinogenesis, 1988 Apr, 9(4), 661 - 4 Epidermal growth factor enhances N-ethyl-N-nitrosourea-induced morphological transformation of Syrian hamster embryo cells; de Kok AJ et al.; To improve the usefulness of the Syrian hamster embryo (SHE) cell transformation system as a short-term test, it was investigated whether the variation in results due to serum variability could be reduced by the addition of epidermal growth factor (EGF) . It was found that EGF-significantly (3-7-fold) enhanced the frequency of morphological transformation induced by N-ethyl-N-nitrosourea or benzo{a}pyrene if added to growth medium supplemented with a batch of serum which had a low ability to support transformation . Furthermore, addition of EGF to the assay medium enabled the demonstration of dose dependence of transformation with relatively small group sizes (up to 2000 colonies) . Finally, it was observed that the transformed phenotype was easier to recognize in the presence of EGF . These data suggest that routine addition of EGF to the assay medium might reduce variability and enhance sensitivity of the SHE transformation assay. Brain Res, 1988 Apr 1, 467(2), 217 - 23 Acetylcholine responses in synaptically active neurons in mouse ventral mesencephalon cultures; Peacock JH et al.; Acetylcholine (ACh) sensitivity was examined in neurons in dissociated cultures from fetal ventral mesencephalon (14-16 days gestational age); many neurons probably originate in substantia nigra . Direct responses to iontophoretically applied ACh were recorded by intracellular microelectrodes in 53 cells and indirect responses were recorded from 58 synaptically connected cells . Cultures (24-50 days old) were studied in growth media enriched with serum (10%) and calcium (6.8 mM) . Direct responses: ACh caused a slow depolarization (seconds) in 94% (50/53); in 30% of these cells (15/50) the depolarization exceeded action potential threshold . Hyperpolarizing responses occurred in 4% (2/53) and another cell showed reduced action potential firing . Indirect responses: Application of ACh to adjacent cells caused an increase in postsynaptic potential (PSP) activity which was excitatory in 66% (38/58) and inhibitory in 17% (10/58) . A reduced level of PSP activity occurred in 7% (4/58) of cells exhibiting excitatory PSPs and in 10% (6/58) of cells with inhibitory connections . Indirect responses were blocked by tetrodotoxin . ACh receptor types: Responses to ACh were predominantly muscarinic (77%, 10/13) . Nicotinic responses were present in the remaining 23% (3/13). Nucleic Acids Res, 1988 Mar 25, 16(6), 2625 - 37 Structure and expression of the PHO80 gene of Saccharomyces cerevisiae; Madden SL et al.; In yeast, the repression of acid phosphatase under high phosphate growth conditions requires the trans-acting factor PHO80 . We have determined the DNA sequence of the PHO80 gene and found that it encodes a protein of 293 amino acids . The expression of the PHO80 gene, as measured by Northern analysis and level of a PHO80-LacZ fusion protein is independent of the level of phosphate in the growth medium . Disruption of the PHO80 gene is a non-lethal event and causes a derepressed phenotype, with acid phosphatase levels which are 3-4 fold higher than the level found in derepressed wild type cells . Furthermore, over-expression of the PHO80 gene causes a reduction in the level of acid phosphatase produced under derepressed growth conditions . Finally, we have cloned, localized and sequenced a temperature-sensitive allele of PHO80 and found the phenotype to be due to T to C transition causing a substitution of a Ser for a Leu at amino acid 163 in the protein product. Appl Environ Microbiol, 1988 Mar, 54(3), 712 - 7 Anaerobic and aerobic metabolism of diverse aromatic compounds by the photosynthetic bacterium Rhodopseudomonas palustris; Harwood CS et al.; The purple nonsulfur photosynthetic bacterium Rhodopseudomonas palustris used diverse aromatic compounds for growth under anaerobic and aerobic conditions . Many phenolic, dihydroxylated, and methoxylated aromatic acids, as well as aromatic aldehydes and hydroaromatic acids, supported growth of strain CGA001 in both the presence and absence of oxygen . Some compounds were metabolized under only aerobic or under only anaerobic conditions . Two other strains, CGC023 and CGD052, had similar anaerobic substrate utilization patterns, but CGD052 was able to use a slightly larger number of compounds for growth . These results show that R . palustris is far more versatile in terms of aromatic degradation than had been previously demonstrated . A mutant (CGA033) blocked in aerobic aromatic metabolism remained wild type with respect to anaerobic degradative abilities, indicating that separate metabolic pathways mediate aerobic and anaerobic breakdown of diverse aromatics . Another mutant (CGA047) was unable to grow anaerobically on either benzoate or 4-hydroxybenzoate, and these compounds accumulated in growth media when cells were grown on more complex aromatic compounds . This indicates that R . palustris has two major anaerobic routes for aromatic ring fission, one that passes through benzoate and one that passes through 4-hydroxybenzoate. Biochem Pharmacol, 1988 Mar 1, 37(5), 881 - 8 Promotion of cystine uptake and its utilization for glutathione biosynthesis induced by cysteamine and N-acetylcysteine; Issels RD et al.; Chinese hamster ovary (CHO) cells obtain a high capacity to utilize cystine from the growth medium by exposure to cysteamine (2-mercaptoethylamine, MEA) or N-acetylcysteine (NAC) . For uptake studies a modified McCoy's 5A medium supplemented with 0.1 mM {35S}cystine was used . The uptake of cystine was dependent on the time of exposure (0-60 min) and the concentrations of MEA or NAC (0-8 mM) . At high concentrations of MEA or NAC, the uptake of cystine became saturated . Half-maximal uptake of cysteine was observed at concentrations of 0.12 mM MEA and 0.66 mM NAC, respectively . Increase in temperature (37-44 degrees) or pH (6.0-8.0) during MEA or NAC exposure further increased the cystine uptake . The increased uptake of cystine was not affected in the presence of glutamate or homocysteate which both inhibited the cystine uptake of control cells . Determination of both reduced (GSH) and oxidized (GSSG) cellular glutathione showed a twofold increase in MEA- or NAC-treated CHO cells . DL-buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH biosynthesis completely blocked the promotion of cystine uptake by MEA and NAC . By further analysis using reversed-phase HPLC of cell extracts, more than 90% of the {35S} radioactive cystine taken up by the cells could be recovered within the pool of GSH . The results demonstrate that exposure of CHO cells with MEA and NAC leads to a promoted uptake of cystine from the culture medium and its rapid utilization for cellular GSH biosynthesis. Cancer Res, 1988 Mar 1, 48(5), 1286 - 94 Identification of a secreted Mr 95,000 glycoprotein in human melanocytes and melanomas by a melanocyte specific monoclonal antibody; Vogel AM et al.; We have isolated a monoclonal antibody, designated HMB-50, that is highly specific for melanomas and melanocyte derived lesions . The antibody recognizes melanomas, neonatal melanocytes, and junctional nevi but does not react with adult melanocytes, dermal nevi, or a variety of non-melanocyte derived neoplasms . In tissue culture, the antibody reacts with five of seven human melanoma lines and neonatal foreskin melanocytes but fails to recognize fibroblasts and a number of different carcinomas . HMB-50 identifies a Mr 95,000 glycoprotein that is released into the growth medium by melanoma cells and neonatal melanocytes in vitro . This molecule is unrelated to antigens recognized by a variety of antimelanoma monoclonal antibodies isolated in other laboratories . The Mr 95,000 glycoprotein has been purified by antibody affinity chromatography and a polyclonal rabbit antiserum raised that exhibits identical specificity to the monoclonal antibody . The Mr 95,000 glycoprotein is rapidly released by melanoma cells (within 60 min) and one line produces relatively large quantities of the molecule (1 microgram/10(6) cells/24 h) . The molecule in normal melanocytes differs slightly in electrophoretic mobility compared to its counterpart in melanomas and this difference appears to result from posttranslational modification. J Cell Biol, 1988 Mar, 106(3), 893 - 904 Effects of heat shock on the expression of thrombospondin by endothelial cells in culture; Ketis NV et al.; Heat-shock proteins from confluent primary cultures of bovine aortic endothelial cells were analyzed by SDS-polyacrylamide gels . In addition to the increased synthesis of the classical heat-shock proteins, there is an increase of a 180,000-mol wt polypeptide in the growth media of heat-shocked cells . Immunoprecipitation with specific antiserum indicates that the 180,000-mol wt polypeptide is thrombospondin . Assay of mRNA levels coding for thrombospondin after brief hyperthermic treatment (45 degrees C, 10 min), followed by a recovery of 2 h at 37 degrees C, results in a twofold increase in mRNA abundance . In contrast, the activation level of the 71,000-mol wt heat-shock protein mRNA occurs at an earlier time than for thrombospondin mRNA . Immunofluorescence microscopy was used to study the intracellular and extracellular distribution of thrombospondin . Thrombospondin is localized to a prominent pattern of granules of intracellular fluorescence in a perinuclear distribution in cells not exposed to heat . Upon heat treatment, the pattern of granules of intracellular fluorescence appears more pronounced, and the fluorescence appears to be clustered more about the nucleus . There are at least three pools of extracellular forms of thrombospondin: (a) the fine fibrillar extracellular matrix thrombospondin; (b) the punctate granular thrombospondin; and (c) the thrombospondin found in the conditioned medium not associated with the extracellular matrix . When bovine aortic endothelial cells are exposed to heat, the extracellular matrix staining of a fibrillar nature is noticeably decreased, with an increase in the number and degree of fluorescence of focal areas where the punctate granule thrombospondin structures are highly localized . No gross morphological changes in extracellular matrix staining of fibronectin was noted . However, the intermediate filament network was very sensitive and collapsed around the nucleus after heat shock . We conclude that the expression of thrombospondin is heat-shock stimulated. Proc Natl Acad Sci U S A, 1988 Mar, 85(5), 1576 - 80 An Escherichia coli mutation preventing degradation of abnormal periplasmic proteins; Strauch KL et al.; A fusion between tsr (encoding the inner membrane protein Tsr) and phoA (encoding the periplasmic protein alkaline phosphatase, AP) generates a membrane-bound hybrid protein (Tsr-AP 2) with AP enzymatic activity . The hybrid protein is proteolytically unstable and is broken down to yield a smaller, soluble species with AP activity . We devised a genetic screen to distinguish between cells containing only membrane-bound AP and those containing soluble AP . The screen depends on diffusion of soluble AP away from cells with a leaky outer membrane to produce a halo of AP activity around colonies on solid growth medium . Several mutants lacking this halo show reduced degradation of Tsr-AP 2 . One mutant is also defective in breakdown of five other abnormal periplasmic proteins but not of two cytoplasmic proteins . The mutation in this strain, degP4::Tn5, defines a locus distinct from previously identified loci that affect protein stability or protease activities . This strain may be useful for preventing the breakdown of unstable foreign proteins in Escherichia coli. Oncogene, 1988 Mar, 2(3), 259 - 65 The transformation of primary and established mouse mammary epithelial cells by p21-ras is concentration dependent; Redmond SM et al.; We constructed a retroviral vector (pZSR) which is capable of simultaneously expressing the neomycin resistance gene and the viral ras oncogene . Primary mammary gland epithelial cells were prepared from mid-pregnant mice and infected with this virus . Cell lines with epithelial cell characteristics could be established with a low frequency . High expression of p21 v-ras was observed in these cells . They are tumorigenic and form soft agar colonies dependent on the presence of EGF and insulin in the growth medium but progress to hormone independent growth at higher passage numbers . A cloned cell line of non-tumorigenic, established mammary epithelial cells (NOG8) was also infected with the v-ras expressing virus . Individual cell clones expressing increasing amounts of p21 v-ras were selected . The level of p21 v-ras expression directly influences the morphology of the epithelial cells in culture, determines their cloning efficiency in soft agar and their tumorigenicity. Proc Natl Acad Sci U S A, 1988 Mar, 85(5), 1534 - 8 Transcellular proton current in Achlya bisexualis hyphae: relationship to polarized growth; Schreurs WJ et al.; Growing hyphae of Achlya bisexualis drive an electric current through themselves, such that positive charge flows into the apical region (the anterior 300 micron) and exits distally along the hyphal trunk . They also generate a gradient of extracellular pH, such that the medium surrounding the apex is slightly alkaline whereas that along the hyphal trunk is acid . To explore the genesis of these gradients and their relationship to polarized extension, we examined the effects of changes in the composition of the growth medium . The transcellular electric current was most pronounced in medium rich in amino acids . In leaner medium, containing limited amounts of amino acids or none at all, the current was attenuated or absent . We interpret the results to mean that inward current represents H+/amino acid symport, mediated by porters that are preferentially localized in the apical region . Apical alkalinity may be due to ammonia production . Outward current, and perhaps also the generation of metabolic acid, reflects the distribution of the H+-ATPase, which is excluded from the apex but is abundant along the hyphal trunk . Thanks to the spatial segregation of transport functions, protons characteristically flow into the apical region . However, since hyphae grow apically and at the same rate despite wide variations in current pattern, the flow of electric charge through the hyphae cannot be required to polarize extension or to localize the tip. Tsitologiia, 1988 Mar, 30(3), 327 - 34 {Growth factor production and autocrine mechanism of cell proliferation regulation in the RPMI-6410t lymphoblastoid line}; Seregina TM et al.; The human lymphoblastoid B-cell line RPMI-6410t was found to synthesize and secrete into the growth medium a factor necessary to maintain the reproduction of these cells . In the condition of low plating density (concentration 1-1000 cells per ml) cell proliferation can be maintained only in the presence of a definite dose of medium conditioned by 6410t cell growth under high concentration . Using such a medium guaranteed almost 100% cloning efficiency of these cells by the method of limiting dilutions . The cloning of 6410t cells in the presence of feeder cells, such as mouse splenocytes and peritoneal cells, failed . The 6410t cells were shown to bind specifically the growth factor secreted by them, thus suggesting the presence of a growth factor acceptor on their surface . With the help of special selective method some clones were derived which did not secrete growth factor but were likely to have growth factor acceptors on their surface . A comparison of growth properties of clones GF- and GF+ supported the idea of autocrine control of proliferation as one of the mechanisms of malignant cell transformation. J Bacteriol, 1988 Mar, 170(3), 1339 - 45 An exocytoplasmic endonuclease with restriction function in Streptomyces antibioticus; de los Reyes-Gavilan CG et al.; Streptomyces antibioticus produces a strong endo-DNase which is located between the cytoplasmic membrane and the cell wall . All DNA substrates assayed, including the chromosomal DNA of this species and several bacteriophage DNAs, were completely degraded in vitro by the enzyme . The rate of synthesis of the nuclease depended