J Assist Reprod Genet, 1993 Apr, 10(3), 192 - 6 Effects of medium composition on murine and human blastocyst formation and hatching rate; Olar TT et al.; PURPOSE: Murine two-cell embryos (n = 5573) were cultured for 96 hr in human tubal fluid (HTF) medium (n = 2709) or alpha modification of minimum essential medium (MEM; n = 2864) through the hatched blastocyst stage from mid-1990 to mid-1991 . An additional 373 embryos were cultured in MEM or HTF with 0, 1, 5, or 10 ng/ml E . coli endotoxin . A total of 17 patients had supernumerary embryos simultaneously cultured in HTF (n = 48) or MEM (n = 61) . Additionally, pregnancy rates were compared for July to December 1990, when MEM was used as growth medium, and for July to December 1989, when HTF was used . RESULTS: Blastocyst formation was higher (P < 0.001) for murine embryos cultured in MEM (blasts = 95%) compared to HTF (blasts = 70%) . When cultured with endotoxin, blastocyst formation was higher (P < 0.01) for embryos cultured in MEM compared with HTF for controls and at each endotoxin level . No difference in human blastocyst development was observed in HTF and MEM . However, more MEM-cultured blastocysts were cryopreserved (P < 0.05) . There also was a lower spontaneous abortion rate and a higher multiple gestation rate when embryos were cultured in MEM . CONCLUSION: Thus, MEM may result in healthier blastocyst development, especially when culture conditions are substandard, although this is not an acceptable substitution for meticulous technique. Mol Microbiol, 1993 Apr, 8(2), 205 - 10 Trehalose metabolism in Escherichia coli: stress protection and stress regulation of gene expression; Strom AR et al.; Endogenously synthesized trehalose is a stress protectant in Escherichia coli . Externally supplied trehalose does not serve as a stress protectant, but it can be utilized as the sole source of carbon and energy . Mutants defective in trehalose synthesis display an impaired osmotic tolerance in minimal growth media without glycine betaine, and an impaired stationary-phase-induced heat tolerance . Mechanisms for stress protection by trehalose are discussed . The genes for trehalose-6-phosphate synthase (otsA) and anabolic trehalose-6-phosphate phosphatase (otsB) constitute an operon . Their expression is induced both by osmotic stress and by growth into the stationary phase and depend on the sigma factor encoded by rpoS (katF) . rpoS is amber-mutated in E . coli K-12 and its DNA sequence varies among K-12 strains . For trehalose catabolism under osmotic stress E . coli depends on the osmotically inducible periplasmic trehalase (TreA) . In the absence of osmotic stress, trehalose induces the formation of an enzyme IITre (TreB) of the group translocation system, a catabolic trehalose-6-phosphate phosphatase (TreE), and an amylotrehalase (TreC) which converts trehalose to free glucose and a glucose polymer. J Neurosci Res, 1993 Apr 1, 34(5), 546 - 61 Manganese induces spreading and process outgrowth in rat pheochromocytoma (PC12) cells; Lin WH et al.; Mn2+ has been shown to promote cell-substrate adhesion and cell spreading in many cell culture systems . In this study, we present data demonstrating that Mn2+ not only promotes spreading, but also induces process outgrowth in rat pheochromocytoma (PC12) cells . In the presence of 1.0 mM MnCl2, cell spreading was apparent by 6 hr, and nearly 50% of the exposed cells extended neurite-like processes . These morphological effects of Mn2+ were both time- and dose-dependent . In the presence of cycloheximide, a protein synthesis inhibitor, both Mn(2+)-induced spreading and neurite outgrowth were prevented, indicating that de novo protein synthesis is required for the effects of Mn2+ to take place . Of the other divalent cations tested, Mg2+, Cd2+, Cu2+, Ni2+, and Zn2+ were ineffective, and only Co2+ partially mimicked the effects of Mn2+ . Although Mn(2+)-induced cell adhesion and spreading have been extensively studied, this is the first report that this divalent cation can cause neurite outgrowth . The neurite outgrowth-promoting effects of Mn2+ were distinct from those of nerve growth factor in that the response to Mn2+ was considerably more rapid, but apparently lacked the ability to sustain continuous outgrowth and networking of neurites . Mn2+ also induced the levels of GAP-43 and peripherin, two proteins associated with neuronal differentiation of PC-12 cells . In cells grown in serum-free defined medium, Mn2+ was capable of promoting neurite outgrowth when the cells were plated on surfaces pretreated with normal growth medium, vitronectin, or fibronectin, while it failed to cause these morphological changes in cells plated on untreated or poly-D-lysine-coated substrata . Similarly, Mn2+ also promoted neurite outgrowth from rat sympathetic neurons attached to laminin-treated substrate, but had no effect on neurons maintained on substrate with polylysine only . The pentapeptide Gly-Arg-Gly-Asp-Ser nearly completely prevented the morphological effects of Mn2+ on PC12 cells . These findings are consistent with a hypothesis that Mn(2+)-mediated alteration of an RGD-dependent extracellular matrix-integrin interaction is responsible for the neuritogenic effects. Appl Microbiol Biotechnol, 1993 Apr, 39(1), 42 - 7 Induction of ajmalicine formation and related enzyme activities in Catharanthus roseus cells: effect of inoculum density; Moreno PR et al.; In Catharanthus roseus cell cultures the time courses of four enzyme activities, tryptophan decarboxylase (TDC), strictosidine synthase (SSS), geraniol-10-hydroxylase (G10H) and anthranilate synthase (AS), and alkaloid accumulation were compared under two different culture conditions (low-inoculum density and high-inoculum density on induction medium) and a control on growth medium . In growth medium a transient increase in TDC activity was first observed after which G10H reached its maximum activity; only tryptamine accumulated, no ajmalicine could be detected . Apparently, a concerted induction of enzyme activities is required for ajmalicine formation . Cells inoculated in induction medium showed such a concerted induction of AS, TDC and G10H activities . After 30 days the low-density culture had accumulated six times more ajmalicine (in mumoles/g) than the high-density culture . Thus, increase in biomass concentration (high-density cultures) did not enhance the total alkaloid production . The major differences observed in enzyme levels between high- and low-density cultures were in the AS and TDC activities, which were two to three times higher in the low-density culture, indicating that there is a positive correlation between ajmalicine formation and AS and TDC activities. Placenta, 1993 Mar-Apr, 14(2), 187 - 201 Colchicine inhibits human trophoblast differentiation in vitro; Douglas GC et al.; When cultured in keratinocyte growth medium, mononuclear cytotrophoblast cells aggregate into multicellular colonies which then fuse to form multinucleated syncytiotrophoblast . In an attempt to characterize better the mechanism of human trophoblast differentiation and to obtain information about the role of the cytoskeleton, experiments were performed using cytoskeletal-disrupting drugs and primary cultures of cytotrophoblast cells from term placentae . Addition of colchicine 6 h after plating permitted aggregation but the cells did not form syncytiotrophoblast, as revealed by staining for desmosomes and nuclei . Staining with an anti-tubulin antibody showed that microtubules were present in untreated control cells but absent in colchicine-treated cultures . If colchicine was added 24 h after plating, the cells also failed to differentiate . When cells were exposed to colchicine for the first 24 h after plating and then cultured in the absence of the drug, differentiation proceeded normally . Cells exposed to colchicine for 48 h and then incubated in the absence of the drug failed to form syncytiotrophoblast . The results suggest that a decision to differentiate is made between 24 and 48 h after plating . The effects of colchicine were observed between 2.5 and 250 microM . Beta-lumicolchicine blocked differentiation at 250 microM but was ineffective at lower concentrations . Colchicine also inhibited HCG secretion in a dose-dependent manner; beta-lumicolchicine only caused inhibition at 250 microM . Staining with antitubulin antibody revealed that lumicolchicine-treated cells had intact microtubules . These results suggest a role for microtubules in trophoblast differentiation. Indian J Exp Biol, 1993 Mar, 31(3), 224 - 30 Modification of radiation damage in transformed mammalian cells by 5-bromo-2-deoxy-uridine and 2-deoxy-D-glucose; Kalia VK et al.; The effects of 2-deoxy-D-glucose (2-DG) and 5-bromo-2-deoxy-uridine (BrdU) on gamma ray (60Co) induced damage were studied in monolayer cultures of transformed mammalian (BHK-21) cells . Micronuclei formation and changes in DNA content dispersion were used as indices of cytogenetic damage . Exposure of cells to BrdU (0.8 microM) for nearly two cell cycles before irradiation significantly increased micronuclei formation in exponentially growing cells . Incubation of irradiated cells under suboptimal growth conditions (in HBSS) for 4 hr, instead of growth medium, decreased the manifestation of damage . However, post-irradiation presence of 2-DG (5 mM, equimolar with glucose; 4 hr) in growth medium or HBSS significantly increased radiation damage . The effects of 2-DG treatment following irradiation in plateau phase were quantitatively less . These results suggest that: (i) radiation induced DNA lesions leading to micronuclei formation in BrdU incorporated cells are partly repairable; (ii) 2-DG could increase radiation induced cytogenetic damage in transformed mammalian cells, possibly by inhibiting the cellular repair processes; and (iii) combination of 2-DG treatment may decrease the BrdU doses required for radiosensitization of proliferating tumour cell populations. Prikl Biokhim Mikrobiol, 1993 Mar-Apr, 29(2), 280 - 5 {The action of exogenous RNAse on the cells of lower eukaryotes}; Il'inskaia ON et al.; We studied the mechanism of growth-stimulating effect of various compounds using an exogenous RNase-micromycete model and found that the treatment with microdoses of RNase intensified the reproduction of the yeast Candida valida and growth of Aspergillus fumigatus . At the same time the respiration activity of yeast cells and fungal mitochondria as well as succinate dehydrogenase activity increased . The secretion of total protein, cellulase and cellobiase changed in the presence of RNase in the growth medium . Electrophoretic mobility of Candida valida cells enhanced by 20% . The RNase stimulating effect on microbial cells seems to result from changes in characteristics of the cell surface. Eur J Haematol, 1993 Mar, 50(3), 127 - 32 Misincorporation of uracil into the DNA of folate- and B12-deficient HL60 cells; Wickramasinghe SN et al.; HL60 cells were cultured for 10 days under various experimental conditions . They were then incubated with 1 mumol/l {5-3H} uridine for 2 hours and their DNA extracted . The DNA was hydrolysed to deoxyribonucleosides with phosphodiesterase and alkaline phosphatase and the hydrolysate subjected to Aminex A6 chromatography . The elution profiles showed that, when compared with control cells, DNA from cells grown in medium deficient in folate, B12 or both folate and B12 contained increased amounts of deoxyuridine (dU) and increased radioactivity in the dU peak . The data demonstrate that misincorporation of uracil into DNA occurs in a myeloid cell line cultured in growth medium deficient in folate, B12 or both folate and B12. J Bacteriol, 1993 Mar, 175(6), 1823 - 30 Bioenergetics of sulfur reduction in the hyperthermophilic archaeon Pyrococcus furiosus; Schicho RN et al.; The bioenergetic role of the reduction of elemental sulfur (S0) in the hyperthermophilic archaeon (formerly archaebacterium) Pyrococcus furiosus was investigated with chemostat cultures with maltose as the limiting carbon source . The maximal yield coefficient was 99.8 g (dry weight) of cells (cdw) per mol of maltose in the presence of S0 but only 51.3 g (cdw) per mol of maltose if S0 was omitted . However, the corresponding maintenance coefficients were not found to be significantly different . The primary fermentation products detected were H2, CO2, and acetate, together with H2S, when S0 was also added to the growth medium . If H2S was summed with H2 to represent total reducing equivalents released during fermentation, the presence of S0 had no significant effect on the pattern of fermentation products . In addition, the presence of S0 did not significantly affect the specific activities in cell extracts of hydrogenase, sulfur reductase, alpha-glucosidase, or protease . These results suggest either that S0 reduction is an energy-conserving reaction, i.e., S0 respiration, or that S0 has a stimulatory effect on or helps overcome a process that is yield limiting . A modification of the Entner-Doudoroff glycolytic pathway has been proposed as the primary route of glucose catabolism in P . furiosus (S . Mukund and M . W . W . Adams, J . Biol . Chem . 266:14208-14216, 1991) . Operation of this pathway should yield 4 mol of ATP per mol of maltose oxidized, from which one can calculate a value of 12.9 g (cdw) per mol of ATP for non-S0 growth . Comparison of this value to the yield data for growth in the presence of S0 reduction is equivalent to an ATP yield of 0.5 mol of ATP per mol of S0 reduced . Possible mechanism to account for this apparent energy conservation are discussed. Arterioscler Thromb, 1993 Mar, 13(3), 360 - 6 A new anti-inflammatory leucine derivative, NPC-15669, inhibits growth of cultured human aortic smooth muscle cells; Bennett RL et al.; We have observed that NPC-15669, a leucine derivative with anti-inflammatory activity, reduced the proliferation of human aortic smooth muscle cells (HASMCs) in culture . We used a colorimetric assay and tritiated thymidine to measure the cell density and proliferation of HASMC cultures treated with this agent . We also studied the effect of NPC-15669 on the proliferation and migration of human aortic endothelial cells (HAECs) . Subconfluent HASMC cultures were growth arrested for 2 days . On the third day, growth was stimulated with either growth media (medium M199 containing 10% fetal bovine serum {FBS}), human platelet-derived growth factor (hPDGF), or fibroblast growth factor (FGF) in the absence or presence of NPC-15669 (0.1-50 microM) . Regardless of the stimulating agent for HASMCs (FBS, hPDGF, or FGF), NPC-15669 at concentrations of 10-25 microM caused a significant reduction in thymidine incorporation (36.7% and 77.2% in 10 microM and 25 microM, respectively; p < 0.005) and cell density (25-87%, p < 0.001) compared with control . NPC-15669 did not, however, have an effect on the rate of proliferation or migration of HAECs, even at concentrations up to 50 microM . Two other anti-inflammatory agents, aspirin and dexamethasone, caused substantially and significantly less inhibition, even at high concentrations (50 and 25 microM, respectively) . This study demonstrates that in vitro, NPC-15669 significantly inhibits HASMC proliferation but has no effect on proliferation or migration of HAECs. FEBS Lett, 1993 Mar 1, 318(2), 186 - 8 Reduction in the amount of 8-hydroxy-2'-deoxyguanosine in the DNA of SV40-transformed human fibroblasts as compared with normal cells in culture; Barciszewski J et al.; DNA damage due to oxidative free radicals is considered to be a major cause of ageing and age-related diseases including cancer . Of more than 20 modifications formed in DNA by the action of hydroxyl radicals, 8-hydroxy-2'-deoxyguanosine (oh8dG) is potentially highly mutagenic and is known to occur most frequently . Using HPLC combined with electrochemical (HPLC/EC) detection of oh8dG, fivefold higher levels of oh8dG are detected in the DNA of cultured normal human skin fibroblasts as compared with SV40-transformed human fibroblasts MRC-5V2 . In comparison, the levels of oh8dG were similar in the growth medium of both types of cells . Applications of this method range from studies on the genomic stability and instability of normal and cancerous cells to the clinical and laboratory testing of toxic substances and drugs. FEBS Lett, 1993 Mar 1, 318(2), 162 - 6 SC-0051, a 2-benzoyl-cyclohexane-1,3-dione bleaching herbicide, is a potent inhibitor of the enzyme p-hydroxyphenylpyruvate dioxygenase; Schulz A et al.; Growth inhibition of Lemna gibba plantlets by the bleaching herbicide, SC-0051 (2-(2-chloro-4-methanesulfonylbenzoyl)-1,3-cyclohexanedione)) was alleviated by the addition of homogentisic acid to the growth medium . Homogentisic acid is a key intermediate in the biosynthesis of tyrosine-derived plant quinones as well as in tyrosine metabolism . The herbicide prevented the incorporation of radioactivity from {14C}tyrosine into lipophilic plant metabolites and, in rat liver extracts, the herbicide inhibited the conversion of tyrosine to homogentisic acid . The enzyme p-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27) from both Zea mays seedlings and liver tissues, was found to be subject to strong inhibition by SC-0051 . Inhibition of plant quinone biosynthesis is a new mode of herbicidal action . One of the consequences of quinone depletion in plants by SC-0051 . Inhibition of plant quinone biosynthesis is a new mode of herbicidal action . One of the consequences of quinone depletion in plants in vivo is apparently an indirect inhibition of phytoene desaturation . The enzyme phytoene desaturase itself, however, is not afflicted by the herbicide. J Immunoassay, 1993 Mar-Jun, 14(1-2), 1 - 19 A capture enzyme-linked immunosorbent assay for virus infectivity titrations as exemplified in an adenovirus system; Everitt E et al.; An enzyme-linked immunosorbent assay (ELISA), employing a capturing antihexon monoclonal antibody specifically recognizing free hexons, was developed for quantitative infectivity titration of adenovirus in a microscale titration assay . The method is based on the quantitative assessment of the total excess production of the major structural protein late in infection in samples consisting of 10(5) virus-infected HeLa cells maintained as stationary suspension cultures . Results are obtained with a coefficient of variation of 10% within 50 hours after virus infection . The method was designed for monitoring substances interfering with viral replication, e.g., neutralizing antibodies or antiviral drugs . Since it measured the total antigen content associated with cells as well as antigens possibly released into the growth medium the general approach should be applicable to any viral system where a structural protein is synthesized in excess. Int J Artif Organs, 1993 Mar, 16(3), 164 - 71 Biomaterials in medicine--a bioengineering perspective; Courtney JM et al.; Biomaterials are considered with an emphasis on those used in artificial organs . Attention is drawn to the importance of the polymeric biomaterials and factors which affect their properties . Functions of membranes, sorbents, blood tubing, ventricular diaphragms and cell culture substrates are examined in order to obtain a summary of fundamental properties . Observations are made on the importance of blood compatibility assessment and its association with a biomaterial structure-property relationship . Blood-biomaterial interactions are discussed in terms of an overall relationship between the three components--blood, biomaterial and antithrombotic agent, with examples given of factors influencing each component . Cell-biomaterial interactions are examined in the areas of toxicity evaluation and the promotion of cell attachment and growth, where an overall relationship is described for the cell, growth medium and growth factors, and the biomaterial acting as a substrate. Plant Physiol, 1993 Mar, 101(3), 773 - 9 An osmotic stress protein of cyanobacteria is immunologically related to plant dehydrins; Close TJ et al.; Dehydrins are a family of desiccation proteins that were identified originally in plants (T.J . Close, A.A . Kortt, P.M . Chandler {1989} Plant Mol Biol 13: 95-108; G . Galau, T.J . Close {1992} Plant Physiol 98: 1523-1525) . Dehydrins are characterized by the consensus amino acid sequence domain EKKGIMDKIKEKLPG found at or near the carboxy terminus; the core of this domain (KIKEKLPG) may be repeated from one to many times within the complete polypeptide . Dehydrins generally accumulate in plants in response to dehydration stress, regardless of whether the stimulus is evaporation, chilling, or a decrease in external osmotic potential . Polyclonal antibodies highly specific to the consensus carboxy terminus of plant dehydrins were used to search for dehydrins in cyanobacteria, many of which are known to survive desiccation . A 40-kD osmotic-stress-induced protein was identified in Anabaena sp . strain PCC 7120 . The 40-kD protein was usually not detected in logarithmic cultures and was induced by shifting the growth medium to higher solute concentrations . Several solutes have inductive effects, including sucrose, sorbitol, and polyethylene glycol (PEG) . Measurements of osmotic potential suggest that a shift of -0.5 MPa (sucrose and PEG) or -1.2 MPa (sorbitol) is sufficient to induce synthesis of the 40-kD protein . Glycerol, which is highly permeable, was not an inducer at -1.2 MPa (0.5 M), nor was the plant hormone abscisic acid . Induction appears to be evoked by a shift in osmotic potential approximately equal in absolute magnitude to the expected turgor pressure of bacterial cells in logarithmic phase growth . A dehydrin-like polypeptide was also identified among osmotically induced proteins from two other filamentous, heterocyst-forming cyano-bacteria . A 40-kD protein was observed in Calothrix sp . strain PCC 7601, and in Nostoc sp . strain Mac-R2, an osmotic-induced doublet at 39 and 40 kD was observed . From these data, it appears that cyanobacteria produce a dehydrin-like protein under osmotic stress. J Neuroimmunol, 1993 Mar, 43(1-2), 69 - 78 Interleukin-3 and encephalitogenic activity of SJL/J myelin basic protein-specific T cell lines; Zhao ML et al.; Myelin basic protein (MBP)-specific SJL/J T cells were cultured in normal growth medium or growth medium supplemented with 10% culture supernatant from WEHI-3 cells, a source of interleukin-3 (IL-3), or with recombinant IL-3 . T cell lines cultured with IL-3 supplementation were more encephalitogenic compared to parallel lines cultured without this supplement . There was little difference in antigen-specific proliferative response or expression of cell surface markers CD3, CD4, CD8, IL-2R, or alpha/beta TCR in the parallel lines . Supernatant fluids from antigen-stimulated T cells from each cycle were tested for the presence of IL-2, IL-3, IL-4, granulocyte macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF alpha/beta) and transforming growth factor-beta (TGF beta) . No significant difference in IL-2, IL-4, GM-CSF, TNF alpha/beta, or TGF beta levels were seen when supplemented and unsupplemented cultures were compared . Supernatant culture fluids contained an activity that was highly stimulatory for the IL-3-dependent mouse mast cell line, MC/9 . This activity was attributable to a combination of at least three factors that varied in relative concentrations throughout the course of the experiments . Based on neutralization by monoclonal antibodies, MC/9 stimulating activity in early passage lines was attributable entirely to IL-3 and GM-CSF . The fraction of the MC/9 stimulatory activity that could be neutralized by monoclonal antibody to IL-3 decreased with increasing stimulation cycle while the fraction neutralized by anti-GM-CSF antibodies remained relatively constant . At the time that the lines lost encephalitogenicity, the activity neutralizable by anti-IL-3 had dropped to low levels in the culture supernatants; however, MC/9 stimulatory activity remained present in the supernatants . This was due to GM-CSF and a third unidentified factor. Mutat Res, 1993 Mar, 299(1), 9 - 18 Cytotoxic and genotoxic properties of tert.-butyl-p-quinone (TBQ) in an in vitro assay system with Chinese hamster V79 cells and in strain D7 of Saccharomyces cerevisiae; Rogers CG et al.; tert.-Butyl-p-quinone (TBQ), a major metabolite of the phenolic antioxidant tert.-butyl-4-hydroxyanisole (BHA), was examined for cytotoxic and genotoxic properties in an in vitro assay system with Chinese hamster V79 cells and in diploid strain D7 of Saccharomyces cerevisiae . TBQ was prepared from BHA by oxidative demethylation with sodium nitrite at low pH . Spectroscopic analyses identified the crystalline reaction product as TBQ with a purity close to 100% . Cytotoxicity of TBQ was determined by cloning efficiency in the absence of hepatocyte activation . TBQ reduced colony size of V79 cells at 0.4 micrograms/ml, prevented growth of 50% of the cells at 0.6 micrograms/ml, and was lethal to 100% of the cells at concentrations above 1.0 micrograms/ml . TBQ was 6-7 times more cytotoxic to V79 cells than TBHQ, a related BHA metabolite, and 100 times more cytotoxic than BHA . At dose levels of 0.2, 0.4 and 0.6 micrograms/ml of medium, TBQ did not increase significantly the frequency of sister-chromatid exchanges (SCE) in V79 cells and did not consistently increase the frequency of mutation to thioguanine resistance (TGR) at the hgprt gene locus either alone or with activation by rat hepatocytes . Incubation with TBQ for 4 h at pH 3.6 without activation resulted in only small increases in the frequency of gene conversion and reverse mutation in strain D7 of Saccharomyces cerevisiae . However, exposure to TBQ alone in growth medium for 24 h, produced inconsistent results . From these studies it was concluded that TBQ was cytotoxic but not genotoxic to V79 cells; and may be weakly genotoxic to strain D7 of S . cerevisiae. Biochim Biophys Acta, 1993 Feb 20, 1172(1-2), 175 - 80 Molecular cloning and expression of rabbit pancreatic cholesterol esterase; Colwell NS et al.; Rabbit pancreatic cholesterol esterase (CEase, carboxyl ester lipase, EC 3.1.1.3) has been cloned from a lambda gt11 library of adult rabbit pancreatic cDNA . The open reading frame consists of 1788 nucleotides which encodes 576 amino acids of the functional protein and a 20 amino acid leader peptide . When compared to other species, the greatest homology is observed between residues 82-248 with little or no homology at the C-terminal end where proline-glutamate-serine-threonine (PEST) segments are a characteristic feature of the human CEase . Rabbit CEase (RCEase) retains the active-site serine (gxsxg), the active-site histidine and the tentative heparin binding site (KKRCLQ) at similar positions in comparison to pancreatic CEases of other species . When rabbit CEase cDNA is expressed in monkey kidney (COS-7) cells, enzymatic hydrolytic activity is detected in the growth medium as is a 67 kDa protein by Western blotting with polyclonal anti-CEase antibody . Northern blot analysis shows two mRNA (2.2 and 3.2 kb) species. Biochim Biophys Acta, 1993 Feb 17, 1175(3), 277 - 82 Increased accumulation of drugs in a multidrug resistant cell line by alteration of membrane biophysical properties; Callaghan R et al.; Growth of CHRC5 multidrug resistant cells in media enriched in a saturated C-17 fatty acid, heptadecanoic acid, resulted in these cells accumulating vinblastine at a rate and to an extent comparable to that of the parental cell line AB1 . The fatty acid-enriched growth media had no effect on the ability of AB1 cells to take up vinblastine . The action of amphiphiles on the uptake of rhodamine dyes by CHRC5 cells was compared with the increased dye accumulation affected by verapamil . Membrane rigidifying agents, such as the saturated fatty acid stearic acid, or the cholesterol derivatives, cholesteryl hemisuccinate and cholesteryl phosphorylcholine, as well as a membrane fluidizing unsaturated fatty acid, linoleic acid, could significantly increase dye uptake, although not as well as verapamil . These results taken in conjunction with other reports in the literature, demonstrate that multidrug resistance is sensitive to alterations of membrane properties . They suggest that perturbation of the membrane to either increased or to decreased membrane fluidity can lower the level of resistance. Biochim Biophys Acta, 1993 Feb 13, 1161(2-3), 249 - 56 The chloroperoxidase from the fungus Curvularia inaequalis; a novel vanadium enzyme; van Schijndel JW et al.; The presence of vanadium-containing bromoperoxidases in various types of seaweed is well-documented . We now report that the terrestrial fungus Curvularia inaequalis excretes a novel chloroperoxidase which also contains vanadium as a prosthetic group . The chloroperoxidase is excreted in the medium as the only protein and is, therefore, almost purely obtained . Atomic absorption spectroscopy measurements showed that the chloroperoxidase contained vanadium, which was essential for enzymatic activity, in a stoichiometry of 1 mol vanadium per mol of enzyme . When the fungus was grown in media containing low concentrations of vanadate (VO4(3-)) or when vanadate was absent, the enzyme was excreted in an apoform . Addition of vanadate to the apoenzyme purified from the medium, dialyzed holo-enzyme or growth medium led to incorporation of the metal and to a subsequent increase in specific activity from 0.7 to about 7.5 units/mg . The reduced enzyme showed an axially symmetric EPR spectrum (g(o) = 1.971, Ao = 91.7 x 10(-4) cm-1) with 16 hyperfine lines that is essentially the same as the EPR spectrum of the vanadium-containing bromoperoxidase of the seaweed Ascophyllum nodosum . This demonstrates that the active sites in the two enzymes are very similar . The chlorinating and brominating activities of the chloroperoxidase from C . inaequalis were also studied and compared to those of the vanadium bromoperoxidase from A . nodosum . The chlorinating reaction catalyzed by the chloroperoxidase had a pH optimum around 5.5 and the Km for Cl- was small (0.25 mM at pH 4.5), but the logarithm of its value increased linearly with increasing pH . At high bromide concentrations, the pH optima of chloroperoxidase and bromoperoxidase in the brominating reaction were about the same (5.5) . However, at low bromide concentrations the pH optimum of the chloroperoxidase was at higher pH values than that of the bromoperoxidase. Cell, 1993 Feb 12, 72(3), 365 - 78 ZIP1 is a synaptonemal complex protein required for meiotic chromosome synapsis; Sym M et al.; ZIP1 is a novel meiosis-specific gene required for chromosome synapsis and cell cycle progression in S . cerevisiae . zip1 strains undergo homologous chromosome pairing, but are defective in synaptonemal complex (SC) formation . The zip1 mutation confers a uniform arrest in meiosis prior to the first division . zip1 strains display nearly wild-type levels of commitment to meiotic recombination; however, mature reciprocal recombinants are not formed until cells are released from meiotic arrest by return to growth medium . DNA sequence analysis of ZIP1 reveals structural homology to a number of proteins containing coiled coils . Immunofluorescence experiments using anti-ZIP1 antibodies demonstrate that the ZIP1 protein localizes to synapsed meiotic chromosomes but not to unsynapsed axial elements . Taken together, these data suggest that ZIP1 is a component of the central region of the SC . We propose a model in which ZIP1 acts as a molecular zipper to bring homologous chromosomes in close apposition. J Surg Res, 1993 Feb, 54(2), 157 - 62 Comparison of techniques for harvesting enterocytes for transplantation; Nguyen BL et al.; Selective enterocyte transplantation is a potential alternative to small intestinal transplantation for treatment of the short bowel syndrome . Our aim was to compare chelation and enzymatic methods of isolating enterocytes with respect to initial cell yield and characteristics and in vitro growth . Enterocytes were harvested from adjacent ileal segments in 35 rabbits using chelation with EDTA and warm trypsinization . Determinations were made of initial viability by trypan blue exclusion, cell yield, and proportion of intact crypts . Cells (5 x 10(6)) were seeded in growth media into culture vessels . Cell attachment was estimated by measuring cells liberated by Dispase at 24 hr . In vitro growth was assessed at 14 and 28 days . Although total cell yield (15.0 +/- 9.9 vs 12.5 +/- 8.3 x 10(6) cells/cm) and intact crypts were similar, the trypsin technique resulted in cells with higher initial viability (86 +/- 7 vs 71 +/- 18%, P < 0.05) . Cell attachment (7 +/- 8 vs 8 +/- 4%) and enterocyte disaccharidase activity were similar using both techniques . While the number of epithelial cell growth foci was not significantly different at 14 days, there was significantly greater surface coverage on both plain (44 +/- 20% vs 1 +/- 0%, P < 0.05) and Matrigel-coated (80 +/- 14 vs 16 +/- 25%, P < .05) vessels at 28 days with trypsin-isolated cells . Trypsinization resulted in a cell population which had a higher percentage of viable cells but a similar proportion of intact crypts and differentiated cells compared to those resulting from the chelation technique . Trypsin-liberated cells had greater capacity for in vitro growth particularly on Matrigel-coated surfaces.(ABSTRACT TRUNCATED AT 250 WORDS) J Dairy Sci, 1993 Feb, 76(2), 600 - 5 Effects of beta-carotene and alpha-tocopherol on rumen bacteria in the utilization of long-chain fatty acids and cellulose; Hino T et al.; Addition of safflower oil to a growth medium depressed the growth of mixed rumen bacteria above 200 mg/L and did not significantly increase bacteria, even at lower concentrations . However, when 10 mg/L of beta-carotene were added to 50 to 100 mg/L of safflower oil, bacterial growth was significantly increased . When more than 200 mg/L of safflower oil were present, beta-carotene markedly restored the growth capacity . alpha-Tocopherol was more effective than beta-carotene, although it inhibited growth at high concentrations . The combination of beta-carotene and alpha-tocopherol (each 5 mg/L) exerted partially additive effects . beta-Carotene plus alpha-tocopherol enhanced bacterial cell yield in the presence of safflower oil, caprate, stearate, or linoleate, suggesting that beta-carotene and alpha-tocopherol increase the utilization of fatty acids . beta-Carotene plus alpha-tocopherol also stimulated cellulose digestion in the presence of 100 mg/L of safflower oil, evidently through the increased growth of cellulolytic bacteria. J Cell Biochem, 1993 Feb, 51(2), 165 - 74 4-Quinolones cause a selective loss of mitochondrial DNA from mouse L1210 leukemia cells; Lawrence JW et al.; The 4-quinolone antibiotics nalidixic acid and ciprofloxacin are potent inhibitors of the bacterial type II topoisomerase DNA gyrase . Treatment of mouse L1210 leukemia cells with these drugs resulted in a delayed inhibition of cell proliferation . Prior to inhibition of cell proliferation, there was a time-dependent decrease in the cellular content of mitochondrial DNA (mtDNA) . The decrease in mtDNA was associated with a decrease in the rate of mitochondrial respiration and an increase in the concentration of lactate in the growth medium . Inhibition of cell proliferation by 4-quinolones was reversible upon drug washout . However, there was a 2- to 4-day lag before the growth rate returned to normal levels . This was preceded by an increase in mtDNA content and mitochondrial respiration . These studies suggest that inhibition of mammalian cell proliferation by 4-quinolone drugs is related to the selective depletion of mtDNA. Int J Cancer, 1993 Feb 1, 53(3), 499 - 505 Persistence of plasmin-mediated pro-urokinase activation on the surface of human monocytoid leukemia cells in vitro; Tapiovaara H et al.; Human leukemia cell lines, unlike those from adherent tumors, have been shown to continuously activate the pro-urokinase (pro-u-PA) they produce . In the present study we found that, in normal cell-culture conditions in 10% FCS the plasminogen activation cascade works continuously on monocytoid leukemia cells, which expressed plasmin activity and active u-PA on their cell surface . This plasmin catalyzed the conversion of the produced pro-u-PA to active 2-chain urokinase (tcu-PA), and was derived from bovine serum plasminogen by the activity of cell-bound tcu-PA . Plasmin generation was abolished and pro-u-PA accumulated in cell cultures that were grown for several days, either in the presence of serum thoroughly depleted of plasminogen, or in the presence of 1 mM tranexamic acid . Plasmin generated on the cell surface was found to be present in 2 enzymatically active fragments, of M(r) 85,000 and M(r) 50,000, which were slowly released into the growth medium . These fragments could activate pro-u-PA in serum-free growth medium . Most of the bound plasmin could be washed off cells with 10 mM tranexamic acid, but complete removal of plasmin from the cell surface required washing of the cells with acid-glycine pH 3.0. Am J Clin Nutr, 1993 Feb, 57(2 Suppl), 313S - 314S Selenium-dependent regulation of type I 5'-deiodinase expression; Oertel M et al.; Selenite concentration regulates activity and expression of the p27 substrate-binding subunit of type I 5'deiodinase (5'-D) and of a protein labeled with bromoacetylthyroxin (BrAcT4), or p30, with yet unknown function in a porcine-kidney epithelial cell line (LLC-PK1) cultured in serum-free medium . p27 is metabolically labeled by 75-selenite and affinity labeled by BrAc{125I}T4 . Compared with glutathione peroxidase, expression of the p27 5'D subunit (5'-DI) is observed at 10-fold-lower concentrations of selenium in the growth medium, suggesting an intracellular hierarchy of selenite utilization . Selenium deficiency retards cell growth and prevents 5'-DI expression and may thus impair thyroid hormone action in vivo. Oncogene, 1993 Feb, 8(2), 503 - 7 Alternative mRNA forms and open reading frames of the max gene; Vastrik I et al.; The max gene encodes a heterodimeric partner of Myc . We have recently identified an alternative max mRNA (delta max) that contains an additional internal exon introducing an in-frame translational termination . Here we have studied the expression of human max mRNAs by Northern blotting analysis . In addition to the major 2.3-kb mRNA form, four bands were identified . Our results indicate that these bands represent differentially spliced mRNA forms, which contain altogether three open reading frames . In addition to the previously identified Max and delta Max proteins, sequence analysis of a 3.5-kb mRNA form predicted a protein that resembles delta Max in structure . Like delta Max, this protein enhanced the number of transformed foci in the ras-myc co-transformation assay . Although the 3.5-kb mRNA represents a minor form in actively proliferating cells, a shift from the major 2.3-kb mRNA to the 3.5-kb form was observed in response to high cell density or acidification of the growth medium . Our results indicate the presence of several differentially spliced mRNA forms of the max gene, and suggest a possible mechanism for the production of functionally distinct Max proteins. Indian J Biochem Biophys, 1993 Feb, 30(1), 71 - 2 Further studies on the influence of aminophylline on lipids of Microsporum gypseum; Bindra A et al.; Aminophylline added to the growth medium of Microsporum gypseum for varying periods exhibited varied effects on lipid synthesis . A decreased incorporation of {14C}acetate into both lipids and phospholipids was initially observed which showed increase after 24 hr of incubation . Short-time exposure of aminophylline also resulted in decreased activity of glycerol-3-phosphate acyltransferase, the key enzyme of lipid synthesis, which, however, got stimulated on longer incubation, supporting the decreased/increased synthesis of lipids during short/long time exposure . Changes in the intracellular levels of cAMP at different time points account for the induced alterations in lipid synthesis, possibly due to formation of metabolites of aminophylline during growth of the organism. J Cell Sci, 1993 Feb, 104 ( Pt 2), 307 - 15 Non-genotoxic hepatocarcinogenesis in vitro: the FaO hepatoma line responds to peroxisome proliferators and retains the ability to undergo apoptosis; Bayly AC et al.; A range of hepatoma cell lines (RH1, HTC, FaO, 7800C1 and MH1C1), has been studied with the aim of establishing an in vitro model to investigate the molecular mechanisms of hepatocarcinogenicity induced by the peroxisome proliferator class of non-genotoxic carcinogens . In view of speculation that peroxisome proliferators suppress hepatocyte apoptosis in vivo, we have placed particular emphasis on evaluating whether hepatoma cell lines retain the ability to undergo apoptotic cell death . Expression of the liver-specific differentiation marker albumin and the peroxisome proliferator-activated receptor (PPAR) was highest in the Reuber hepatoma cell line, FaO . This cell line also demonstrated the most marked response to the peroxisome proliferator nafenopin with a 2.2-fold induction of the microsomal enzyme cytochrome p450IVA1 . This response was found to display intercellular heterogeneity by immunocytochemistry . Thus, the FaO cell line maintained characteristics of hepatocytes, both in vivo and in vitro, in terms of expression of constitutive and inducible markers . However, none of the cell lines tested mirrored the hyperplastic response of hepatocytes to nafenopin, since no increase in cell growth kinetics was observed on addition of nafenopin to the growth medium . The mode of cell death in confluent FaO cultures was characterised as apoptosis, by fluorescence microscopy and agarose gel electrophoresis of extracted DNA . Cells detaching from confluent FaO cultures exhibited chromatin condensation and DNA fragmentation patterns characteristic of cels undergoing apoptotic death.Interestingly, no apoptosis was seen in monolayer cells, suggesting that apoptosis in vitro is associated with cell shrinkage and detachment similar to that documented for the liver in vivo.(ABSTRACT TRUNCATED AT 250 WORDS) Arch Biochem Biophys, 1993 Feb 1, 300(2), 734 - 7 The expression of human relaxin in yeast; Yang S et al.; The chemically synthesized DNA-coding sequence for an artificial single chain human relaxin consisting of a B chain, an Arg-Arg-Glu-Phe-Lys-Arg-connecting peptide, followed by the A chain, was used to construct two plasmids which were introduced into Saccharomyces cerevisiae . Expression of the relaxin-coding sequence was under the control of either the yeast TDH3 promoter or the CUP1 promoter . The yeast alpha-factor signal sequence was used to direct the protein into the secretory path, and the appearance of human relaxin in the growth medium was confirmed by radioimmunoassay and immunoblotting . Partially purified human relaxin from yeast was biologically active in the mouse symphysis pubis assay and radioreceptor assay. Int J Cancer, 1993 Feb 1, 53(3), 478 - 85 Distinct P-glycoprotein expression in two subclones simultaneously selected from a human colon carcinoma cell line by cis-diamminedichloroplatinum (II); Yang LY et al.; Two drug-resistant sublines, CP2.0 and RT, were simultaneously selected by cis-diamminedichloroplatinum (CDDP) from the human colon carcinoma cell line LoVo by the conventional method of continuous drug exposure . The 2 sublines differed in morphology, growth kinetics and pattern of gene expression . Genetic signature analysis indicated that the lines were independent subclones but that both arose from LoVo . These sublines were maintained in a growth medium containing 2.0 micrograms/ml CDDP . However, CP2.0 cells were 3 times more resistant to CDDP than were RT cells . Although both were cross-resistant to mustargen and 5-fluorouracil, only CP2.0 was resistant to Adriamycin and vincristine . Western-blot analysis, immunocytochemical staining and in vitro phosphorylation experiments indicated that the level of P-glycoprotein was significantly elevated in CP2.0 but not in RT . Despite the differences between these sublines, they possess similar CDDP-resistance mechanisms, including decreased intracellular CDDP accumulation, elevated levels of glutathione and metallothionein-like proteins, increased glutathione transferase-pi mRNA, and enhanced susceptibility to CDDP cytotoxicity after treatment with DL-buthionine-{S,R}-sulfoximine . Nevertheless, our results suggest that, in certain tumor types, P-glycoprotein-mediated multi-drug resistance and CDDP-resistance phenotypes can coexist in cells with primary resistance to CDDP. In Vitro Cell Dev Biol, 1993 Feb, 29A(2), 127 - 34 A cobblestone cell isolated from the human omentum: the mesothelial cell; isolation, identification, and growth characteristics; Pronk A et al.; Normal human mesothelial cells (NHMC) were isolated from pieces of human omentum . The cell yield was approximately one million cells per square centimeter omentum . The mesothelial cells were identified by their positive staining with monoclonal antibodies against cytokeratins 6 and 18 . Transmission electronmicroscopy of cultured NHMC revealed many microvilli on the apical surface and many mitochondria and pinocytotic vesicles in the cytoplasm, indicating active transmembrane transport . Growth of NHMC was directly related to the concentration of human serum or of fetal bovine serum in the growth medium . Addition of epidermal growth factor with or without hydrocortisone resulted in a significant increase of NHMC growth; when endothelial cell growth factor, insulin, or hydrocortisone were added no such increase was observed . Seeding NHMC at densities less than 3000/cm2 did not result in monolayer formation . The mesothelial cells were serially passed in growth medium M199 with added 10% fetal bovine serum up to 7 passages . However, after Passage 4 the cells changed into giant cells with an irregular pattern, and a lack of intracellular cytokeratin expression was observed for most of the cells. Carcinogenesis, 1993 Feb, 14(2), 285 - 9 Inactivation of a tumor suppressor function in immortal Syrian hamster cells by N-methyl-N'-nitro-N-nitrosoguanidine and by 5-aza-2'-deoxycytidine; West RW et al.; Clonal lines of immortal Syrian hamster cells were previously isolated that either suppressed (supB+) tumorigenicity in hybrids with a malignant hamster cell line (BP6T) or had lost this suppression ability (supB-) . Neither line was tumorigenic or showed anchorage-independent growth in normal growth medium . SupB- cells, but not supB+ cells, grew in agar supplemented with the growth factors EGF, PDGF and insulin (EPI), providing a selective assay for the supB- phenotype . After treatment of supB+ cells with either N-methyl-N'-nitro-N-nitrosoguanidine (10-300 ng/ml) or 5-aza-2'-deoxycytidine (25-250 ng/ml), and an expression period of 4-8 weeks, a dose-dependent increase in altered cells that grew in agar supplemented with EPI was observed . Cell lines derived from colonies in agar showed persistent EPI-stimulated growth in agar, and decreased suppression of growth in agar for hybrids with BP6T cells . Thus, carcinogen-induced loss of the tumor suppressor phenotype has been demonstrated. Brain Res, 1993 Jan 29, 602(1), 41 - 4 Effect of ascorbate on Na(+)-independent and Na(+)-dependent uptake of {3H}norepinephrine by rat primary astrocyte cultures from neonatal rat cerebral cortex; Kimelberg HK et al.; We have previously reported that primary astrocyte cultures prepared from neonatal rat brains show Na(+)-dependent, tricyclic antidepressant-sensitive, high-affinity uptake of {3H}norepinephrine ({3H}NE) . Other workers, however, using primary astrocyte cultures from neonatal mice, have failed to find such uptake . This prompted us to examine possible reasons for the variability of the uptake in primary astrocyte cultures such as growth conditions and the effect of ascorbic acid . The presence of ascorbic acid increased the Na(+)-dependent uptake of NE by inhibiting the Na(+)-independent component . Na(+)-dependent uptake in rat cultures occurs when either fetal bovine or horse serum are present in the growth media, but not in a serum-free growth medium . Other workers have shown a species difference such that, even under optimal uptake conditions where rat astrocyte cultures exhibit Na(+)-dependent {3H}NE uptake, mouse astrocyte cultures do not. FEBS Lett, 1993 Jan 25, 316(2), 181 - 5 Molecular weight-determination of biosynthetically modified monomeric and oligomeric muropeptides from Escherichia coli by plasma desorption-mass spectrometry; Caparros M et al.; The presence of certain D-amino acids in the growth media of Escherichia coli results in the accumulation of 2 major and 3-5 minor new muropeptides in the murein sacculus . Preliminary data suggested that the major muropeptides correspond to a monomer and a cross-linked dimer with one residue of D-amino acid per molecule . We have analyzed several D-amino acid-modified muropeptides by plasma desorption-mass spectrometry . Our results confirmed that the general structures of the major modified muropeptides are: GlucNAc-MurNAc-L-Ala-D-Glu-m-A2pm-D-X, and GlucNAc-MurNAc-L-Ala-D-Glu-m-A2pm-D-Ala; GlucNAc-MurNAc-L-Ala-D-Glu-m-A2pm-D-X, being X a residue of the D-amino acid . These results corroborate the usefulness of this technique for the structural analysis of muropeptides. Gene, 1993 Jan 15, 123(1), 51 - 8 Analysis of hemolysin operons in Actinobacillus pleuropneumoniae; Frey J et al.; Among the twelve different serotypes of Actinobacillus pleuropneumoniae, the causative agent of swine pleuropneumonia, a strongly active hemolysin I (HlyI) is produced by serotypes which are particularly virulent, and less active hemolysin II (HlyII) is produced by all serotypes except type 10 . In the serotypes 1, 5a, 5b, 9, 10 and 11, which produce HlyI, the hemolysin (hly) operon consists of a structural hlyIA gene, encoding pre-HlyI, an activator gene, hlyIC, necessary for the activation of pre-Hly to active Hly, and two genes, hlyIB and hlyID, involved in Hly secretion . These genes are clustered in the order, hlyICABD . This is characteristic to RTX toxin (repeats in the structural toxin) operons . The HlyII operons in all serotypes producing HlyII consist only of the pre-HlyII-encoding gene, appA, and its activator gene, appC . The serotypes, which produce HlyII, but not HlyI, contain a truncated HlyI operon, with the promoter, hlyIB and hlyID, and a small segment of the C terminus of hlyIA . This partial HlyI operon might have been formed by deletion of hlyIC and most of hlyIA . In serotype 3, which produces HlyII, but no HlyI, and which releases only minute amounts of this Hly into the growth medium, none of the hlyI genes and consequently no Hly secretion genes were found . The above results postulate that HlyII is secreted via the products of hlyIB and hlyID, and explain the low amount of HlyII secreted by serotype 3 . Cloning and analysis of the structural genes encoding pre-HlyI and pre-HlyII among the different serotypes revealed differences in the hlyIA genes which are highly similar in the serologically related serotypes 1, 9 and 11, and differ from the serotypes, 5a, 5b and 10 . The hlyIIA genes, in contrast, seem to be conserved in all serotypes. Cancer Res, 1993 Jan 15, 53(2), 393 - 400 8-Chloroadenosine mediates 8-chloro-cyclic AMP-induced down-regulation of cyclic AMP-dependent protein kinase in normal and neoplastic mouse lung epithelial cells by a cyclic AMP-independent mechanism; Lange-Carter CA et al.; The 8-chloro analogue of the regulatory molecule, cyclic AMP (cAMP), modulates the intracellular concentrations of cAMP-dependent protein kinases (PKA) and inhibits both in vitro and in vivo growth of several neoplastic cell types . Because 8-chloro-cyclic AMP (8-Cl-cAMP) can be converted to 8-chloroadenosine (8-Cl-adenosine) by serum enzymes contained in cell growth media, we tested whether 8-Cl-cAMP effects were mediated by its adenosine metabolite in normal and neoplastic cell lines of mouse lung epithelial origin . 8-Cl-adenosine, directly added to cells or derived from exogenously applied 8-Cl-cAMP, specifically decreased the intracellular concentration of the type I isozyme of cAMP-dependent protein kinase (PKA I) . 8-Cl-adenosine and 8-Cl-cAMP were equipotent at inhibiting cell growth, and elicited similar changes in the proportion of cells in the G1, S, and G2-M phases of the cell cycle . The presence of adenosine deaminase, which converts 8-Cl-adenosine to 8-chloroinosine, completely prevented growth inhibition by 8-Cl-cAMP and the concomitant diminution of PKA I . 8-Cl-cAMP had no discernible effect on cells when its conversion into 8-Cl-adenosine was prevented by 3-isobutyl-1-methyl-xanthene, an inhibitor of phosphodiesterase . 6-(p-Nitrobenzyl)-thioinosine, an inhibitor of adenosine uptake, protected cells from cytostasis, indicating that 8-Cl-adenosine acts intracellularly . 8-Cl-adenosine greatly decreased RI (regulatory subunit of PKA I) and PKA catalytic (C) subunit protein concentrations without affecting RII (regulatory subunit of the PKA type II isozyme) or intracellular cAMP levels . Northern blot analysis of PKA subunit mRNAs following treatment of each cell line with 8-Cl-adenosine demonstrated decreased C alpha mRNA expression, increased RII alpha mRNA, and no change in RI alpha mRNA abundance . Our results indicate that 8-Cl-adenosine inhibits lung cell growth and induces PKA I down-regulation via a cAMP-independent mechanism. Science, 1993 Jan 8, 259(5092), 241 - 4 Tyrosine phosphorylation of actin in Dictyostelium associated with cell-shape changes; Howard PK et al.; When Dictyostelium cells that have initiated their developmental program upon starvation are returned to growth medium, there is a rapid and transient de novo tyrosine phosphorylation of a 43-kilodalton protein . This protein was found to be actin . Most of the phosphorylation occurred in a single, minor acidic isoform of actin . Developing cells that had been returned to growth medium lost their pseudopod extensions, became round, and had reduced adhesion to the substratum . These effects occurred with kinetics that matched the increase in tyrosine phosphorylation of actin . In mutant cell lines in which the gene for the phosphotyrosine phosphatase PTP1 had been disrupted, tyrosine phosphorylation of actin was rapid and more prolonged . These cells responded with proportionally accelerated kinetics of cell rounding . Cell lines overexpressing PTP1 had diminished amplitude and duration of actin tyrosine phosphorylation and exhibited diminished cell-shape change and an accelerated return to the extended cell-shape morphology seen in starved cells. Arch Tierernahr, 1993, 43(1), 45 - 51 Heat-induced formation of soluble Maillard reaction products and its influence on utilization of glucose by rumen bacteria; Marounek M et al.; In a series of experiments with pure strains of rumen bacteria the effect of heat-induced formation of soluble Maillard reaction products on the utilization of glucose was examined . Maillard reaction products were prepared from glucose and amino acids, which were dissolved in a growth medium and autoclaved at 100 degrees C and 120 degrees C . Glucose was utilized almost completely in all cultures, no matter whether it was bound in Maillard products or not . The complexing of glucose with amino acids lowered the growth rate and growth yields in most rumen bacteria studied . In some strains, however, the effect of the Maillard reaction was small . It was concluded that the heat damage of carbohydrates in feedstuffs could be attributed to final stages of the Maillard reaction (formation of insoluble polymers), rather than to initial ones. Agents Actions, 1993 Jan, 38(1-2), 44 - 7 Release of polyamines in cultures of rat parotid and liver cells; Nilsson BO et al.; Rat parotid gland and liver cells were cultured for 6 and 24 h . The cells as well as their growth medium were analyzed on their content of the polyamines putrescine, spermidine and spermine . In control medium the content of polyamines was very low but already after 6 h substantial amounts of all three polyamines under study had been released into the medium from parotid as well as from liver cells . The release was much more pronounced from parotid compared to liver cells . Putrescine was accumulated in parotid cells as well as in the medium indicating that a new synthesis of this amine occurred in these cells. J Craniofac Genet Dev Biol, 1993 Jan-Mar, 13(1), 6 - 17 Effects of 5-fluorouracil on collagen synthesis during quail secondary palate development; Benkhaial GS et al.; A study was undertaken to examine the involvement of collagen synthesis during palate development in quail where mammalian-type shelf reorientation is absent . Teratological observation showed that 100 micrograms 5-fluorouracil (FU) on day 4 of incubation increased the gap between the palatal shelves . Light microscopic observation indicated that the quail palatal shelves develop as horizontal ridges on day 5 and approximate on day 8 of incubation but never fuse . FU treatment affected the approximation of palatal shelves . In separate experiments, both the control and FU-treated quail embryonic palates, which were dissected between days 5 and 10 of incubation, were incubated in a growth medium supplemented with 14C-proline . The rate of collagen synthesis, total protein, hydroxyproline (HYP) levels, and collagen isotype were determined . The result showed that in control palates the rate of collagen synthesis increased fivefold between days 6 and 8 of incubation but dropped thereafter . In FU-exposed palates, the rate of collagen synthesis was lower than that in controls . It increased threefold between days 7 and 8 of incubation . High performance liquid chromatographic measurement of HYP levels indicated that, in comparison to controls, HYP accumulation in FU-treated palates was reduced by 50% on day 6 and 75% on day 8 of incubation . Total protein content in FU-treated palate were also 50-70% lower than those in their control counterparts between days 5 and 10 of incubation . Gel electrophoresis showed that only type I collagen was synthesized in the developing palate of both the control and FU-treated quail embryo . An analysis of results of the present study, along with the data from literature on mammals, corroborate the proposition that an increased collagen synthesis may contribute to the acquisition of volume of vertebrate's secondary palatal shelf for its continuing morphogenesis. Boll Chim Farm, 1993 Jan, 132(1), 23 - 8 {Microbiological validation of a film system for monitoring total aerobic bacteria on surfaces and laboratory garments in controlled areas}; Dal Maso G et al.; The microbiological control of the environment is a real issue inside the pharmaceutical production plants . Actually the most used methods are the Contact Plates and the Swabs . The purpose of this study is to evaluate the performance of a new "ready to use" method called Petrifilm SM (produced by 3M Laboratories) . Petrifilm consists of a dry-rehydratable growth medium coated on a flexible film . Petrifilm is tested in comparison with Swabs and Contact Plates to monitor the microbiological pollution on labs surfaces and operator's overalls . The results obtained are comparable with the above mentioned methods, but with important advantages being less bulky, considerably time saving and highly reproducible . Hence, we think that Petrifilm S, can be successfully used instead of the other two more traditional methods. Arch Virol, 1993, 129(1-4), 251 - 63 Exchange of the cellular growth medium supplement from fetal bovine serum to Ultroser G increases the affinity of adenovirus for HeLa cells; Blixt Y; A comparative investigation was performed on the process of attachment of adenovirus type 2 to HeLa cells cultivated in the presence of 3.5% fetal bovine serum (FBS-cells) or 2% Ultroser G (USG-cells) . The initial rates of virus attachment were markedly higher at temperatures between 10 and 35 degrees C for the virus binding to USG-cells than to FBS-cells . This was not caused by a higher amount of available virus-recognizing cellular receptor sites or cellular receptor units recognizing the viral fiber, but could be explained by a higher affinity of virions for USG-cells as compared to FBS-cells . Studies of virus attachment to cells, pretreated with neuraminidase and/or wheat germ agglutinin, suggested that the cellular receptor sites of FBS-cells were masked to a higher extent by sialic acid than the cellular receptor sites of USG-cells. Arch Toxicol, 1993, 67(1), 76 - 8 On the toxicity of low doses of tetrasodium-ethylenediamine-tetraacetate (Na-EDTA) in normal rat kidney (NRK) cells in culture; Hugenschmidt S et al.; The toxicity of tetrasodium-ethylenediaminetetraacetate (Na-EDTA) was investigated in normal rat kidney cells (NRK-52E, American Type Culture Collection) in culture . Cell death and reduced colony-forming ability were observed at Na-EDTA concentrations < 100 microM, that is at concentrations lower than that required to chelate all the Ca2+ in the growth medium . No toxicity was observed when cells were exposed to 100 microM Zn- or Fe-EDTA, but the same concentration of Cu-EDTA was as toxic as Na-EDTA . Continuous exposure of the cell cultures to 5 microM Na- or Zn-EDTA for up to 7 weeks yielded no indications of toxicity. Neuroscience, 1993 Jan, 52(2), 333 - 46 Grafting in acute spinal cord injury: morphological and immunological aspects of transplanted adult rat enteric ganglia; Jaeger CB et al.; We have studied allogeneic transplants of adult rat enteric ganglia in order to evaluate their use as donor tissue for eventual autografts in rodent spinal cord injury models . Female Sprague-Dawley rats of similar weights served either as transplant donors or as recipients . A glass micropipette of 0.8 mm diameter was used to create a local penetrating injury of the lower thoracic spinal cord and the transplant material was pressure injected through the pipette within the neural parenchyma . Ganglia of the myenteric plexus adhering to the stratum longitudinal muscularis were dissected from portions of the jejunum and ileum . Following partial enzymatic digestion and mechanical disruption of the myenteric plexus and muscle tissue (labeled with adherent rhodamine conjugated microbeads), reaggregates of myenteric plexus and muscle were suspended in growth medium and cultured in vitro for one to two days prior to transplantation . Transplants were examined at three, four, six, and eight weeks after surgery . Some of the donor tissue was grown in vitro, in order to determine its cellular composition . These cultured explants were fixed after 10 days, and like myenteric plexus and muscle grafts, were stained histochemically for acetylcholinesterase and observed by fluorescence and light microscopy . At the earlier post-transplantation periods, grafts contained several clusters of enteric ganglion cells that were positive for acetylcholinesterase and exhibited ultrastructural features characteristic of the enteric nervous system . They had well-defined boundaries . Reactive astrocytes and their processes remained located within the host spinal cord adjacent to the boundary region of the grafts . Likewise, macrophages were located in areas abutting the graft . Newly formed vasculature penetrated the graft interior and appeared to be continuous with the host vessels . Grafts grown for at least eight weeks were characterized by interdigitating boundaries . Finger-like protrusions of graft tissue containing fibroblasts and collagen intermixed with adjacent gray and white matter of the host cord . Such transplants also had reactive astrocytes and ED1-positive macrophages . At this later stage, several groups of ganglion cells were identified that were intensely acetylcholinesterase-positive; however, only two of four grafts were recovered, whereas two of the transplants degenerated . We postulate that degeneration of allogeneic grafts may occur as a result of ongoing immune responses of the host which could be prevented by use of autogeneic enteric ganglia . Our studies show that fully differentiated enteric ganglia can survive transplantation to acutely injured spinal cord of adult rats. Cell Prolif, 1993 Jan, 26(1), 77 - 88 The in vitro response of human tumour cells to desferrioxamine is growth medium dependent; Voest EE et al.; Iron chelating agents have been demonstrated to inhibit tumour cell growth . However, in vitro and in vivo results using desferrioxamine a hexadentate iron chelating agent, for anti-cancer treatment are not always in agreement . Therefore, we have studied the response of three human tumour cell lines (HL-60 promyelocytic leukaemia, MCF-7 breast cancer and HepG2 hepatoma), grown in culture medium supplemented with either human pooled (HPS) or fetal bovine serum (FBS), to desferrioxamine . Desferrioxamine, at micromolar concentrations, induced severe cytotoxicity in all tumour cell lines grown in FBS medium . When grown in HPS medium, comparable desferrioxamine cytotoxicity was observed in the millimolar range . The addition of 50% saturated human transferrin to FBS medium resulted in protection against desferrioxamine cytotoxicity . HL-60 cells were further studied for iron metabolism characteristics . HL-60 cells, grown in medium with FBS, were found to have an 8.4 fold increase in surface transferrin receptor (TfR) expression (P < 0.001) as compared with HL-60 cells grown in medium with HPS . However, iron uptake of HPS cultured HL-60 cells, after incubation with saturated human transferrin, was higher, resulting in a higher concentration of iron in HPS cultured HL-60 cells as compared with FBS cultured cells (1.72 +/- 0.02 mumol/g protein v . 1.32 +/- 0.14 mumol/g protein; P < 0.001) . Using desferrioxamine it was shown that TfR expression is dependent on the biological availability of iron in the cell . Consistent with the lower iron content in FBS cultured cells, we conclude that the cytotoxicity of desferrioxamine is dependent on the ability of cells to replenish cellular iron stores from the culture medium . Cells grown in FBS medium lack this ability and are therefore more susceptible to desferrioxamine. Mol Microbiol, 1993 Jan, 7(1), 151 - 7 Definition of nitrite and nitrate response elements at the anaerobically inducible Escherichia coli nirB promoter: interactions between FNR and NarL; Tyson KL et al.; Transcription initiation at the Escherichia coli nirB promoter is induced by anaerobic growth and further increased by the presence of nitrite or nitrate in the growth medium . Expression from this promoter is totally dependent on the transcription factor, FNR, which binds between positions -52 and -30 upstream of the transcription startsite . The 20 base pairs from position -79 to -60 contain an inverted repeat of two 10-base sequence elements that are related to sequences at the NarL-binding site at the E . coli narG promoter . Comparison of these, and sequence elements at other promoters regulated by NarL, suggests a consensus NarL-binding sequence . Mutations in the putative NarL-binding site at the nirB promoter decrease FNR-dependent anaerobic induction, suggesting that NarL acts as a helper to FNR during transcription activation . These mutations also suppress induction by nitrite: single mutations at symmetry-related positions have similar effects, whilst double mutations have more severe effects, probably because two NarL subunits bind to the inverted repeat . Disruption of narL decreases nitrite induction of the nirB promoter whilst not suppressing induction by nitrate, suggesting that there may be a second nitrate-responsive factor . Nitrate induction was, however, suppressed by double mutations at symmetry-related positions in the NarL-binding site, suggesting that this putative second factor may bind to sequences similar to those recognized by NarL. J Bacteriol, 1993 Jan, 175(1), 214 - 21 Osmotic repression of anaerobic metabolic systems in Escherichia coli; Gouesbet G et al.; The influence of the osmolarity of the growth medium on anaerobic fermentation and nitrate respiratory pathways was analyzed . The levels of several enzymes, including formate dehydrogenase, hydrogenase, and nitrate reductase, plus a nickel uptake system were examined, as was the expression of the corresponding structural and regulatory genes . While some functions appear to be only moderately affected by an increase in osmolarity, others were found to vary considerably . An increase in the osmolarity of the medium inhibits both fermentation and anaerobic respiratory pathways, though in a more dramatic fashion for the former . fnr expression is affected by osmolarity, but the repression of anaerobic gene expression was shown to be independent of FNR regulatory protein, at least for hyd-17 and fdhF . This repression could be mediated by the intracellular concentration of potassium and is reversed by glycine betaine. J Virol, 1993 Jan, 67(1), 277 - 87 Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation; Ensoli B et al.; During acute human immunodeficiency virus type 1 (HIV-1) infection or after transfection of the tat gene, Tat protein is released into the cell culture supernatant . In this extracellular form, Tat stimulates both HIV-1 gene expression and the growth of cells derived from Kaposi's sarcoma (KS) lesions of HIV-1-infected individuals (AIDS-KS cells) . Tat protein and its biological activities appear in the cell supernatants at the peak of Tat expression, when the rate of cell death is low (infection) or cell death is undetectable (transfection) and increased levels of cytoplasmic Tat are present . Tat-containing supernatants stimulate maximal AIDS-KS cell growth but only low to moderate levels of HIV-1 gene expression . This is due to the different concentrations of exogenous Tat required for the two effects . The cell growth-promoting effects of Tat peak at between 0.1 and 1 ng of purified recombinant protein per ml in the cell growth medium and do not increase with concentration . In contrast, both the detection of nuclear-localized Tat taken up by cells and the induction of HIV-1 gene expression or replication require higher Tat concentrations (> or = 100 ng/ml), and all increase linearly with increasing amounts of the exogenous protein . These data suggest that Tat can be released by a mechanism(s) other than cell death and that the cell growth-promoting activity and the virus-transactivating effect of extracellular Tat are mediated by different pathways. Yeast, 1993 Jan, 9(1), 77 - 84 In Saccharomyces cerevisiae, protein secretion into the growth medium depends on environmental factors; Rossini D et al.; In the budding yeast Saccharomyces cerevisiae the cell wall, mainly composed of mannoproteins and glucans, constitutes a barrier to protein excretion in the growth medium . In this paper we have studied the effects of different environmental parameters on excretion of Escherichia coli beta-galactosidase obtained by exploiting the glucoamylase II signal sequence . Excretion of the unglycosylated beta-galactosidase was detectable only in cells grown in rich medium, was affected by temperature (36 degrees C > 30 degrees C >> 24 degrees C) and slightly stimulated by reducing agents . On the contrary, glycosylated proteins, such as alpha-galactosidase and glucoamylase II, were excreted to a good extent under all tested conditions of medium composition, growth temperature and pH . These data indicate that optimization of environmental parameters may help the excretion of heterologous proteins, offering advantages for protein purification. J Pharmacol Exp Ther, 1993 Jan, 264(1), 463 - 8 Tetrahydroaminoacridine is neurotrophic and promotes the expression of muscarinic receptor-coupled phosphoinositide turnover in differentiating cerebellar granule cells; Sunaga K et al.; We have investigated whether 9-amino-1,2,3,4-tetrahydroacridine (THA), a drug with potential antidementia activity, has a trophic action on differentiating cerebellar granule cells by using the method of {3H}inositol incorporation into inositol-containing phospholipid . Addition of THA (30-50 microM) prevented the extensive neuronal degeneration which occurred in the growth medium containing "low" K+ (15 mM) . These effects were similar to the neuroprotective action caused by the presence of 100 microM N-methyl-D-aspartate (NMDA) . Neurotrophic effects of THA and NMDA on cells grown in low K+ were also demonstrated by direct microscopic examination of cellular morphology . Measurement of phosphoinositide (PI) response in the rescued cells indicated that NMDA modestly promoted the PI response to carbachol and norepinephrine but markedly stimulated the activity induced by glutamate . In contrast, although THA had little or no influence on the maturation of the norepinephrine- and glutamate-induced PI response, it selectively enhanced the activity stimulated by carbachol . Furthermore, the THA treatment drastically increased the Vmax value of carbachol-induced PI turnover with no significant alteration in the EC50 value . Scatchard analysis of the binding of N-{3H}methylscopolamine to intact granule cells indicated a selective increase in the maximum binding value in cells grown in THA-supplementing medium . These observations suggest that THA seems to selectively up-regulate muscarinic cholinergic receptors. Reg Immunol, 1993 Jan-Feb, 5(1), 18 - 27 Characterization of an inhibitory factor derived from epithelial cells of the small intestine; Llana T et al.; Rat small intestine epithelial cell (EC) culture conditioned media (ECCM) contains a factor that has inhibitory activity against: a) the response of freshly isolated lymphocytes to Concanavalin A (Con A) or IL-2, b) the proliferative response to antigen of primed lymphocytes, and c) the normal growth of transformed cell lines derived from several species . Inhibition is reversible, and not the result of a cytotoxic effect . The inhibitory activity is enterocyte derived, effective at low concentration (1-2% in growth media), and can be derived from freshly isolated EC . Biochemical analysis indicates the inhibitory activity is associated with a protein with an approximate molecular weight of 32 kd, and an isoelectric point (PI) in the range of 3-5 . The protein is trypsin sensitive, and labile with prolonged heating at 56 degrees C . The described activity differs from previously reported mucosally-derived inhibitory activities on the basis of molecular weight, and its ability to inhibit the growth of several cell lines . We suggest that this factor can provide the immunomodulatory activity necessary to produce the low level of intestinal T cell reactivity that is observed in vivo. Microbios, 1993, 74(298), 23 - 8 The in vitro lysozyme susceptibility of Candida species cultured in sucrose supplemented media; Samaranayake YH et al.; The in vitro sensitivity to lysozyme of twelve isolates of Candida (three isolates each of C . albicans, C . tropicalis, C . glabrata and C . krusei) pre-incubated in sucrose supplemented media, was determined by a growth inhibition assay . In general, C . albicans and C . tropicalis exhibited increased susceptibility to lysozyme as the sucrose concentration in the growth medium was reduced . Candida krusei demonstrated the reverse effect and C . glabrata isolates showed different trends . Although generally the Candida species examined were susceptible to lysozyme in the decreasing order of C . glabrata, C . albicans, C . tropicalis and C . krusei, differences in susceptibility were noted among different isolates within a given species . These results imply that salivary lysozyme and dietary carbohydrates may exert selective pressure on oral colonization by Candida species. Arch Dermatol Res, 1993, 285(7), 385 - 92 Growth and differentiation of normal human melanocytes in a TPA-free, cholera toxin-free, low-serum medium and influence of keratinocytes; Donatien P et al.; Melanocyte cultures were obtained from a modification of the keratinocyte culture system MCDB153 . Either promelanocytes or mature melanocytes were selected from epidermal cell primary cultures . Pure subcultures of actively dividing melanocytes of both types were grown in a low-serum medium totally deprived of TPA and cholera toxin called melanocyte growth medium (MGM) . Early passaged cells from MGM primary cocultures were similar to normal adult human melanocytes in vivo, exhibiting numerous melanosomes, strong dopa positivity and a high dendricity . The ability of MGM to support melanocyte growth was mainly a consequence of its basic composition, combined with a low serum concentration . Bovine pituitary extract significantly enhanced melanocyte growth . Using complete MGM, in the absence of mitogens and keratinocytes, cell growth was maintained, but the differentiation of melanocytes decreased . The presence of keratinocytes was found to promote melanocyte growth . The coculture system used strongly suggests the action of soluble keratinocyte-derived factors . Keratinocyte contact was necessary to sustain melanocyte dendricity and melanization . Melanization and dendricity behaved mostly as independent features when keratinocyte influence was withheld . Our results underline the essential role of keratinocytes in the regulation of melanocyte growth and differentiation in a physiological culture system. C R Acad Sci III, 1993, 316(5), 469 - 73 {A second gene involved in the formation of disulfide bonds in proteins localized in Escherichia coli periplasmic space}; Belin P et al.; A novel mutant of Escherichia coli was isolated whose phenotype is similar to those of dsbA strains . For instance, it is unable to express pH 2.5 acid phosphatase, glucose-1-phosphatase and alkaline phosphatase in the periplasmic space . The mutation lies at min 26.2 of the linkage map, and does not affect expression of DsbA . Addition of oxidized glutathione to the growth medium restores the wild-type phenotype in the mutant while this is not the case in a dsbA strain . The product of this new gene dsbX is thus actually involved in the formation of disulfide bridges in the periplasmic space but can only operate if DsbA is functional. Adv Exp Med Biol, 1993, 343, 319 - 26 IGFs and muscle differentiation; Florini JR et al.; The Role of IGFs in Myogenesis . Thus we are now convinced that the control of myogenesis by IGFs is a general phenomenon that occurs in all skeletal muscle cells, whether or not IGFs are added to the "differentiation" medium . We believe that several medium components contribute to the suppression of IGF-II expression in myoblasts incubated in high serum "growth" medium, and conclude that the IGF-I receptor mediates the feedback inhibition of IGF-II gene expression in muscle cells . Mechanism of Induction of Myogenesis by IGFs . The observations summarized here now permit a reasonably coherent overview of the stimulation of myogenic differentiation by the IGFs . It seems clear that all IGFs act by binding to the Type I IGF receptor, and that this process is inhibited to a significant extent by IGF binding proteins secreted by the target myoblasts . A major, but possibly not the only relevant effect of this binding is the induction of expression of the myogenin gene; this induction appears to require the presence of myf-5 protein, at least during the early part of the response . Cells capable of a mitogenic response undergo a round of division in response to IGF-I, thus delaying their entry into the final processes of postmitotic terminal differentiation . Other laboratories have shown that myogenin complexes with one or more widely occurring proteins such as E12 or E47 to form an active complex that interacts with CAnnTG elements in muscle specific genes, turning on expression of those genes and thus initiating the phenotype associated with terminally differentiated skeletal muscle. Neurotoxicology, 1993 Winter, 14(4), 381 - 6 S9 liver fraction is cytotoxic to neurons in dissociated culture; Kohn J et al.; Methods to evaluate the neurotoxicity of chemicals in vitro must take into account that many xenobiotics exert their toxic effects through metabolites . S9 fraction of liver homogenate has been used in cultures of bacterial and mammalian cells as a system for metabolic activation . The suitability of the S9 activation system for long-term neurotoxicity studies in vitro was investigated in dissociated cultures of murine spinal cord-dorsal root ganglia . Exposure to amounts of S9 greater than 0.07 mg S9 protein/ml of culture medium for 4 days or longer was cytotoxic to all types of neurons in the cultures . Non-neuronal cells tolerated higher exposures, but contained numerous cytoplasmic inclusions when 0.35 mg S9 protein was included in the medium . It was demonstrated that cytotoxicity was caused by the particulate, microsomal fraction of S9 . It is concluded that direct incorporation of S9 fraction in the growth medium (0.07 mg S9 protein/ml or greater) is not a suitable method of generating metabolites in dissociated cultures of central nervous system when several days are required to elicit a biological response . Cytotoxicity can be prevented by using tissue culture inserts to separate cultured cells from S9 particulate fraction by a microporous membrane. Arch Dermatol Res, 1993, 285(6), 347 - 51 Corticosteroids induce proliferation but do not influence TNF- or IL-1 beta-induced ICAM-1 expression of human dermal microvascular endothelial cells in vitro; Hettmannsperger U et al.; The effects of hydrocortisone, dexamethasone, betamethasone 17-valerate and clobetasol propionate at concentrations of 10(-5)-10(-12) M on the proliferation of human dermal microvascular endothelial cells (HDMEC) were studied in vitro . In addition, confluent HDMEC were treated with TNF (1000 U/ml) or IL-1 beta (1000 U/ml) alone or in combination with the corticosteroids (10(-5) M, 10(-6) M) for 24 h, and cytokine-induced expression of intercellular adhesion molecule-1 (ICAM-1) was assessed by immunocytochemistry . Controls were treated either with growth medium or with the corticosteroids alone . All tested corticosteroids stimulated HDMEC growth after 4 and 6 days of treatment in a dose-dependent manner, as assessed by 3H-thymidine incorporation and the 4-methyl-umbelliferyl heptanoate (MUH) assay . The minimal effective concentration was 10(-9) M for hydrocortisone, 10(-10) M for dexamethasone and betamethasone, and 10(-12) M for clobetasol . In untreated and in corticosteroid-treated cultures, less than 5% of the cells expressed ICAM-1 . TNF and IL-1 beta were strong inducers of ICAM-1 expression on 74% and 82% of the cells, respectively . None of the tested corticosteroids significantly influenced cytokine-induced ICAM-1 expression, suggesting that the anti-inflammatory effects of corticosteroids are not related to ICAM-1 modulation on HDMEC. Drug Metab Dispos, 1993 Jan-Feb, 21(1), 56 - 61 Inducibility of enzyme activities associated with the cytochrome P-450 1A family, ethoxyresorufin O-deethylase, and methoxyresorufin O-demethylase in human hepatocyte lines derived from normal liver tissue; Roberts EA et al.; Three human hepatocyte cultures have been developed from specimens of normal human liver, in each case from an infant or child, by coculture with liver epithelial cells from 6-day-old rat pups in a complex growth medium . In the established cultures hepatocytes predominate and maintain typical hepatocellular morphology by light microscopy and albumin secretion into supernatant medium . The activity of ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) basally and after treatment with polycyclic aromatic hydrocarbons was measured spectrofluorometrically in cell homogenates from each culture . Very low levels of EROD and MROD activity were found in each culture without induction {EROD: 1.78 +/- 0.71 (mean +/- SE) pmol/min/mg protein; MROD: 1.33 +/- 0.10 pmol/min/mg protein} . After treatment with 10 microM dibenz (a,h)anthracene x 48 hr, EROD and MROD activities rose approximately 20- to 50-fold . When the basal and induced enzyme activities were remeasured 2 months later, results were essentially the same . Incubation with 10 microM benz(a)anthracene x 48 hr also led to induction of EROD and MROD activities . We believe that these cultures can be regarded as human hepatocyte lines, which conserve human hepatic polycyclic aromatic hydrocarbon-inducible P-450s, most likely including P-450 1A2. Eur J Immunol, 1993 Jan, 23(1), 103 - 9 Rapid re-expression of CD45RC on rat CD4 T cells in vitro correlates with a change in function; Sarawar SR et al.; Rat CD4+ T cells are divided phenotypically by the anti-CD45RC monoclonal antibody OX22 into subsets with contrasting functions . Stimulation of T cells in vitro is known to induce a change in isoform from CD45RC+ to CD45RC- . We have investigated the in vitro conditions which promote a switch in isoform in the opposite direction . We observed that a majority of CD45RC- CD4 T cells (> 90%) spontaneously re-expressed CD45RC during the first 1-3 days of culture in both the presence and absence of alloantigen . The T cells remained CD45RC+ when cultured for 7 days in serum-free growth medium . However, alloantigen-activated lymphocytes, expressing the interleukin-2 receptor (IL-2R), downregulated CD45RC by day 4 and remained CD45RC- during the course of the experiment . Using mixtures of allotype-marked CD45RC+ and CD45RC- T cells, it was demonstrated that each subset showed comparable survival, IL-2R expression and time courses of activation in response to alloantigen . The repertoire of neither subset was, therefore, deficient in terms of allorecognition . The rapid re-expression of CD45RC in culture was accompanied by a change in function: CD45RC+ "converts", obtained by overnight culture of CD45RC- T cells, induced significantly higher graft-versus-host responses . Thus, the transition in culture from CD45RC- to CD45RC+ reflects a major functional reprogramming of the cell and not a trivial modulation of a surface antigen. Rom J Physiol, 1993 Jan-Jun, 30(1-2), 65 - 71 The effect of pulsed electromagnetic field (Diapulse) on cellular systems; Badea MA et al.; The effect of a 27.12 MHz pulsed electromagnetic field (Diapulse) on microbial growth is investigated . A strain of K 12 E . coli grown in complete Pennassay medium is subjected to Diapulse action for 30 min, at 8 hrs and 12 hrs of growth . In this experimental set-up, designed to be closed to the physiological conditions of open wounds, the Diapulse action does not promote any increase of cell population, indicating the safety of this type of therapy for wound healing process . The same K 12 E . coli strain grown in Pennassay medium for 2 hours is inoculated into a minimal growth medium and the lagless exponential growth thus obtained is followed by a spectrophotometric method . Diapulse field is applied to this lagless phase of cellular cultures at 30, 60, and 90 minutes after inoculation . A slight increase in the number of cells was observed at 2 and 4 hours after the Diapulse application, when the cultures were previously subjected to Diapulse action between the period of 60 and 90 minutes of their growth . A possible molecular mechanism for these effects is discussed. Cytotechnology, 1993, 13(3), 185 - 93 Dielectric spectroscopy of mammalian cells . 1 . Evaluation of the biomass of HeLa- and CHO cells in suspension by low-frequency dielectric spectroscopy; Cerckel I et al.; The capacitance of suspensions of CHO and HeLa cells (0.5-3 x 10(6) cells/ml) has been measured between 0.2 and 10 MHz . As frequencies decrease, there is a continuous increase in capacitance of both the cell suspension and the spent growth medium free of cells, a phenomenon which is partially attributed to an increased polarisation of the electrodes . At a given frequency, subtraction of the capacitance of the spent medium from that of the cell suspension allows one to determine the capacitance of the cells only . The intensity of this signal varies linearly with the biomass and cell size . At low frequencies such as those used in this study (0.25 MHz), where sensitivity is the highest, concentrations as low as 0.5 x 10(6) cells/ml can be accurately measured . Suggestions are made how to make these measures on-line, non-invasive and in real time. Mutat Res, 1993 Jan, 285(1), 13 - 8 The influence of growth medium on the yield of X-ray-induced chromatid exchanges in the presence and absence of aphidicolin; Moore RC et al.; The frequency of exchanges in JU56 cells irradiated in the G2 phase in the presence and absence of the polymerase inhibitor aphidicolin (APC), and in the presence of a range of concentrations of cysteine was measured . It was found that in the absence of cysteine, incubation for 2 h with APC had no effect on the yield . Addition of cysteine at concentrations of 50-250 mg/l reduced the frequency of exchanges, and at these concentrations the frequency was increased by incubation with APC . At higher concentrations, the yield was reduced and incubation with APC did not elevate it . In following experiments, it was found that incubation with cysteine for a period of longer than 10 minutes was necessary before APC affected the yield of exchanges. Cytotechnology, 1993, 13(3), 195 - 202 Dielectric spectroscopy of mammalian cells . 2 . Simultaneous in situ evaluation by aperture impedance pulse spectroscopy and low frequency dielectric spectroscopy of the biomass of HTC cells on Cytodex 3; Degouys V et al.; Low-frequency dielectric spectroscopy has been used in situ, i.e . while the cells are still attached to their microsupport, to monitor the changes of biomass accompanying the growth of anchorage-dependent cells . This method, when compared to Aperture Impedance Pulse Spectroscopy (also called electronic sizing), is characterized by a somewhat lower degree of resolution . Suggestions are made on how to determine the capacitance of the spent growth medium alone, still keeping the probe inserted in the bioreactor . This will make dielectric spectroscopy the first truly in situ, on-line, in real time, non-invasive measure of the biomass. Proc Natl Acad Sci U S A, 1992 Dec 15, 89(24), 11881 - 5 1H NMR studies on the catalytic subunit of aspartate transcarbamoylase; Cohen RE et al.; The 1H NMR spectrum of the catalytic subunit of Escherichia coli aspartate transcarbamoylase (EC 2.1.3.2) was simplified by using strains auxotrophic for the aromatic amino acids and a growth medium containing fully deuterated Trp, Phe, and His and partially deuterated Tyr . 1H resonances for Tyr in the catalytic trimer (M(r) = 10(5)) were partially resolved into five peaks at 27 degrees C, which above 50 degrees C were further resolved to give a distinct resonance for each of the eight Tyr residues in the polypeptide chain . Experiments on chemically modified catalytic subunits and on a mutant form in which Tyr-165 was converted to Ser-165 led to the assignment of resonances for Tyr-165, Tyr-240, and Tyr-185 . Binding of the substrate, carbamoyl phosphate, caused shifts of two of the unassigned resonances, and the subsequent binding of the aspartate analog succinate perturbed the resonances corresponding to Tyr-165 and Tyr-240 . The bisubstrate analog N-(phosphonacetyl)-L-aspartate produced a spectrum differing considerably from that caused by the combination of carbamoyl phosphate and succinate . The NMR spectrum for the Tyr-165-->Ser mutant trimer showed clearly that the single amino acid substitution caused conformational changes affecting the environment of residues remote from the position of the replacement . In contrast, the inactive mutant subunit in which Gly-128 was replaced by Asp exhibited a spectrum virtually identical to that of the wild-type protein . However, addition of the substrate carbamoyl phosphate caused a marked change in the spectrum of the mutant enzyme, whereas that of the wild-type trimer was altered only slightly, showing that the effect of the amino acid substitution was manifested in the NMR spectrum only with the liganded enzyme. J Biol Chem, 1992 Dec 15, 267(35), 25264 - 73 Distinct sterol and nonsterol signals for the regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase; Roitelman J et al.; The in vivo turnover rate of the endoplasmic reticulum protein 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the mevalonate (MVA) pathway, is accelerated when excess MVA or sterols are added to the growth medium of cells . As we have shown recently (Roitelman, J., Bar-Nun, S., Inoue, S., and Simoni, R . D . (1991) J . Biol . Chem . 266, 16085-16091), perturbation of cellular Ca2+ homeostasis abrogates the MVA-accelerated degradation of HMG-CoA reductase and HMGal . Here we show that, in contrast, the sterol-accelerated degradation of HMG-CoA reductase is unaffected by Ca2+ perturbation achieved either by Ca2+ ionophore or by inhibitors of the endoplasmic reticulum Ca(2+)-ATPase . The differential effects of Ca2+ perturbation can be attributed neither to global alteration in protein synthesis nor to inhibition of MVA conversion to sterols . Yet, such manipulations markedly reduce the incorporation of MVA into cellular macromolecules, including prenylated proteins . Furthermore, we directly demonstrate that MVA gives rise to at least two distinct signals, one that is essential to support the effect of sterols and another that operates independently of sterols . Our results indicate that the cellular signals operating in the MVA-accelerated turnover of HMG-CoA reductase are distinct from those involved in the sterol-regulated degradation . A working model for the degradation pathway is proposed. Proc Natl Acad Sci U S A, 1992 Dec 1, 89(23), 11327 - 31 Stable heparin-producing cell lines derived from the Furth murine mastocytoma; Montgomery RI et al.; Stable cell lines that synthesize heparin have been established from the Furth murine mastocytoma . The parental line (MST) divides in suspension every 14-18 h in growth medium supplemented with fetal bovine serum or defined growth factors . Adherent subclones were selected by adhesion to plastic culture vessels . Both adherent and nonadherent cells contain about 0.4 micrograms of glycosaminoglycan hexuronic acid per 10(6) cells, composed of 80% heparin and 20% chondroitin sulfate E . Deaminative cleavage of MST heparin by HNO2 at pH 1.5 released disaccharides that were similar in composition to those obtained from commercial heparin, except that disaccharides containing 3,6-O-desulfated GlcN units were not found . Greater than 90% of the glycosaminoglycans were stored in cytoplasmic granules, and challenge of the cells with dinitrophenylated bovine serum albumin and anti-dinitrophenyl IgE released a portion of the stored material . Growth studies of subclones showed that MST cells tolerate a 10-fold variation in glycosaminoglycan content . Incubation of cells with sodium chlorate reduced glycosaminoglycan sulfation by > 95% without affecting cell growth . Thus, granule glycosaminoglycans appear to be nonessential for growth of MST cells. Gene, 1992 Dec 1, 122(1), 219 - 23 An inducible expression system for the production of human lactoferrin in Aspergillus nidulans; Ward PP et al.; The production and secretion of human lactoferrin (hLF) in Aspergillus nidulans is described . The hLF cDNA was expressed under the control of the strong ethanol-inducible alcohol dehydrogenase (alcA) promoter . Recombinant hLF (re-hLF) is produced at levels up to 5 micrograms/ml . Approximately 30% of the re-hLF produced in this system is secreted into the growth medium . The re-hLF is indistinguishable from native hLF with respect to size and immunoreactivity . Furthermore, re-hLF is functional by the criterion of iron-binding capacity . The A . nidulans expression system offers an inexpensive, convenient method for the controlled production of mg amounts of biologically active mammalian glycoproteins. J Invest Dermatol, 1992 Dec, 99(6), 683 - 90 HMEC-1: establishment of an immortalized human microvascular endothelial cell line; Ades EW et al.; The study of human microvascular endothelial cells has been limited, because these cells are difficult to isolate in pure culture, are fastidious in their in vitro growth requirements, and have a very limited lifespan . In order to overcome these difficulties, we have transfected human dermal microvascular endothelial cells (HMEC) with a PBR-322-based plasmid containing the coding region for the simian virus 40 A gene product, large T antigen, and succeeded in immortalizing them . These cells, termed CDC/EU.HMEC-1 (HMEC-1), have been passaged 95 times to date and show no signs of senescence, whereas normal microvascular endothelial cells undergo senescence at passages 8-10 . HMEC-1 exhibit typical cobblestone morphology when grown in monolayer culture, express and secrete von Willebrand's Factor, take up acteylated low-density lipoprotein, and rapidly form tubes when cultured on matrigel . HMEC-1 grow to densities three to seven times higher than microvascular endothelial cells and require much less stringent growth medium . HMEC-1 will grow in the absence of human serum, whereas microvascular endothelial cells require culture medium supplemented with 30% human serum . These cells express other cell-surface molecules typically associated with endothelial cells, including CD31 and CD36 and epitopes identified by monoclonal antibodies EN4 and PAL-E . They also express the cell adhesion molecules ICAM-1 and CD44 and following stimulation with interferon-gamma express major histocompatibility complex class II antigens . HMEC-1 specifically bind lymphocytes in cell adhesion assays . Thus HMEC-1 is the first immortalized human microvascular endothelial cell line that retains the morphologic, phenotypic, and functional characteristics of normal human microvascular endothelial cells. J Bacteriol, 1992 Dec, 174(23), 7743 - 9 Escherichia coli is able to grow with negligible sodium ion extrusion activity at alkaline pH; Ohyama T et al.; The Escherichia coli mutant NM81, which is deficient in the nhaA gene for the sodium/proton antiporter, still has a sodium ion extrusion activity because of a second antiporter encoded by nhaB (E . Padan, N . Maisler, D . Taglicht, R . Karpel, and S . Schuldiner, J . Biol . Chem . 264:20297-20302, 1989) . By chance, we have found that E . coli pop6810 already contains a mutation affecting the sodium ion circulation, probably in or near nhaB, and that its delta nhaA mutant, designated RS1, has no sodium ion extrusion activity at alkaline pH . The growth of RS1 was inhibited completely by 0.1 M sodium, whereas growth inhibition of NM81 was observed only at sodium concentrations greater than 0.2 M . RS1 grew at a normal rate in an alkaline medium containing a low sodium concentration . Furthermore, RS1 grew with a negligible proton motive force in the alkaline medium containing carbonyl cyanide m-chlorophenylhydrazone . The transport activities for proline and serine were not impaired in RS1, suggesting that these transport systems could be driven by the proton motive force at alkaline pH . These findings led us to conclude that the operation of the sodium/proton antiporter is not essential for growth at alkaline pH but that the antiporter is required for maintaining a low internal sodium concentration when the growth medium contains a high concentration of these ions. Yonsei Med J, 1992 Dec, 33(4), 344 - 50 Culture of melanocytes obtained from normal and vitiligo subjects; Im S et al.; The development of human melanocyte culture in vitro from normal adult skin and uninvolved skin of vitiligo patients is essential to investigate the mechanism of depigmentation in vitiligo and other pigmentary dermatoses . By using selective growth and long-term maintenance conditions, we selectively cultured melanocytes derived from normal foreskins and arm skins, and uninvolved foreskins and arm skins of vitiligo patients . The melanocytes of the arm skins were successfully cultured from the roofs of suction blisters . Melanocyte Growth Media (MGM) consisting of MCDB-153 formulation with basic fibroblast growth factor (bFGF), bovine pituitary extract (BPE), insulin, hydrocortisone, phorbol 12-myristate 13-acetate (PMA) and 10% human AB serum was sufficient to grow the melanocytes from normal and vitiligo donors . Melanocytes from uninvolved skin of vitiligo donors showed no different morphologic features, initial seeding capacity and population doubling time compared with those from normal skin . Melanocytes from both cell types grew without any lag period for more than 6 months (6-11 passages) . Melanocytes obtained from foreskins had higher initial seeding capacity and shorter population doubling time than those obtained from arm skins using suction-blistered roofs . Our results suggest that the culture method using suction blisters may be a simple and easy way to obtain melanocytes . In addition, vitiligo melanocytes can be successfully cultured with appropriate growth conditions and may show no defective growth patterns . This culture system will be applied to investigate the basic pathophysiology of vitiligo and other various pigmentary dermatoses. Biofactors, 1992 Dec, 4(1), 47 - 9 Variable alpha-tocopherol stimulation and protection of glutathione peroxidase activity in established and malignant fibroblasts; Hu WL et al.; Studies on glutathione (GSH) metabolism in an established baby hamster kidney fibroblast cell line (BHK-21/C13) and in its polyoma virus-transformed counterpart (BHK-21/PyY) have revealed a significant stimulation of intracellular GSH peroxidase (GSHpx) activity (selenium-independent plus selenium-dependent) by alpha-tocopherol supplementation (14 microM) . This stimulation was found to be much greater in the transformed cells . Other GSH-requiring enzyme activities (i.e . GSH reductase and GSH S-transferase) were unaltered by alpha-tocopherol treatment, suggesting a degree of specificity in its action on GSHpx . In unsupplemented growth media, the GSHpx activity in both cell lines was significantly decreased by oxidative stress . However, the same stress applied to the alpha-tocopherol-supplemented cells had no effect on the stimulated GSHpx activity, suggesting that some protection was afforded by the alpha-tocopherol. Melanoma Res, 1992 Dec, 2(5-6), 321 - 6 Potentiation of radiation lethality in mouse melanoma cells by mild hyperthermia and chloroquine; Djordevic B et al.; To test the hypothesis that radiosensitization by combined mild hyperthermia and chloroquine may be increased by the presence of melanin in treated cells, Cloudman melanotic mouse melanoma S91/6 cells, and the amelanotic S91/amel cells were incubated during a 3 h post-irradiation period with 0.03 mM chloroquine at 41 degrees C . A considerable increase in radiation lethality was observed (radiation potentiation factor > 1.6) in both cases . Addition of 0.1 mM isobutyl-methyl xanthine (IBMX), a promoter of melanin synthesis, to the growth medium of S91/6 cells 10 days before irradiation, did not further increase the lethality of radiation followed by combined heat and chloroquine treatment . Under these conditions, toxicity to unirradiated cells was slight . On the other hand, 10 microM chloroquine showed similar toxicity to unirradiated B-16 mouse melanoma cells, but did not increase radiation lethality . Factors other than melanin content therefore play a role in the potentiation of radiation lethality by mild hyperthermia and chloroquine. Mutat Res, 1992 Dec, 298(2), 97 - 103 Mutagenesis and comutagenesis by lead compounds; Roy NK et al.; We have previously reported that lead(II) is weakly mutagenic to Chinese hamster V79 cells . A transgenic cell line G12 containing a single copy of the E . coli gpt gene was developed in this laboratory from Chinese hamster V79 cells . The gpt locus in the G12 cells is more mutable by radiation and oxidative agents compared with the endogenous hprt locus of wild-type V79 cells . We have investigated the mutagenicity of two lead compounds at the gpt locus in G12 cells . Only at a toxic dose is lead acetate significantly mutagenic to G12 cells . Lead nitrate is not significantly mutagenic at any dose . Although both compounds are water-soluble, lead acetate, but not lead nitrate, forms a fine white insoluble precipitate upon addition to growth medium . A nick translation assay on cells treated with lead compounds and then permeabilized indicated that lead nitrate and, to a greater extent, lead acetate causes the appearance of nicks in chromosomal DNA . Lead ions in the presence of hydrogen peroxide, but not alone, introduced nicks into supercoiled plasmid DNA in vitro, suggesting that lead ions can partake in a Fenton reaction and thereby damage DNA . At lower nonmutagenic concentrations, lead acetate enhances the mutagenicity of MNNG and ultraviolet light . DNA damage by ultraviolet light is not enhanced by lead ions in vitro . Our data support the concept that non-toxic concentrations of lead(II) can inhibit DNA repair . Thus, at biologically relevant doses, lead(II) could act as a comutagen and possibly a cocarcinogen, but is not likely to act as an initiating genotoxic carcinogen. Proc Natl Acad Sci U S A, 1992 Nov 15, 89(22), 10807 - 11 Covalent modification of proteins by ligands of steroid hormone receptors; Takahashi N et al.; Retinoylation, acylation with retinoic acid (RA), is a covalent modification of proteins occurring in a variety of eukaryotic cell lines . In this study, we found that proteins in HL-60 cells were labeled by 17 beta-{3H}estradiol (E2), {3H}progesterone (Pg), 1 alpha,25-dihydroxy{3H}vitamin D3 {1,25(OH)2D3}, {125I}triiodothyronine (T3), {125I}thyroxine (T4), and {3H}prostaglandin E2 (PGE2) . All of these hormones, except PGE2, are ligands of the steroid hormone receptor family . Addition to the growth medium of 5 microM ketoconazole, an inhibitor of cytochrome P450-dependent enzymes, increased about 2-fold the labeling of proteins by T3, T4, 1,25(OH)2D3, and PGE2 . In contrast, ketoconazole did not change markedly the extent of labeling by RA, E2, or Pg . Alkaline methanolysis, which cleaves ester bonds, released variable percentages of the radioactive ligands bound to protein . These values were about 80% for RA and PGE2; 50% for T3, T4, and Pg; and 20% for E2 and 1,25(OH)2D3 . Treatment with thioether-cleavage reagents, iodomethane or Raney nickel catalyst, released < 2% of the covalently bound ligands . Two-dimensional polyacrylamide gel electrophoresis patterns of labeled proteins were unique for each ligand . Proteins of M(r) 47,000 and 51,000 were labeled by RA, E2, T3, and T4 . These proteins had the same mobilities as RI and RII, the cAMP-binding regulatory subunits of type I and type II cAMP-dependent protein kinases . 1,25(OH)2D3 also bound to proteins of M(r) 47,000 and 51,000 . However, these proteins had pI values different from those of RI or RII . These results suggest that some activities of ligands of the steroid hormone receptor family and of PGE2 may be mediated by their covalent modification of proteins. J Biol Chem, 1992 Nov 15, 267(32), 23261 - 8 Processing and secretion of tumor necrosis factor alpha in endotoxin-treated Mono Mac 6 cells are dependent on phorbol myristate acetate; Pradines-Figueres A et al.; Lipopolysaccharide (LPS, endotoxin) is a potent stimulator of tumor necrosis factor alpha (TNF alpha) synthesis and secretion in mouse macrophage tumor cells (Golenbock, D . T., Hampton, R . Y., Qureshi, N., Takayama, K., and Raetz, C . H . R . (1991) J . Biol . Chem . 266, 19490-19498) . In contrast, addition of LPS (10 ng/ml) to human monomyelocytic (Mono Mac 6) cells induces very little production of TNF alpha, as judged by immunoassay of the growth medium . When 30 ng/ml 4-beta-phorbol-12-myristate 13-acetate (PMA) is added together with LPS, large amounts of TNF alpha are secreted . PMA alone is inactive . Maximal TNF alpha levels in the medium are achieved at 1 ng/ml of LPS . Protein kinase C inhibitors, such as H7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine), staurosporine, and sphingosine, reduce TNF alpha secretion stimulated by PMA . The effect of PMA has been investigated at each stage of TNF alpha biogenesis . Treatment of Mono Mac 6 cells with LPS alone results in rapid, transient, and full expression of TNF alpha mRNA . Concomitant addition of PMA does not increase TNF alpha mRNA synthesis any further, but it prolongs the half-life of TNF alpha mRNA about 3-fold . However, mRNA stabilization does not account for the striking effect of PMA on TNF alpha secretion . Analysis of TNF alpha synthesis and secretion by immunoprecipitation indicates that LPS alone is fully effective in stimulating the formation of the intracellular 26-kDa TNF alpha precursor . LPS alone is not sufficient to allow processing of the precursor and secretion of mature 17-kDa TNF alpha . The rate of TNF alpha secretion observed immediately after the addition of PMA to LPS-pretreated cells is similar to the maximum rate from LPS/PMA-treated cells, but without the lag observed in cells after being exposed to LPS and PMA simultaneously . In summary, PMA is required for the completion of TNF alpha precursor processing and secretion in LPS-treated human Mono Mac 6 cells, whereas murine RAW cells are able to complete the terminal steps of TNF alpha processing in the absence of PMA. J Chromatogr, 1992 Nov 6, 582(1-2), 253 - 7 High-performance liquid chromatographic monitoring of metabolic products resulting from the treatment of mouse hepatoma cells with N6-cycloalkylated nucleosides; Thedford R et al.; High-performance liquid chromatography was used to analyze cell lysates and growth medium of mouse hepatoma cells, separately treated with N6-cyclopropyl-, N6-cyclobutyl-, and N6-cyclopentyladenosines, in an effort to gain insight into the mechanism by which these modified nucleosides exert their cytotoxic effect(s) . The corresponding 5'-monophosphate of the respective modified nucleoside was detected in the separate cell lysate samples . Both the modified nucleoside and its corresponding 5'-monophosphate were detected in the separate growth medium samples and their relative concentrations therein were determined . These results indicate that the cytotoxicity of these N6-cycloalkylated nucleosides may be attributed to their 5'-monophosphates within the cells. In Vitro Cell Dev Biol, 1992 Nov-Dec, 28A(11-12), 730 - 4 Relative promoter activity in human mammary epithelial cells assayed by transient expression; Huper G et al.; Chimeric DNA expression vectors containing regulatory sequences proximal to the 5' end of coding sequences for mammalian genes provide valuable tools to study gene expression . Genes coding for easily measured products (reporter genes) can be used to study promoter strength and regulation of gene expression after transient expression of promoter-reporter constructs in mammalian cells . To determine the strength of a variety of mammalian and viral promoter-enhancer sequences in primary cultures of human mammary epithelial cells (HMEC), these sequences were fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and transfected into HMEC using strontium phosphate . The long terminal repeat (LTR) of the endogenous murine leukemia virus AKR-623 was the most potent promoter of transient CAT expression in HMEC . A number of commonly available promoter sequences displayed a wide range of activities in these cells . The glucocorticoid responsive LTR promoter from the murine mammary tumor virus modulated expression of CAT and was sensitive to the concentration of dexamethasone in the growth media . In a similar fashion, the regulatory sequences from the murine metallothionein-1 gene retained responsiveness to zinc concentration in the growth media. Mol Gen Genet, 1992 Nov, 235(2-3), 242 - 6 Leucine and serine induce mecillinam resistance in Escherichia coli; Bouloc P et al.; We have previously shown that resistance to the beta-lactam mecillinam in Escherichia coli can be brought about by a high ppGpp pool, as observed under conditions of partial amino acid starvation and RelA-dependent induction of the stringent response . We show here that our E . coli wild-type strain, which is sensitive to mecillinam on minimal glucose plates, becomes resistant in the presence of L-leucine or L-serin