Hyaluronate synthetase inhibition by normal and transformed human fibroblasts during growth reduction

To establish the relation of glycosaminoglycan synthesis to cell proliferation, we investigated the synthesis of individual glycosaminoglycan species by intact cells and in a cell-free system, using normal and transformed human fibroblasts under differing culture conditions. Reducing serum concentration brought about a marked decline in the synthesis of hyaluronate (HA) as well as cell proliferation on both normal and transformed cells. Both HA synthesis and proliferation decreased with increasing cell densities markedly (in inverse proportion to cell density) in normal cells but gradually in transformed cells. This noticeable congruity of the changes in HA synthesis and proliferation indicates that the change in HA synthesis is related primarily to cell proliferation rather than to cell density or cellular transformation. Examination of HA synthesis in a cell-free system demonstrated that the activity of HA synthetase also fluctuated in conjunction with cell proliferation. Furthermore, growth-reduced cells (except crowded transformed cells) inhibited cell-free HA synthesis and this inhibition was induced coincidentally with a decrease in both HA synthetase activity and proliferation. These findings suggest that the change in HA synthesis is significant in the regulation of cell proliferation.     

Serine transhydroxymethylase in methionine biosynthesis in Saccharomyces cerevisiae

Serine transhydroxymethylase appears to be the first enzyme in the synthesis of the methyl group of methionine. Properties of serine transhydroxymethylase activity as assayed by the production of formaldehyde were correlated with properties of cell-free extracts for the methylation of homocysteine deriving the methyl group from the beta-carbon of serine. The reaction required pyridoxal phosphate and tetrahydrofolic acid, and was characterized in cell-free extracts with respect to Michaelis constant, pH optimum, incubation time, and optimal enzyme concentration. The activity was sensitive to inhibition by methionine, and to a much greater extent by S-adenosylmethionine. Serine transhydroxymethylase and the methylation of homocysteine reactions were not repressed by methionine and were stimulated by glycine. The activities of cell-free extracts for these reactions were significantly higher in cells in exponential than in stationary growth. When cells were grown in 10 mm glycine, the activities remained high throughout the culture cycle. The data indicated that glycine rather than methionine is involved in the control of the formation of the enzyme.     

Reversal of 2-bromoethanesulfonate inhibition of methanogenesis in Methanosarcina sp.

2-Bromoethanesulfonate (BES) inhibition of methanogenesis from methanol by resting-cell suspensions or cell extracts of Methanosarcina was reversed by coenzyme M. BES inhibition of methylcoenzyme M methylreductase activity in cell-free extracts was reversed by methylcoenzyme M but not by coenzyme M. Methanol/coenzyme M methyltransferase activity was not inhibited by 10 microM BES. Inhibition of methylreductase by BES and 3-bromopropionate was competitive with methylcoenzyme M, but inhibition by 2-bromoethanol exhibited mixed kinetics. The Ki values for the inhibitors in cell-free extracts were similar to the concentrations which inhibited intact cells. BES-resistant mutants of strain 227 were apparently permeability mutants because in vitro assays showed that mutant and parent strain methylreductases were equally sensitive to BES.     

Mechanism of Interferon Action: Inhibition of Viral Messenger Ribonucleic Acid Translation in L-Cell Extracts

Encephalomyocarditis (EMC) virus ribonucleic acid (RNA) stimulated the incorporation of (14)C-amino acids into polypeptides in cell-free systems using preincubated S10 extracts from L cells. Incorporation was linear for over 2 hr. Analysis of the tryptic peptides derived from the polypeptide products formed in response to EMC RNA showed them to be virus specific. The major product, a polypeptide of 140,000 in molecular weight, migrated on sodium dodecyl sulfate-polyacrylamide gels with one of the virus-specific polypeptides present in EMC-infected cells. A minor component of molecular weight about 230,000 may correspond to the product of complete translation of the EMC virus genome. Little or no effect of interferon or vaccinia virus infection was observed in the preincubated, cell-free system. The EMC RNA-stimulated incorporation of (14)C-amino acids into polypeptides was not inhibited in extracts derived from L cells early in virus infection, from interferon-treated cells, or from cells subjected to both treatments. Interferon treatment did appear to have a slight inhibitory effect on chain elongation in this system. However, treatment of cells with highly purified interferon before virus infection caused a decrease of about 80% in the capacity of non-preincubated cell extracts to translate added EMC RNA. This effect did not extend to the translation of polyuridylic acid and could be reversed by preincubation of the extracts at 37 C for 20 min. The inhibition of translation was manifest at interferon concentrations as low as 5IU/ml, and in this respect closely paralleled the inhibition of virus growth. Inactivation of the antiviral activity of the interferon by heating or digestion with trypsin also abolished the effect on cell-free protein synthesis. The EMC-specific polypeptides formed in reduced amounts in extracts of interferon-treated vaccinia-infected cells were smaller than those formed in extracts of untreated, vaccinia-infected cells. Thus, inhibition of initiation or elongation of polypeptides, or both, can be demonstrated in cell-free systems employing non-preincubated extracts from interferon-treated, virus-infected cells. These results indicate that antiviral activity of interferon is directed against the translation of viral messenger RNA.     

Urea-hydrolyzing activity of a T-strain mycoplasma: Ureaplasma urealyticum.

The urea-hydrolyzing activity of a T-strain mycoplasma was studied in experiments using whole cells and cell-free enzyme preparations by measuring the release of 14CO2 from [14C]urea. Under the conditions used, the urea concentration optimum is approximately 5.6 X 10(-3) M urea. The activity is soluble and not membrane bound. It is stable at -70 C for several weeks but is more labile at higher temperatures. The pH optimum is between 5.0 and 6.0. The effect of several inhibitors on the activity was tested and revealed similarities, as well as differences, between T-strain mycoplasma urease activity and the urease activity of other organisms and plants.     

The recA+ gene product is more important than catalase and superoxide dismutase in protecting Escherichia coli against hydrogen peroxide toxicity.

Various deoxyribonucleic acid repair-deficient strains of Escherichia coli K-12 were exposed to hydrogen peroxide under anaerobic conling of the strains was determined. The level of catalase, peroxidase, and superoxide dismutase in cell-free extracts of the strains as well as the capacity of intact cells to decompose hydrogen peroxide were assayed, recA strains were more rapidly killed than other strains with deoxyribonucleic acid repair deficiencies. There was no correlation between the killing rate of the strains and the capacity of intact cells to decompose hydrogen peroxide or the level of catalase and superoxide dismutase in cell-free extracts. The level of peroxidase in cell-free extract was too low to be determined.     

Ribonucleic acid synthesis in streptomyces antibioticus: Stable ribonucleic acid species synthesized by young and old cells

In studies of RNA synthesis by intact cells and cell-free extracts of $\text{Streptomyces antibioticus}$, it has been found that 48 hr cells (producing actinomycin) and cell-free extracts are less efficient than 12 hr cells (not producing actinomycin) and extracts in the synthesis of RNA. Analysis of the products of “in vivo” and “in vitro” RNA synthesis by sucrose gradient centrifugation reveals that both 12 and 48 hr cultures and cell-free extracts synthesize ribosomal RNA as well as RNA species of higher and lower molecular weights. However, 50–60% of the ³H-uridine labelled RNA synthesized by intact cells sediments as rRNA as compared with only 5–10% of the cell-free product. The addition of 2 × 10^?5 M actinomycin D to incubation mixtures for cell-free RNA synthesis does not significantly alter the relative amounts of the various RNA species synthesized by 12 or 48 hr extracts.     

Urease testing and yeast taxonomy

When urease production was assayed by the hydrolysis of [14C]urea, all basidiomycetous yeasts tested, including the Cryptococcus vishniacii complex (previously reported urease negative), produced significant amounts of 14CO2. The Schizosaccharomycetaceae were the only urease-positive ascomycetous yeasts tested. Yarrowia lipolytica was urease negative. The stoichiometry of [14C]urea hydrolysis paralleled by Roberts' rapid urea hydrolysis (RUH) test indicated that causes of anomalous results in conventional urease testing include acidification and alkalinization of the test medium by products of endogenous metabolism and autolysis rather than urease activity. Anomalous results also occurred when cells were grown on media containing the chelating agent ethylenediaminetetraacetic acid (EDTA) prior to RUH. The addition of EDTA to a complex natural medium inhibited urease production in all yeasts reportedly growing at 35 degrees C (and all other yeasts tested), except Filobasidiella (Cr.) neoformans var. neoformans (NIH 12). The RUH test could differentiate at the varietal level: Fil. (Cr.) neoformans var. neoformans was about 10 times more resistant to EDTA in media used for the growth of cells prior to RUH testing than was Fil. neoformans var. bacillispora (Cr. neoformans var. gattii) (NIH 191). Urease production by Fil. neoformans var. bacillispora was specifically restored to half maximal activity by the addition of 22 microM Ni+2 (as NiCl2) to a growth medium containing 0.100 mM EDTA.     

Hexavalent Chromium Reduction by Immobilized Cells and the Cell-Free Extract of Bacillus sp. ES 29

Bacillus sp. ES 29 (ATCC: BAA-696) is an efficient chromate reducing bacterium. We evaluated hexavalent chromium (Cr[VI]) reduction by immobilized intact cells and the cell-free enzyme extracts of Bacillus sp. ES 29 in a bioreactor system. Influences of different flow rates (3 to 14 mL h-1), Cr(VI) concentration (2 to 8 mg L-1), and immobilization support materials (Celite, amberlite, and Ca-alginate) on Cr(VI) reduction were examined. Both immobilized intact cells and the cell-free extract of Bacillus sp. ES 29 displayed substantial Cr(VI) reduction. Increasing flow rates from 3 to 6 mL h-1 did not affect the rate of Cr(VI) reduction, but above 6 mL h-1, the Cr(VI) reducing capacity of the immobilized intact cells and cell-free extract of Bacillus sp. ES 29 decreased. With both intact cells and the cell-free extracts, the rate of Cr(VI) reduction was inversely related to the concentration. Intact cells immobilized to Celite displayed the highest rate (k = 0.443 at 3 mL h-1) of Cr(VI) reduction. For the immobilized cell-free extract, maximal reduction (k = 0.689 at 3 mL h-1) was observed with Ca-alginate. Using initial Cr(VI) concentrations of 2 to 8 mg L-1 at flow rates of 3 to 6 mL h-1 both immobilized intact cells and the cell-free extracts reduced 84 to 98% of the influent Cr(VI). Results indicate that immobilized cells and the cell-free extracts of Bacillus sp. ES 29 could be used for large-scale removal of Cr(VI) from contaminated water and waste streams in containment systems.     

Hexavalent Chromium Reduction by Immobilized Cells and the Cell-Free Extract of Bacillus sp. ES 29

Bacillus sp. ES 29 (ATCC: BAA-696) is an efficient chromate reducing bacterium. We evaluated hexavalent chromium (Cr[VI]) reduction by immobilized intact cells and the cell-free enzyme extracts of Bacillus sp. ES 29 in a bioreactor system. Influences of different flow rates (3 to 14 mL h?1), Cr(VI) concentration (2 to 8 mg L?1), and immobilization support materials (Celite, amberlite, and Ca-alginate) on Cr(VI) reduction were examined. Both immobilized intact cells and the cell-free extract of Bacillus sp. ES 29 displayed substantial Cr(VI) reduction. Increasing flow rates from 3 to 6 mL h?1 did not affect the rate of Cr(VI) reduction, but above 6 mL h?1, the Cr(VI) reducing capacity of the immobilized intact cells and cell-free extract of Bacillus sp. ES 29 decreased. With both intact cells and the cell-free extracts, the rate of Cr(VI) reduction was inversely related to the concentration. Intact cells immobilized to Celite displayed the highest rate (k = 0.443 at 3 mL h?1) of Cr(VI) reduction. For the immobilized cell-free extract, maximal reduction (k = 0.689 at 3 mL h?1) was observed with Ca-alginate. Using initial Cr(VI) concentrations of 2 to 8 mg L?1 at flow rates of 3 to 6 mL h?1 both immobilized intact cells and the cell-free extracts reduced 84 to 98% of the influent Cr(VI). Results indicate that immobilized cells and the cell-free extracts of Bacillus sp. ES 29 could be used for large-scale removal of Cr(VI) from contaminated water and waste streams in containment systems.     

A Possible Role for Urease as a Storage Protein in Aspergillus tamarii

A marked decrease in mycelial urease activity during the endogenousphase of undifferentiated Aspergillus tamariicultures was foundto be independent of preparative procedures but related to thedepletion of external nutrients. The enzyme, which was synthesizedduring the active growth stage, was produced in similar quantitieswith ammonium or urea as sole nitrogen source and at its peakrepresented c. 8·5 per cent of the total soluble proteinpool of the mycelium. It was found to show maximum activityat pH 8·20–8·65 when measured in cell-free,phosphate-buffered extracts. Isolation of urease from differentstages of the endogenous phase by affinity chromatography hasshown that the observed decrease in activity was due to breakdownof the enzyme protein in mature cultures, followed by the progressivedeactivation of residual enzyme during the autolytic stage.Since selective inhibition of 80–90 per cent of activityby acetohydroxamic acid in media containing urea as the onlynitrogen source or total repression of urease synthesis by L-histidinein ammonium-grown cultures did not interfere with normal growth,it was concluded that in A. tamarii urease fulfils the functionof a storage protein with a measure of catalytic activity. Aspergillus tamarii, urease, storage protein, nitrogen metabolism     

Inhibition of growth of mammalian cell cultures by extracts of arginine-utilizing mycoplasmas

Mechanisms of the inhibition of growth of mammalian cell cultures caused by mycoplasmal infection were investigated by using cell-free extracts of 14 species of mycoplasmas. In four mammalian cell lines tested, the growth of two cell lines, FM3A and MDCK, was inhibited by the extracts of arginine-utilizing mycoplasmas, whereas that of the other two cell lines, Vero and LLC-MK2, was not inhibited by extracts of either arginine- or glucose-utilizing mycoplasmas. These results suggest that there are two types of cell cultures, one susceptible and the other insusceptible to arginine-utilizing mycoplasmas. In a series of experiments using FM3A cells, it was found that the growth inhibition caused by the extracts of arginine-utilizing mycoplasmas was due to removal of arginine from the medium by the action of arginine deiminase present in the extracts and that none of the metabolic products of arginine had any effect on the growth. A highly positive correlation (r = 0.96, P less than 0.01) was observed between the activity of arginine deiminase and the growth-inhibiting activity of extracts of arginine-utilizing mycoplasmas.     

Ureolytic activity of Nitellopsis obtusa (Characeae)

The decomposition of urea by Nitellopsis obtusa from Characeae was investigated. The intact cells were exposed to the inhibition by two typical urease inhibitors: boric acid and fluoride ion, used as a criterion to define if urease or UAL-ase is responsible for the ureolytic activity of the algae. It was found that boric acid and fluoride ion are simple competitive and slow-binding competitive inhibitors of Nitellopsis obtusa enzyme respectively, which is the response characteristic of urease. The inhibition constants equal to 2.3 and 0.1 mM for boric acid and fluoride ion, when compared to those of jack bean urease, indicate that in the observed kinetic behaviour of Nitellopsis obtusa urease partake transport processes taking place in the intact cells.     

Arachidonate activation of the neutrophil NADPH-oxidase. Synergistic effects of protein phosphatase inhibitors compared with protein kinase activators.

Arachidonate activation of the NADPH-oxidase in intact neutrophils and in a cell-free O2- generation system was compared to synergistic activation in response to arachidonate and agents that effect protein phosphorylation. In intact neutrophils, suboptimal doses of retinal which increase protein phosphorylation, or 4B-phorbol 12-myristate 13-acetate (PMA) an activator of protein kinase C, induced minimal O2- release, but primed neutrophils to release enhanced amounts of O2- in response to 2.5 microM arachidonate. In contrast to retinal or PMA, okadaic acid, a specific inhibitor of serine/threonine protein phosphatases, did not induce any release of O2-, but significantly increased the maximal rate and duration of O2- release in response to arachidonate. In the cell-free system, only arachidonate induced O2- generation. Consistent with previous findings, activation of the cell-free system was dependent of the presence of light membranes, cytosol, NADPH, Mg2+, and 82 microM arachidonate. Pretreatment of neutrophils with suboptimal doses of PMA or retinal had little effect on the arachidonate-stimulated release of O2- in cell-free preparations of these cells. However, cytosol (but not light membranes) from PMA or retinal-primed neutrophils was more effective in completing resting membrane NADPH-oxidase activity when compared to cytosol from resting cells. The addition of protein kinase C inhibitors staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine decreased the effectiveness of PMA-primed cytosol to complete the cell-free system, but had little effect on cytosol obtained from cells primed with retinal. The addition of protein phosphatase inhibitors, p-nitrophenyl phosphate or okadaic acid to neutrophil cavitates increased 3-fold the release of O2- in cell-free preparations of these cells. Okadaic acid and p-nitrophenyl phosphate also increased the effectiveness of both cytosol and light membranes to complete the cell-free system when combined with cytosol or light membranes from resting neutrophils, respectively, indicating that both fractions are affected by the inhibition of protein phosphatase activity. These data indicate that increases in protein phosphorylation alone do not lead to the activation of the NADPH-oxidase, but in addition to the requirement of an anionic amphiphile, the release of O2- from intact neutrophils or in the cell-free system is increased by stimulus activation of protein kinase C or more impressively by inhibition of protein phosphatase activity.     

Inhibition of bacterial bioluminescence by chlorophenols

Photobacteria were used as a test object for rapid monitoring of ecotoxicants. Specific inhibitory effects of phenol and its chlorinated derivatives (2-chlorophenol, 2,3-dichlorophenol, pentachlorophenol, 2,4-dichlorophenoxyacetic acid, and 2,4,5-trichlorophenoxyacetic acid) on bioluminescence and respiration of intact cells, as well as on the emission activity of the bioluminescence system and luciferase itself, were studied. The toxic effect on the photobacterial cells was found to increase as the number of chlorine atoms in the chlorophenol molecule increases. However, this trend was not observed in cell-free systems (purified luciferase or the protein fraction of a cell-free extract treated with (NH)4SO4 at 40-75% saturation). Bacterial cells have a higher threshold sensitivity to chlorophenols in comparison to the sensitivity of the bioluminescence enzyme system or luciferase. Neutral phenols inhibit luciferase by competing with decanal, whereas a mixed mechanism of inhibition with this substrate is typical of phenoxyacetic acids. With respect to FMNH2, all chlorophenols tested in this work were uncompetitive inhibitors. Oxygen uptake by photobacteria was shown to be insensitive to chlorophenols, at least within the concentration range that was effective in bioluminescence inhibition. The results of this study suggest that bacterial bioluminescence system is not the primary target of the chlorophenol-induced effect on photobacteria.     

Cordycepin analogs of ppp5'A2'p5'A2'p5'A (2-5A) inhibit protein synthesis through activation of the 2-5A-dependent endonuclease

Analogs of the triphosphate 2'-5'-linked adenylate trimer (ppp5'A2'p5'A2'p5'A, called 2-5A) which contain 3'-deoxyadenosine (cordycepin) instead of adenosine either in positions one and two, or in all three positions, are 10-100-fold less potent than is parent 2-5A in inhibition of protein synthesis in intact cells, when utilizing calcium co-precipitation techniques to introduce the 5'-triphosphate oligonucleotides into the cells. That the inhibition of protein synthesis was a consequence of activation of the 2-5A-dependent endonuclease by the 3'-deoxyadenosine analogs of 2-5A was demonstrated in obtaining the ribosomal RNA cleavage pattern that is characteristic of endonuclease activation by parent 2-5A. Additional results (i.e. lack of activity by the dimer species ppp5'(3'dA)2'p5'-(3'dA) or the monomer 3'dA) as well as kinetic analysis both in intact cells and in cell-free extracts provided further evidence that the inhibition of protein synthesis observed with these 3'-deoxyadenosine 2-5A analogs was not due to their degradation to the antimetabolite monomer unit 3'-deoxyadenosine.     

Induction and general properties of beta-galactosidase and beta-galactoside permease in Pseudomonas BAL-31.

The hydrolysis of o-nitrophenyl-beta-D-galactopyranoside (ONPG) by BAL-31, a marine Pseudomonas that acts as a host for bacteriophage PM2, was studied with intact cells and with cell-free extracts. A transport system for ONPG in whole cells and a beta-galactosidase activity in extracts were evident for cells grown on lactose minimal medium. It was found that the addition of isopropylthio-beta-D-galactopyranoside (IPTG) to cells growing in rich medium induced an ONPG hydrolytic activity detectable in cell extracts but cryptic in whole cells. The existence of a transport system for IPTG, which remained cryptic for ONPG, became apparent from studies of the rates of induction of beta-galactosidase as a function of cell mass at different concentrations of IPTG. The main properties of beta-galactosidase and the lactose transport system of BAL-31 were studied in terms of how they were affected by pH, temperature, or by the presence of several sugars. IPTG competitively inhibits the hydrolysis of ONPG by cell extracts. In cells pregrown on lactose, IPTG slightly inhibits the transport of ONPG. Glucose, and with less efficiency lactose, also inhibits the hydrolysis of ONPG in cell extracts. The growth of cells on lactose minimal medium was inhibited by the addition of IPTG. A mechanism for this inhibition and for the inhibition of ONPG transport by IPTG is discussed.     

Structural alterations of peripheral nerve monogalactosylceramides during development and Wallerian degeneration

Reaction products of selenite with thiols were tested for an inhibitory effect on amino acid incorporation in a cell-free system derived from rat liver and on protein synthesis in intact P815 and L1210 cells. In the cell-free system maximum inhibition, up to 96%, was reached at about 10 microM selenium. In intact cells inhibitory effect varied depending on which reaction product or cell line was used. Maximum inhibition was obtained after 30 min of incubation with selenium concentrations ranging from 0.25 microM to over 7 microM. Selenite itself also inhibited protein synthesis of L1210 cells, but only after 90 min of incubation and starting at selenium concentrations of 2 microM. Inhibition of protein synthesis in intact cells was followed by cell death. Pre-incubation of the reaction products of a monothiol (2-propanethiol) and of a vicinal dithiol (2,3-dimercapto-1-propanol) in culture medium showed a rapid decrease of the inhibitory capability of the product from the monothiol, but not of the product from the dithiol. The results indicate that selenite and a thiol react to form products which have differential toxic effects to cells in vitro.     

Relationship between uridine kinase activity and rate of incorporation of uridine into acid-soluble pool and into RNA during growth cycle of rat hepatoma cells

Uridine kinase activity measured in cell-free extracts of Novikoff rat hepatoma cells grown in suspension culture fluctuates about 10 fold during the growth cycle of the cells. Maximum specific activity (units/10⁶ cells) is observed early in the exponential phase and then decreases progressively until the stationary phase. The rate of incorporation of uridine into the acid-soluble pool by intact cells fluctuates in a similar manner and both the rate of uridine incorporation by intact cells and the uridine kinase actvity of the cells increase several fold before cell division commences following dilution of stationary phase cultures with freshmedium. Regardless of the stage of growth, uridine is rapidly phosphorylated to the triphosphate level by the cells. The rates of incorporation of uridine into the nucleotide pool and into RNA by intact cells fluctuate in a similar manner during the growth cycle. However, evidence is presented that indicates that alterations in the rate of incorporation of uridine into RNA are not simply due to changes in the rate of phosphorylation of uridine, but are regulated independently. Inhibition of protein synthesis by treating cells with puromycin or actidione causes a marked inhibition of incorporation of uridine into RNA, but has little effect on the phosphorylation of uridine to UTP for several hours. Thus the depression of incorporation of uridine into RNA probably reflects a decrease in the rate of RNA synthesis as a result of inhibition of protein synthesis. Inhibition of RNA synthesis by treating cells with actinomycin D does not affect the rate of conversion of uridine to UTP and thus results in the accumulation of labeled UTP in treated cells.     

The effect of parabens on DNA, RNA and protein synthesis in Escherichia coli and Bacillus subtilis

The effect of methyl, propyl and butyl esters of p-hydroxybenzoic acid on DNA and RNA synthesis has been tested in toluenized cells of Escherichia coli and Bacillus subtilis. Both RNA and DNA synthesis of these bacteria were inhibited. The inhibitory concentrations were higher than those previously reported for growth inhibition. Protein synthesis in cell-free extracts (S-30 fraction) of B. subtilis was even more sensitive to parabens than DNA and RNA synthesis, while protein synthesis in Esch. coli was largely unaffected.