Bacillus thiaminolyticus sp. nov., nom. rev

The name "Bacillus thiaminolyticus" Kuno 1951 was not included on the Approved Lists of Bacterial Names and has lost standing in bacteriological nomenclature. The genetic homogeneity of "Bacillus thiaminolyticus" was assessed by determining guanine-plus-cytosine contents by the buoyant density method and by measuring DNA relatedness by using spectrophotometric reassociation procedures. Of the 26 strains which I studied, 24 had guanine-plus-cytosine contents in the range from 52 to 54 mol%. The consistently high DNA relatedness values of 60 to 100% of these 24 strains to the type strain indicated that the "B. thiaminolyticus" group is genetically homogeneous. Low DNA relatedness values of 20 to 31% showed that "B. thiaminolyticus" is genetically unrelated to Bacillus alvei, "Bacillus aneurinolyticus," "Bacillus apiarius," Bacillus larvae, Bacillus laterosporus, Bacillus macerans, and Bacillus stearothermophilus. In general, the "B. thiaminolyticus" group was highly homogeneous for 49 phenotypic characteristics and clearly distinguishable from B. alvei, with which it was allegedly synonymous. On the basis of these findings, revival of the name Bacillus thiaminolyticus is proposed.     

Inhibition of glutathione S-transferase by bile acids.

The effects of bile acids on the detoxification of compounds by glutathione conjugation have been investigated. Bile acids were found to inhibit the total soluble-fraction glutathione S-transferase activity from rat liver, as assayed with four different acceptor substrates. Dihydroxy bile acids were more inhibitory than trihydroxy bile acids, and conjugated bile acids were generally less inhibitory than the parent bile acid. At physiological concentrations of bile acid, the glutathione S-transferase activity in the soluble fraction was inhibited by nearly 50%. This indicates that the size of the hepatic pool of bile acids can influence the ability of the liver to detoxify electrophilic compounds. The A, B and C isoenzymes of glutathione S-transferase were isolated separately. Each was found to be inhibited by bile acids. Kinetic analysis of the inhibition revealed that the bile acids were not competitive inhibitors of either glutathione or acceptor substrate binding. The microsomal glutathione S-transferase from guinea-pig liver was also shown to be inhibited by bile acids. This inhibition, however, showed characteristics of a non-specific detergent-type inhibition.     

Studies on the bacterial spore coat. 6. Effects of alkali extraction on the spore of Bacillus thiaminolyticus

Thin sections of the spore of Bacillus thiaminolyticus Matsukawa and Misawa show a characteristic surface structure with five ridges, and a series of three distinct layers. The outer layer of the spore coat was peeled off by SDS sonic treatment, and then the middle layer was solubilized by alkali extraction of the SDS sonic-treated spore. The spores subjected to these treatments were still refractile, heat resistant, and contained dipicolinic acid, but lost their resistance to mechanical shock.     

NATURE OF THE INHIBITION OF PROTEIN SYNTHESIS BY ETHIONINE AND AMINO ACID UPTAKE IN EARLY SEA URCHIN EMBRYOS

The inhibition of protein synthesis by ethionine reported previously was found to be apparent, and ethionine inhibited only amino acid uptake like other usual amino acids. Even under such strong inhibition of the uptake, the syntheses of protein and DNA remained almost undiminished. The uptake of amino acid mixture by sea urchin embryos in the early cleavage stage was found to be carried out by active transport, since it was temperature-sensitive and was inhibited by 2,4-dinitrophenol. The uptake of an amino acid mixture or of single amino acids, e.g., valine, leucine and phenylalanine, was inhibited nonspecifically by an excess amount of other single amino acids added exogenously. Reflecting the inhibition of amino acid uptake, in vivo incorporation of amino acids into the protein fraction was apparently inhibited by excess amounts of other amino acids. As far as tested, the inhibition seems to be nonspecific and competitive for all amino acid species. The uptakes of leucine and phenylalanine were inhibited mutually by competition, with almost the same Km and Ki.     

Long chain fatty acid inhibition of sodium plus potassium-activated adenosine triphosphatase from rat heart.

Long chain fatty acids were found to inhibit (Na+ + K+)-ATPase prepared from rat heart. Unsaturated and polyunsaturated fatty acids were more inhibitory than saturated fatty acids with myristic acid being the most inhibitory saturated fatty acid tested and linoleic the most inhibitory unsaturated fatty acid. As an example of fatty acid modification of the enzyme, inhibition of (Na+ + K+)-ATPase by oleate was examined. When compared to ouabain, inhibition of (Na+ + K+)-ATPase by oleate was found to be similar in that both were dependent on K+ concentration, but, in contrast to the almost instantaneous inhibition by ouabain, oleate inhibition was a slow process requiring over 20 min incubation at 37 degrees to produce maximum inhibition. Inhibition of rat heart (Na+ + K+)-ATPase by oleate was found to be readily reversible by washout. In the presence of albumin an oleate/albumin molar ratio greater than 7.5 was required for inhibition to occur. The activity of rat heart (Na+ + K+)-ATPase had a temperature optimum above 40 degrees and a discontinuous Arrhenius' plot with a transition temperature of 25 degrees. In the presence of oleate, however, the enzyme's optimum temperature decreased to below 40 degrees, the activation energy of the reaction at temperatures below 25 degrees was lowered from 24.7 kcal/mol to 12.6 kcal/mol and the enzyme had a linear Arrhenius' plot. The possibility of in vivo inhibition of cardiac (Na+ + K+)-ATPase under conditions of elevated fatty acids is discussed.     

Unsaturated fatty acids inhibit ADP- arachidonate-induced platelet aggregation without affecting thromboxane synthesis

The inhibitory effects of three cis-unsaturated C18 fatty acids (oleic, linoleic, and linolenic acids, sodium salts) on ADP- and sodium-arachidonate-induced aggregation of washed rabbit platelets were investigated. When the platelets were suspended in protein-free medium containing dextran, it was found that these fatty acids at very low concentrations (2-45 microM) were potent inhibitors of platelet responsiveness and the inhibitory effect occurred within seconds. The inhibition of ADP-induced aggregation was not affected by abolishing the activity of platelet cyclooxygenase using aspirin. Human serum albumin relieved the inhibition caused by fatty acids for both ADP- and arachidonate-induced aggregation. The inhibitory effect of fatty acids does not seem to be due to decreased thromboxane formation (except possibly in the case of linolenate), and the relief of fatty acid inhibition by albumin does not potentiate thromboxane B2 formation from exogenous arachidonate. It is suggested that the inhibitory effect of polyunsaturated fatty acids on platelet aggregation is specific and not related to a general surfactant effect, since inhibition occurs far below the critical micelle concentration of fatty acid soaps.     

Polyunsaturated fatty acids inhibit mouse hepatic glucocorticoid receptor activation in vitro

The effect of saturated fatty acids (SFAs) stearic and palmitic acids and polyunsaturated fatty acids (PUFAs) oleic, linoleic and arachidonic acids was studied on in vitro heat activation of mouse hepatic glucocorticoid receptor (GR) complex, as assessed by binding to DNA-cellulose and purified nuclei. Significant dose-dependent inhibition of heat activation of hormone-receptor complex by the PUFAs was observed. Linoleic and arachidonic acids were found to be more potent (caused approximately 70% inhibition maximally at 160 microM) inhibitors of GR heat activation, compared to oleic acid (approximately 38% inhibition at 40 microM). However, stearic and palmitic acids were unable to modulate GR heat activation, suggesting that the unsaturated moieties in PUFAs are possibly the important determinants of receptor activation. Thus, our study shows an inhibitory effect of PUFAs on in vitro hepatic GR activation.     

Effect of alanine-containing dipeptides on germination of Bacillus thiaminolyticus spores.

Stereoisomeric alanylalanine (Ala-Ala) derivatives were examined for their effects on germination of Bacillus thiaminolyticus spores. L-Ala-L-Ala and L-Ala-glycine were effective in inducing germination, and their activities were completely inhibited by D-Ala. L-Ala-D-Ala and glycine-D-Ala competitively prevented L-Ala-induced germination. Sarcosine- or beta-Ala-containing L-alanyldipeptides and eight kinds of alanyltripeptides did not show any detectable effect on germinability or any inhibitory effect. No detectable amounts of Ala were found in germination exudates when alanylpeptides were incubated with spores. The ability of these peptides to induce or inhibit germination depends on their steric conformation and a certain distance between the primary amino group and the free carboxyl groups. Involvement of L-Ala dehydrogenase in the initiation of germination is unlikely because L-Ala-L-Ala was not a substrate and L-Ala-D-Ala was not an effective inhibitor of enzyme activity.     

Studies on the bacterial spore coat 6 effects of alkali extraction on the spore of Bacillus thiaminolyticus.

Thin sections of the spore of Bacillus thiaminolyticus Matsukawa and Misawa show a characteristic surface structure with five ridges, and a series of three district layers. The outer layer of the spore coat was peeled off by SDS sonic treatment, and than the middle layer was solubilized by alkali extraction of the SDS sonic-treated spore. The spores subjected to these treatments were still refractile, heat resistant, and contained dipicolinic acid, but lost their resistance to mechanical shock.     

Effect of cerulenin on the growth and differentiation of Dictyostelium discoideum.

The growth of Dictyostelium discoideum Ax-2 was inhibited completely by cerulenin at a concentration of 5 mug/ml. This inhibition of growth was found to be due to the inhibition of fatty acid synthesis. Acetate incorporation into a long-chain fatty acid was inhibited completely by cerulenin, and the growth inhibition could be reversed by inclusion of certain saturated fatty acids in the medium. Unsaturated fatty acids and sterols failed to reverse the inhibitory effect. The fatty acid and sterol compositions of cerulenin-treated cells were determined to establish whether the drug could be used to manipulate the organism's lipid composition. Only relatively small manipulations were obtained under the conditions employed in this study. Cerulenin inhibited differentiation but only at high concentrations (150 mug/ml). This inhibition could be reversed by palmitic acid, suggesting that the prime cause of the inhibition was an inhibition of fatty acid synthesis. Thus, it appears that continued fatty acid synthesis is required for the cellular process of differentiation in D. discoideum.     

Effect of organic acids found in cottonseed hull hydrolysate on the xylitol fermentation by Candida tropicalis

Five organic acids (acetic, ferulic, 4-hydroxybenzoic, formic and levulinic acids) typically associated in the hemicellulose hydrolysate were selected to study their effects on the xylitol fermentation. The effects of individual and combined additions were independently evaluated on the following parameters: inhibitory concentration; initial cell concentration; pH value; and membrane integrity. The results showed that the toxicities of organic acids were related to their hydrophobility and significantly affected by the fermentative pH value. In addition, it was revealed that the paired combinations of organic acids did not impose synergetic inhibition. Moreover, it was found that the fermentation inhibition could be alleviated with the simple manipulations by increasing the initial cell concentration, raising the initial pH value and minimizing furfural levels by evaporation during the concentration of hydrolysates. The proposed strategies for minimizing the negative effects could be adopted to improve the xylitol fermentation in the industrial applications.     

On-line extraction of volatile fatty acids in acidogenic chemostat culture using a supported liquid membrane

An on-line extraction of volatile fatty acids (acetic and butyric acids) from acidogenic fermentation in chemostat cultures using a supported liquid membrane (SLM) was investigated in order to overcome end-product inhibition. By using SLM, the high-cell-density retaining dilution rate of the chemostat could be increased, thus enhancing the microbial acidogenesis. To further understand this phenomenon, the growth and extraction kinetics were studied. The effect of substrate concentration was found to obey the Haldane equation. Regarding the inhibition by volatile fatty acids, it turned out that undissociated butyric acid rather than acetic acid severely inhibited the growth, corresponding to non-competitive kinetics. The extraction rates of the acids were proportional to their undissociated concentration as well as to the SLM area/broth volume, and butyric acid extraction was easier than that of acetic acid.     

Alkaline phosphatase: affinity chromatography and inhibition by phosphonic acids.

Five phosphonic acid derivatives were synthesized, coupled to agarose, and tested for affinity chromatographic binding of alkaline phosphatase from bovine intestine. Agarose coupled to L-histidyldiazobenzylphosphonic acid was found to be a highly effective adsorbent. In order to understand the large differences in binding capacity observed with derivatized agaroses, inhibition of alkaline phosphatase by phosphonic acid ligands, and related phosphonic acids, was measured. The results of affinity chromatography and inhibition studies were in good agreement, demonstrating that phosphonic acids with large aromatic/hydrophobic, carboxylate substituents bind strongly and competitively to the enzyme active site.     

Batch and continuous anaerobic toxicity of resin acids from chemithermomechanical pulp wastewater

Chemithermomechanical pulp (CTMP) wastewaters are being treated anaerobically, in full scale operations, in spite of the presence of inhibitory compounds. These compounds are neither well identified nor are they placed in an order of priority in terms of inhibition to anaerobic bacteria. In this study batch anaerobic toxicity assays were used to quantify the relative toxicity of resin acids to the total toxicity in hydrogen peroxide bleached CTMP (BCTMP) wastewater. Resin acids are partitioned between the soluble wastewater and the insoluble fiber fractions. Therefore fiber toxicity was also compared to raw BCTMP wastewater toxicity. Resin acids were found to be toxic to anaerobic bacteria but were not found to be responsible for all the toxicity in BCTMP effluents. Toxicity associated with the fiber was soluble in methanol. The bulk of the fiber which was not methanol soluble exhibited no toxicity to the anaerobic cultures used. Batch results were compared to continuous toxicity assays using an upflow anaerobic sludge blanket reactor. The continuous reactor was relatively unaffected by short term or long term exposure to resin acids at concentrations that invoked inhibition in batch tests. Inferences from these tests indicated that batch results may be unreliable when applied to continuous systems.     

Manipulation of fatty acid composition of membrane phospholipid and its effects on cell growth in mouse LM cells

Fatty acid composition of the phospholipids of mouse LM cells grown in suspension culture in serum-free chemically defined medium was modified by supplementing the medium with various fatty acids bound to bovine serum albumin.Following supplementation with saturated fatty acids of longer than 15 carbons (100 μM) profound inhibition of cell growth occurred; this inhibitory effect was completely abolished when unsaturated fatty acids were added at the same concentration. Supplementing with unsaturated fatty acids such as linoleic acid, linolenic acid or arachidonic acid had no effect on the cell growth.Fatty acid composition of membrane phospholipids could be manipulated by addition of different fatty acids. The normal percentage of unsaturated fatty acids in LM cell membrane phospholipids (63%) was reduced to 35–41% following incorporation of saturated fatty acids longer than 15 carbon atoms and increased to 72–82% after addition of unsaturated fatty acids.A good correlation was found between the unsaturated fatty acid content of membrane phospholipids and cell growth. When incorporated saturated fatty acids reduced the percentage of unsaturated fatty acids in membrane phospholipids to less than 50%, severe inhibition of the cell growth was found. Simultaneous addition of an unsaturated fatty acid completely abolished this effect of saturated fatty acids.     

Fatty acid synthesis in Escherichia coli is indirectly inhibited by phenethyl alcohol.

Experiments were performed to determine how phenethyl alcohol inhibits phospholipid synthesis in E. coli. At a nonbacteriostatic concentration, the drug reduces the rate of de novo fatty acid and phospholipid synthesis by 60 to 70%. The inhibition of fatty acid synthesis was found to be a secondary consequence of the inhibition of phospholipid synthesis. Phenethyl alcohol reduces the rate of incorporation of exogenous fatty acids into the phospholipids of a fatty acid auxotroph by 60%. These results indicate that this drug controls phospholipid synthesis beyond the level of fatty acid synthesis. Phenethyl alcohol inhibits the synthesis of phospholipids containing saturated fatty acids to a greater extent than it does the synthesis of phospholipids containing unsaturated fatty acids. It controls the synthesis of phospholipids containing saturated fatty acids at both the level of fatty acid synthesis and the level of incorporation of the saturated fatty acids into phospholipids. The synthesis of phospholipids containing unsaturated fatty acids is inhibited at the level of incorporation of the fatty acids into phospholipids.     

Toxic effects of fatty acids on yeast cells: dependence of inhibitory effects on fatty acid concentration.

The yeasts Saccharomyces cerevisiae, Candida utilis, and Candida lipolytica were used to investigate the action of different concentrations of fatty acids (from acetic to myristic acid) on cell growth, division, uptake of inorganic phosphate, and substrate oxidation. The former two yeasts were found to undergo an inhibition of growth, cell division, and phosphate uptake at lower acid concentrations and to experience the inhibition of substrate oxidation at higher acid concentrations. The concentration dependence of the action of fatty acids can be classified into four categories: 1) subthreshold concentrations which do not inhibit growth and have either no effect on, or stimulate, oxygen consumption; 2) threshold concentrations which lower the rate of growth, cell division, and phosphate uptake but do not inhibit the oxidation of carbon substrate; 3) above-threshold concentrations which inhibit partially even substrate oxidation, and 4) microbicide concentrations. Candida lipolytica displays the same sensitivity toward the action of fatty acids as the above yeast species; however, the threshold concentrations are higher and can be quickly lowered owing to oxidation by the yeast. The concentrations of fatty acids found in the medium after cultivations of yeast with n-alkanes are of the same order as limiting concentrations; the formation of acids with twelve and less carbons in the molecule can thus be assumed to be one of the basic reasons for lowering of biomass yields during cultivations on these hydrocarbons.     

Cell-Bound Thiaminase I of Bacillus thiaminolyticus

The distribution of the extracellular enzyme, thiaminase I, was determined for logarithmically growing cultures of Bacillus thiaminolyticus. About 60% of the enzyme is associated with the cells throughout the growth cycle. The remainder of the enzyme is in the culture medium. The release of the cell-bound thiaminase I is examined under a variety of conditions. The rate and extent of release is dependent on the pH and the nature of the incubation solution. The release process appears to be relatively independent of de novo protein synthesis, energy derived from oxidative phosphorylation, or divalent metal ions. The absence of carbon or nitrogen sources has little effect on the release of the enzyme. Cell-bound thiaminase I probably is the immediate precursor for extracellular thiaminase I found in the culture medium. Washed cells continue to release thiaminase I at the expense of cell-bound enzyme. In addition, purified cell-bound thiaminase I is indistinguishable from purified extracellular thiaminase I by a number of physical and kinetic criteria.     

Ethanol-induced inhibition of testosterone biosynthesis in rat Leydig cells: role of culture medium composition

The biochemical mechanisms responsible for the ethanol-induced inhibition of testicular testosterone synthesis were studied in isolated rat Leydig cells in vitro. This inhibition was removed when HAM-F12 nutrient mixture was added to the DME culture medium. The components of HAM-F12, i.e. vitamins, amino acids and other supplements, were tested individually and the amino acids L-glutamate (Glu) and L-aspartate (Asp) were found to potentiate strongly the hCG stimulated testosterone synthesis. None of the other components of HAM-F12 had any effect upon testosterone synthesis or its ethanol-induced inhibition. Moreover, Glu, but not Asp, effectively reversed the acute inhibition of steroidogenesis by ethanol. These results demonstrate the importance of the composition of the culture media and provide the first piece of evidence that the metabolic stress in rat Leydig cells in vitro induced by the metabolism of ethanol can be overcome by proper culture medium supplementation.     

EFFECT OF PHENYL AND PHENOLIC ACIDS ON MEVALONATE-5-PHOSPHATE KINASE AND MEVALONATE-5-PYROPHOSPHATE DECARBOXYLASE OF THE RAT BRAIN

Abstract— Phenyl and phenolic acids are known to inhibit metabolism of mevalonate in rat brain. The site of inhibition has been found to be mevalonate-5-pyrophosphate decarboxylase. Phenolic acids also inhibited mevalonate-5-phosphate kinase on preincubation. The kinetics showed that p -coumaric acid and isoferulic acid were competing with substrates, mevalonate-5-phosphate or mevalonate-5-pyre phosphate, whereas others showed an uncompetitive type of inhibition. Chlorophenoxyisobutyrate, a hypocholesterolaemic drug, had no effect on these enzymes. An improved method for the synthesis of mevalonate-5-phosphate and mevalonate-5-pyrophosphate, labeled at carbon-1, is described.