Dihydrosphingosine growth inhibition and repression of 3-ketodihydrosphingosine synthetase activity in Bacteroidesmelaninogenicus

Addition of dihydrosphingosine or 3-ketodihydrosphingosine to growing cultures of $\text{Bacteroides}\text{melaninogenicus}$ markedly reduced the activity of 3-ketodihydrosphingosine synthetase in extracts. Neither compound reduced the activity of a previously solubilized preparation of the enzyme. Dihydrosphingosine and synthetic acetyl ceramides inhibited the growth of the microorganism; 3-ketodihydrosphingosine was not inhibitory and reversed the growth inhibition caused by dihydrosphingosine. This reversal may indicate a preferential utilization of 3-ketodihydrosphingosine over dihydrosphingosine by the cells for the biosynthesis of complex sphingolipids.     

The 3-ketodihydrosphingosine synthetase of Bacteroides melaninogenicus: Induction by vitamin K

An enzyme present in cell-free extracts of B. melaninogenicus grown with vitamin K is described which catalyzes the synthesis of 3-ketodihydrosphingosine from palmitoyl CoA and l-serine. Activity of the 3-ketodihydrosphingosine synthetase was measured as a function of time, palmitoyl CoA concentration, and serine concentration. The Bacteroides synthetase differs from corresponding enzymes from brain microsomes and from yeast in that it is present in the 100,000g supernatant of sonicated cells and is not associated with any particulate fraction.Extracts prepared from cells depleted of vitamin K showed only slight 3-ketodihydrosphingosine synthetase activity. Neither vitamin K₁, menadione, nor pyridoxal phosphate were effective in enhancing the activity in cell-free extracts of vitamin K-depleted B. melaninogenicus. However, induction of the enzyme activity in intact cells was demonstrated by the addition of vitamin K to a vitamin K-depleted culture. Synthetase activity was found to be increased 15 min following the addition of the vitamin, reached a maximum at 75 min, and thereafter remained constant. Both puromycin and rifampcin inhibit induction of the enzyme by vitamin K₁ suggesting that vitamin K induces de novo synthesis of the synthetase.     

Biosynthesis of methionine in mouse cells cultured in vitro

In the mouse cell-lines cultured in vitro, viz. L-cells and mouse embryo fibroblasts, the methylation of homocysteine to methionine is carried out by vitamin B12-dependent 5-methyltetrahydrofolate:L-homocysteine methyltransferase only. In these cells grown in the standard Eagle medium, the activity of another methyltransferase, which utilizes betaine as the methyl donor, was not detected. The high activity of the vitamin B12-dependent methionine synthetase is typical for mouse cells from the logarithmic phase of growth. In L-cells 60%, and in the mouse fibroblasts 30% of the enzyme exist in the holo-form; the ratio between the holo- and apoenzyme activity remains stable in cells from logarithmic and stationary cultures. The level of the activity of methionine synthetase strongly depends on the presence of vitamin B12, folate and methionine in the culture medium and is greater after prolonged contact of the cells with these agents.     

Inhibition of Bacillus megaterium by a Trimethylamine Oxide-Associated Browning Reaction Product

Heated combinations of trimethylamine oxide (TMAO) and culture media (tryptone, glucose, yeast extract broth or a defined minimal medium), or heated TMAO and glucose, contained substance(s) that inhibited growth of Bacillus megaterium. Inhibition was expressed primarily as an increase of the lag phase of growth; the logarithmic growth rate was comparable to control cultures. The addition of unheated TMAO to the culture media had no effect on growth. Results suggested that TMAO was decomposed during heating and that dimethylamine, one of the degradation products, reacted with glucose by a Maillard-Amadori reaction to produce the inhibitory substance(s).     

Spectroscopic and molecular dynamics simulation studies of the interaction of insulin with glucose

The interaction between monomeric insulin and monosaccharides has been investigated through circular dichroism, fluorescence spectroscopy and two dimensional nuclear magnetic resonance. CD spectra indicate that D-glucose interacts with monomeric insulin whereas D-galactose, D-mannose and 2-deoxy-D-glucose have a lower effect. Fluorescence emission was quenched at sugar concentrations of 5-10 mM. Titration with the different sugars produces a quenching of the tyrosine spectrum from which a binding free energy value for the insulin-sugar complexes has been evaluated. Transfer nuclear Overhauser enhancement NMR experiments indicate the existence of dipolar interactions at short interatomic distances between C-1 proton of D-glucose in the beta form and the monomeric insulin. Further, NMR total correlation spectra experiments revealed that the hormone is in the monomeric form and that upon addition of glucose no aggregation occurs. The interaction does not involve relevant changes in the secondary structure of insulin suggesting that the interaction occur at the side chain level. Molecular dynamics simulations and modeling studies, based on the dynamic fluctuations of potential binding moiety sidechains, argued from results of NMR spectroscopy, provide additional informations to locate the putative binding sites of D-glucose to insulin.     

Determination of monosaccharides and sugar alcohols in tissues from diabetic rats by high-performance liquid chromatography with pulsed amperometric detection.

A sensitive and simple high-performance liquid chromatographic method has been developed to determine the concentration of monosaccharides and sugar alcohols in animal tissues. Five neutral monosaccharides (D-glucose, D-galactose, D-mannose, D-fructose, and D-ribose) and three neutral sugar alcohols (myo-inositol, glycerol, and D-sorbitol) predominate in the renal cortices and sciatic nerves of rats. These monosaccharides and sugar alcohols were extracted with distilled water, purified by deproteinization with ethanol, a Sep-Pak C18 cartridge, and columns of Dowex 50W-X8 and Amberlite CG-400, then separated on Ca2+ and Pb2+ cation-exchange columns, eluted with deionized distilled water at 80 degrees C, and detected using integrated pulsed amperometry. About 10 pmol of each sugar was detectable with a signal-to-noise ratio of 10:1. D-Glucose, D-fructose, D-sorbitol, and D-mannose were higher in both the renal and sciatic tissues of diabetic rats than in those of normal animals. D-Ribose and glycerol were higher in the renal cortex of diabetic animals.     

The saccharide components of wood hydrolysates--a constituent part of the fermentation media for the biosynthesis of beta-lactam antibiotics

Spruce and ash wood were subjected to acid hydrolysis. The hydrolysates were paper chromatographed for the presence of saccharides. The following monosaccharides were detected: D-xylose, D-mannose, D-glucose, D-galactose and L-arabinose. The monosaccharides and L-rhamnose in addition in the form of concentrated solutions were used as part of lactose in production of penicillin V. Similarly, the whole amounts of D-glucose and saccharose were substituted in biosynthesis of cephalosporin C. With regard to the results it follows that hydrolysates of the conifers and especially of the hardwood can substitute part of the fermentation medium components in production of penicillin V and cephalosporin C.     

Effect of carbon source on the accumulation of cytochrome P-450 in the yeast Saccharomyces cerevisiae.

The appearance of cytochrome P-450 in the yeast Saccharomyces cerevisiae depended on the substrate supporting growth. Cytochrome P-450 was apparent in yeast cells grown on a strongly fermentable sugar such as D-glucose, D-fructose or sucrose. When yeast was grown on D-galactose, D-mannose or maltose, where fermentation and respiration occurred concomitantly, cytochrome P-450 was also formed. The cytochrome P-450 concentration was maximal at the beginning of the stationary phase of the culture. Thereafter the concentration decreased, reaching zero at a late-stationary phase. When the yeast was grown on a medium that contained lactose or pentoses (L-arabinose, L-rhamnose, D-ribose and D-xylose), cytochrome P-450 did not occur. When a non-fermentable energy source (glycerol, lactate or ethanol) was used, no cytochrome P-450 was detectable. Transfer of cells from D-glucose medium to ethanol medium caused a slow disappearance of cytochrome P-450, although the amount of the haemoprotein still continued to increase in the control cultures. Cytochrome P-450 appeared thus to accumulate in conditions where the rate of growth was fast and fermentation occurred. Occurrence of this haemoprotein is not necessarily linked, however, with the repression of mitochondrial haemoprotein synthesis.     

Benzeneboronic acid selectively inhibits sporulation of Bacillis subtilis.

m-Aminobenzeneboronic acid at levels of 0.2 mM in nutrient broth medium selectively inhibited sporulation without appreciably altering vegetative growth. Significant inhibitory effects were seen even when it was added as late as 6 h after the end of logarithmic growth. The pH changes associated with growth and sporulation of Bacillus subtilis in nutrient broth were not significantly altered by the inhibitor. When it was present in cultures of actively growing cells, its inhibitory effect could not be reversed by simple dilution. The compound caused extensive clumping, of cells, which appeared not to be related to the ability of boronates to esterify to diols.     

Inhibition of intestinal sugar transport by phenformin.

The effect of phenformin on the absorption of D-glucose and D-galactose by hamster and rat intestine, was studied. Phenformin did not affect D-glucose absorption by rat intestine, but it inhibited at 10(-3) to 10(-2) M the absorption of D-glucose and D-galactose by hamster intestine. The inhibition was higher when D-glucose was tested. Phenformin also inhibited active accumulation of these sugars by rings of hamster small intestine, in vitro; this effect was greater when D-glucose was utilized. The drug inhibits the oxygen uptake in the tissue in the absence or in the presence of added substrate. Phenformin, as previously suggested, does not seem to act as a specific inhibitor on D-glucose transport, but most likely by its inhibitory effect on mitochondrial respiration.     

Characteristics of D-galactose transport systems by luminal membrane vesicles from rabbit kidney

The characteristics of renal transport of D-galactose by luminal membrane vesicles from either whole cortex, pars recta or pars convoluta of rabbit proximal tubule were investigated by a spectrophotometric method using a potential-sensitive carbocyanine dye. Uptake of D-galactose by luminal membrane vesicles prepared from whole cortex was carried out by an Na+-dependent and electrogenic process. Eadie-Hofstee analysis of saturation-kinetic data suggested the presence of multiple transport systems in vesicles from whole cortex for the uptake of D-galactose. Tubular localization of the transport systems was studied by the use of vesicles derived from pars recta and pars convoluta. In pars recta, Na+-dependent transport of D-galactose and D-glucose occurred by means of a high-affinity system (half-saturation: D-galactose, 0.15 +/- 0.02 mM; D-glucose, 0.13 +/- 0.02 mM). These results indicated that the "carrier' responsible for the uptake of these hexoses does not discriminate between the steric position of the C-4 hydroxyl group of these two isomers. This is further confirmed by competition experiments, which showed that D-galactose and D-glucose are taken up by the same and equal affinity transport system by these vesicle preparations. Uptake of D-galactose and D-glucose by luminal membrane vesicles isolated from pars convoluta was mediated by a low-affinity common transport system (half-saturation: D-galactose, 15 +/- 2 mM; D-glucose, 2.5 +/- 0.5 mM). These findings strongly suggested that the "carrier' involved in the transport of monosaccharides in vesicles from pars convoluta is specific for the steric position of the C-4 hydroxyl group of these sugars and presumably interacts only with D-glucose at normal physiological concentration.     

Effect of glucose and pyruvate metabolism on membrane potential in synaptosomes

Fluorescence changes of rhodamine 6G in synaptosomal suspension, which are correlated to changes in membrane potential in synaptosomes, were measured in the presence of various monosaccharides and organic acids. Addition of D-glucose, D-mannose, pyruvate and L-lactate hyperpolarized the membrane potential, whereas D-fructose, L-glucose, D-galactose, citrate, succinate and L-glutamate were without effect on the membrane potential. Hyperpolarization induced by D-glucose was inhibited by cytochalasin B, phloretin, iodoacetate, F- and 2-deoxy-D-glucose, but not inhibited by oligomycin or phlorizin. On the other hand, hyperpolarization induced by pyruvate was inhibited by alpha-cyanocinnamate or phloretin, but not inhibited by cytochalasin B or F-. Elimination of Na+ in physiological saline depressed hyperpolarization of membrane potential induced by addition of D-glucose, L-lactate or pyruvate. These results suggest that the activity of (Na+ + K+)-ATPase in plasma membranes of synaptosomes is increased by ATP formed by glycolysis, and that the accumulated K+ in synaptosomes hyperpolarizes the membrane potential.     

Multiphoton imaging of the effect of monosaccharide diffusion and formation of fluorescent advanced end products in porcine aorta

Prolonged exposure of tissues to elevated blood sugar levels lead to the formation of advanced glycation end products (AGEs), thus contributing to diabetic complications. Since the vascular system is in immediate contact with blood, diabetic effects on aorta is a major health concern. However, the relative effect of the diffusion of sugar molecular through the vascular wall and the rate of AGE formation is not known. In this study, we aim to address this issue by incubating excised porcine aorta in D-glucose, D-galactose, and D-fructose solutions for different periods. The tissue specimens were then excised for multiphoton imaging of autofluorescence intensity profiles across the aorta wall. We found that for Days 4 to 48 incubation, autofluorescence is constant along the radial direction of the aorta sections, suggesting that monosaccharide diffusion is rapid in comparison to the rate of formation of fluorescent AGEs (fAGEs). Moreover, we found that in porcine aorta, the rate of fAGE formation of D-fructose and D-glucose are factors 2.08 and 1.14 that of D-galactose. Our results suggest that for prolonged exposure of the cardiovascular system to elevated monosaccharides 4 days or longer, damage to the aorta is uniform throughout the tissues.     

The role of intercellular contacts in the initiation of growth and in the development of a transiently nonculturable state by the cultures of Rhodococcus rhodochrous grown in poor media

It was found that the growth of Rhodococcus rhodochrous cells in modified Saton's medium strongly depends on the rate of culture agitation in the flask: an agitation at 250 rpm in flasks with baffles stops cell multiplication, whereas slight agitation leads to pronounced culture growth. The growth retardation phenomenon was reversible and did not manifest itself in exponential-phase cultures or when the cells were grown in a rich medium; furthermore, it was not connected with the degree of culture aeration. When agitated at a moderate rate, the bacterial cells formed aggregates in the lag phase, which broke up into single cells in the exponential phase. The inhibitory effect of vigorous agitation was removed by the addition to the medium of the supernatant (SN) of a log-phase culture grown in the same medium with moderate agitation. Vigorous agitation is thought to interfere with the cell contacts, whose establishment is necessary for the development of an R. rhodochrous culture in a poor medium, which occurs in the form of (micro) cryptic growth. When grown in modified Saton's medium, R. rhodochrous cells were capable of transition, in the prolonged stationary phase, to a resting and transiently nonculturable state. Such cells could be resuscitated by incubation in a liquid medium with the addition of the supernatant or the Rpf secreted protein. The formation of transiently nonculturable cells was only possible under the conditions of a considerable agitation rate (250-300 rpm), which prevented secondary (cryptic) growth of the culture. This circumstance indicates the importance of intercellular contacts not only for the initiation of growth but also for the transition of the bacteria to a dormant state.     

Characterization of IL-2-dependent cytotoxic T-cell clones. III. Inhibition of killing activity by monosaccharides

The effects of monosaccharides on the cytotoxic activity of cytotoxic T lymphocytes (CTL) and three cloned long-term cytotoxic T-lymphocyte lines (CTLL) are compared. Uncultured CTL and clones CTLL-A2 and CTLL-A11 were derived from the peritoneal cavity of C57BL/6 mice immunized against the H-2Dd determinants on the BALB/c sarcoma Meth A. Clone CTLL-R5 was derived from spleen of (BALB/c X C57BL)F1 mice immunized against a unique determinant on the BALB/c radiation-induced leukemia RL male 1. The cell-surface phenotype of the clones is Lyt-1+,2+,3+. Cytotoxic activity of CTLL-A2 and CTLL-R5 as determined by a 4-hr 51Cr-release assay was inhibited over 50% by 1 mM 2-deoxy-D-glucose. CTLL-A11 and the uncultured cytotoxic T cells were more resistant to inhibition by 2DG (40% at 20 mM). Surprisingly, it was found that the addition of D-mannose, D-galactose, D-glucose, L-fucose, alpha-methyl-D-mannose, and N-acetyl-D-glucosamine also inhibited, in a dose-related manner, the cytotoxicity of CTLL-A2 and CTLL-A11. CTLL-R5 showed a more restricted inhibition pattern: only D-mannose and D-galactose were inhibitory. The mechanism of inhibition remains to be clarified.     

Effect of culture conditions on batch growth ofPseudomonas fluorescens on olive oil

Summary Batch cultures ofPseudomonas fluores-cens were grown in minimal medium with olive oil as the sole carbon source. When olive oil me-dium was inoculated with cells from nutrient broth there was an initial lag phase followed by logarithmic growth. The duration of the lag phase was influenced by the incubation temperature and the growth phase of the inoculum. Both factors are known to affect lipase induction during growth in fat-free media. Maintenance of condi-tions reported to be conducive to lipase produc-tion in cultures used for inoculation ensured a minimal lag before logarithmic growth com-menced on olive oil. Growth on oil occurred when the culture was maintained at pH 6 or 7, but did not occur at pH 5 or 8.     

Chemoattractants elicit methylation of specific polypeptides in Spirochaeta aurantia

On the basis of this investigation, chemotaxis in Spirochaeta aurantia correlates with methylation of specific polypeptides which are presumed to be analogous to the methyl-accepting chemotaxis proteins (MCPs) in bacteria such as Escherichia coli. The polypeptides exhibited apparent molecular weights in the range of 55,000 to 65,000. Generally, two major presumptive MCP bands and three minor bands were observed on sodium dodecyl sulfate-polyacrylamide gels. Upon addition of D-glucose to S. aurantia cells, methylation of the presumptive MCPs increased for 10 to 12 min to a level greater than 4 times the level of methylation in the absence of D-glucose. Removal of D-glucose resulted in a decrease in methylation of the presumptive MCPs to a level similar to that in unstimulated cells. All attractants tested, including a non-metabolizable attractant (alpha-methyl-D-glucoside) stimulated methylation of the presumptive MCPs (from 1.7 to 4.3 times the level of methylation in unstimulated cells). D-Mannitol, a metabolizable sugar which is not an attractant for S. aurantia, did not stimulate methylation. Stimulation of methylation by D-galactose occurred in cells induced for D-galactose taxis but not in uninduced cells. These data are indicative of an evolutionary relationship between the chemotaxis systems of spirochetes and of flagellated bacteria.     

A study into the role of L-aspartic acid on the metabolism of L-malic acid and D-glucose by Oenococcus oeni

AIMS: The purpose of this work was to study the effect of L-aspartic acid concentration on bacterial growth, D-glucose fermentation and L-malic acid consumption of Oenococcus oeni NCFB 1707. METHODS AND RESULTS: Bacterial cultures were performed in synthetic media. Bacterial growth, D-glucose fermentation and L-malic acid consumption were reduced when L-aspartic acid concentration became excessive. This inhibitory effect of high concentrations of L-aspartic acid on bacterial growth was also observed with several Oenococcus oeni strains, except O. oeni BL01. The L-aspartic acid inhibitory effect on bacterial growth could be reduced by increasing the concentration of L-glutamic acid. L-glutamic acid transport was found to be competitively inhibited by L-aspartic acid. In addition, an excessive amount of L-aspartic acid modified D-glucose metabolism, with an overproduction of acetic acid and reduced ethanol production. CONCLUSION: Since L-glutamic acid is an essential amino acid for the bacterial strain used, the L-aspartic acid inhibitory effect on bacterial growth could be linked to its involvement in an antagonistic interaction with L-glutamic acid. SIGNIFICANCE AND IMPACT OF THE STUDY: Such antagonistic interactions between amino acids in O. oeni strains could be another explanation for the difficulties of inducing malolactic fermentation in wines.     

Growth ofCandida utilis on a mixture of monosaccharides,acetic acid and ethanol as a model of waste sulphite liquor

Candida utilis cultivated under batch conditions in a synthetic medium with a mixture of different carbon sources utilized first D-glucose and then D-galactose, D-mannose, D-xylose, L-arabinose, ethanol and acetic acid. The effect of acetic acid was primarily a function of pH and the physiological state of the inoculum. At pH 4.5, acetic acid at a concentration of 1 g/l increased the specific growth rate, reduced time of cultivation and increased yield of the yeast dry weight. The yield from acetic acid was 61%. In the presence of a higher content of acetic acid (3--6 g/l) the yield was only 18--26%. The yield calculated only from monosaccharides increased but the yield with respect to total carbon sources was lower. The specific growth rate decreased as well. The addition of ethanol also resulted in an increase of the production and yield of the yeast dry weight but the cultivation time was prolonged. The simultaneous utilization of carbon sources of the studied mixture modelling a sulphite fermentation medium with ethanol is advantageous. However, due to physiology of the yeast, it is most suitable to cultivate a strain adapted to utilizable carbon sources in a continuous way, in the presence of their limiting concentrations in the cultivation medium.     

Preliminary studies on excretory components of a virulent strain (F-2) of Pseudomonas solanacearum

The excretory material (EM) was isolated from the culture medium of a virulent strain (F-2) of Pseudomonas solanacearum. Electrophoretic studies depicted the heterogeneous nature of EM and the presence of released lipopolysaccharide in it. Both the exopolysaccharide and the released LPS contained D-galactose as the major sugar constituent together with D-glucose, N-acetyl-D-galactosamine and N-acetyl-D-glucosamine. In addition, L-rhamnose was present as a constituent sugar of the released LPS.