Microbial and chemical conversion of antibiotic K41. II. Preparation of K41-DA1, -DA2 and -DA3 deamicetosyl derivatives of antibiotic K41.
(49/840)
Antibiotic derivative K41-DA1, -DA2 and -DA3 (2 approximately 4), deamicetosyl derivatives of antibiotic K41 (1), were prepared by acidic degradation of K41 and following hydrogenation reaction. K41-DA2 (3) showed comparable antimicrobial activities to K41 in vitro but not in vivo. (+info)
Control of glycolytic dynamics by hexose transport in Saccharomyces cerevisiae.
(50/840)
It is becoming accepted that steady-state fluxes are not necessarily controlled by single rate-limiting steps. This leaves open the issue whether cellular dynamics are controlled by single pacemaker enzymes, as has often been proposed. This paper shows that yeast sugar transport has substantial but not complete control of the frequency of glycolytic oscillations. Addition of maltose, a competitive inhibitor of glucose transport, reduced both average glucose consumption flux and frequency of glycolytic oscillations. Assuming a single kinetic component and a symmetrical carrier, a frequency control coefficient of between 0.4 and 0.6 and an average-flux control coefficient of between 0.6 and 0.9 were calculated for hexose transport activity. In a second approach, mannose was used as the carbon and free-energy source, and the dependencies on the extracellular mannose concentration of the transport activity, of the frequency of oscillations, and of the average flux were compared. In this case the frequency control coefficient and the average-flux control coefficient of hexose transport activity amounted to 0.7 and 0.9, respectively. From these results, we conclude that 1) transport is highly important for the dynamics of glycolysis, 2) most but not all control resides in glucose transport, and 3) there should at least be one step other than transport with substantial control. (+info)
Preparation and chemical composition of the cell walls of Streptococcus mutans.
(51/840)
Purified cell walls from Streptococcus mutans strain BHT were prepared without the use of proteolytic enzymes in order to retain all cell wall constituents for chemical analysis. Of four methods employed, the Ribi cell fractionator produced disrupted cell suspensions which could be most thoroughly purified on sucrose gradients. Results of chemical analyses on purified cell walls prepared in this 8.9% glycerol teichoic acid, 33.6% non-peptidoglycan polysaccharide, and 49.9% peptidoglycan. (+info)
Isolation and characterization of sulfhydryl oxidase from bovine milk.
(52/840)
A method is described for purification of sulfhydryl oxidase from bovine milk which consistently yields preparations with greater than 3000-fold purification over skim milk. A concentration-dependent association-dissociation of the enzyme was adapted to the development of an isolation procedure. Purified preparations exhibited two zones, both of which displayed activity, upon polyacrylamide disc gel electrophoresis, but only one zone following disc gel electrophoresis in sodium dodecyl sulfate. Its mobility indicated a subunit weight of 89,000. Several lines of evidence suggest that iron is an integral part of the enzyme. Treatment of the enzyme with EDTA resulted in complete loss of activity which could be subsequently restored by dialysis against 1 muM ferrous sulfate. Furthermore, atomic absorption analysis and neutron activation analysis of separate enzyme preparations each indicated 0.5 atom of iron per subunit. Chemical analyses of sulfhydryl oxidase accounted for 97% of the sample weight, of which 89% could be attributed to amino acid residues and 11% to carbohydrate residues. Five half-cystine residues per subunit were indicated by cysteic acid analysis and by sulfhydryl group determination following reaction with sodium borohydride. Comparison of this value to the total sulfhydryl groups without reduction tentatively suggests the presence of one disulfide bond. Sulfhydryl oxidase was found to catalyze the oxidation of sulfhydryl groups in both small compounds and proteins, using O2 as oxidant and producing, in equimolar quantities, H2O2 and the corresponding disulfide. A Michaelis constant of 90 muM was obtained using reduced glutathione as substrate, under conditions of optimal pH and temperature, viz., pH 7.0 and 35 degrees. Substrate inhibition was apparent at GSH concentrations above 0.8 mM. In the presence of sulfhydryl oxidase, reductively denatured RNase was reoxidized and fully reactivated within 1 hour, whereas in the absence of the oxidase under otherwise identical conditions, full recovery of RNase activity required 24 hours. The presence of reducing agent was not required for this activity, nor was prior reduction of the sulfhydryl oxidase. Based on the observed activity, it appears that the enzyme could be involved in the biosynthesis of disulfide bonds in certain proteins. (+info)
Formation pathways for lysine-arginine cross-links derived from hexoses and pentoses by Maillard processes: unraveling the structure of a pentosidine precursor.
(53/840)
Covalently cross-linked proteins are among the major modifications caused by the advanced Maillard reaction. So far, the chemical nature of these aggregates and their formation pathways are largely unknown. Synthesis and unequivocal structural characterization are reported for the lysine-arginine cross-links N(6)-(2-([(4S)-4-ammonio-5-oxido-5-oxopentyl]amino)-5-[(2S,3R)-2,3,4- trihydroxybutyl]-3,5-dihydro-4H-imidazol-4-ylidene)-l-lysinate (DOGDIC 12), N(6)-(2-([(4S)-4-ammonio-5-oxido-5-oxopentyl]amino)-5-[(2S)-2,3-dihydroxypropyl]- 3,5-dihydro-4H-imidazol-4-ylidene)-l-lysinate (DOPDIC 13), and 6-((6S)-2-([(4S)-4-ammonio-5-oxido-5-oxopentyl] amino)-6-hydroxy-5,6,7,7a-tetrahydro-4H-imidazo[4,5-b] pyridin-4-yl)-l-norleucinate (pentosinane 10). For these compounds, as well as for glucosepane 9 and pentosidine 11, the formation pathways could be established by starting from native carbohydrates, Amadori products, and 3-deoxyosones, respectively. Pentosinane 10 was unequivocally proven to be an important precursor of pentosidine 11, which is a well established fluorescent indicator for advanced glycation processes in vivo. The Amadori products are shown to be the pivots in the formation of the various cross-links 9-13. The bicyclic structures 9-11 are directly derived from aminoketoses, whereas 12 and 13 stem from reaction with the 3-deoxyosones. All products 9-13 were identified and quantified from incubations of bovine serum albumin with the respective 3-deoxyosone or carbohydrate. From these results it seems fully justified to expect both glucosepane 9 and DOGDIC 12 to constitute important in vivo cross-links. (+info)
Multisite control of the Crabtree effect in ascites hepatoma cells.
(54/840)
AS-30D hepatoma cells, a highly oxidative and fast-growing tumor line, showed glucose-induced and fructose-induced inhibition of oxidative phosphorylation (the Crabtree effect) of 54% and 34%, respectively. To advance the understanding of the underlying mechanism of this process, the effect of 5 mM glucose or 10 mM fructose on the intracellular concentration of several metabolites was determined. The addition of glucose or fructose lowered intracellular Pi (40%), and ATP (53%) concentrations, and decreased cytosolic pH (from 7.2 to 6.8). Glucose and fructose increased the content of AMP (30%), glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate (15, 13 and 50 times, respectively). The cytosolic concentrations of Ca2+ and Mg2+ were not modified. The addition of galactose or glycerol did not modify the concentrations of the metabolites. Mitochondria isolated from AS-30D cells, incubated in media with low Pi (0.6 mM) at pH 6.8, exhibited a 40% inhibition of oxidative phosphorylation. The data suggest that the Crabtree effect is the result of several small metabolic changes promoted by addition of exogenous glucose or fructose. (+info)
Semicarbazide-sensitive amine oxidase substrates stimulate glucose transport and inhibit lipolysis in human adipocytes.
(55/840)
Semicarbazide-sensitive amine oxidases (SSAO) are widely distributed enzymes scavenging biogenic or exogenous amines and generating hydrogen peroxide. We asked whether human adipose tissue could express SSAO. Since hydrogen peroxide exhibits pharmacological insulin-like effects, we also tested whether its endogenous production by SSAO could mimic several insulin effects on adipocytes, such as stimulation of glucose uptake and inhibition of lipolysis. The benzylamine oxidation by human adipose tissue was inhibited by semicarbazide or hydralazine and resistant to pargyline or selegiline. It was due to an SSAO activity localized in adipocyte membranes. A protein of 100-kDa and a 4-kb mRNA corresponding to SSAO were identified in either mammary or abdominal subcutaneous fat depots. In isolated adipocytes, SSAO oxidized similarly benzylamine and methylamine that dose dependently stimulated glucose transport in a semicarbazide-sensitive manner. Antioxidants also inhibited the benzylamine and methylamine effects. Moreover, the ability of diverse substrates to be oxidized by adipocytes was correlated to their effect on glucose transport. Benzylamine and methylamine exerted antilipolytic effects with a maximum attained at 1 mM. These results show that human adipocytes express a membrane-bound SSAO that not only readily oxidizes exogenous amines and generates H(2)O(2), but that also interplays with glucose and lipid metabolism by exerting insulin-like actions. Based on these results and the fact that variations in plasma levels of the soluble form of SSAO have been previously reported in diabetes, we propose that determination of adipocyte SSAO, feasible on subcutaneous microbiopsies, could bring relevant information in pathologies such as obesity or diabetes. (+info)
Differential effect of hexoses on hamster embryo development in culture.
(56/840)
The effects of glucose, fructose, and galactose on hamster embryo development in the absence of phosphate were studied in culture. One- and two-cell embryos were cultured to the blastocyst stage in HECM-9 medium without hexose or in medium with increasing concentrations of hexoses. Embryo development, cell number, and cell allocation were assessed in blastocysts. Blastocyst viability was determined by transfer to pseudopregnant recipients. Although 0.25 mM fructose increased mean cell number, low glucose concentrations had no stimulatory effect on development to blastocyst. Both galactose and 5.0 mM glucose were detrimental to embryos. Addition of 0.5 mM glucose increased implantation and fetal viability as compared with controls. Compared with 0.5 mM glucose, treatment with 0.25 mM fructose gave similar implantation and fetal viability, whereas 5.0 mM glucose tended to decrease implantation and significantly decreased fetal development. These data demonstrate that morphology is a poor indicator of embryo viability and that exposure of preimplantation embryos to glucose or fructose is important for embryo viability post-transfer. Although no difference in blastocyst viability was detected between embryos cultured with 0.25 mM fructose and those cultured with 0.5 mM glucose, increased cell numbers obtained with fructose suggest that fructose may be more appropriate than glucose for inclusion in culture medium. (+info)