Effects of sucrose feeding on the intestinal transport of sugars in two strains of rats.
(73/1049)
The rates of intestinal transport of dietary monosaccharides and disaccharides were determined in Wistar rats and the carbohydrate-sensitive BHE rats fed either a stock diet or a 65% sucrose diet. Sucrose-fed rats of both strains generally showed large and significant increases in the rates of glucose, alpha-methylglucose, fructose, and sucrose transport. The transport of galactose, maltose, and lactose did not show consistent increases due to sucrose feeding. Although the magnitude of the increases in sugar transport due to sucrose feeding was only slightly greater in BHE rats than in Wistar rats, BHE rats tended to exhibit a greater rate of sugar trnasport when fed both strains and in the BHE rats fed the stock diet. Lipogenic enzyme activity was greatly increased as a result of sucrose feeding; however, BHE rats did not show greater levels of enzyme activity than did Wistar rats. Liver lipids were increased in both the Wistar and the BHE sucrose-fed rats and in BHE rats fed either diet. (+info)
Phosphorylation of the yeast phospholipid synthesis regulatory protein Opi1p by protein kinase C.
(74/1049)
Opi1p is a negative regulator of expression of phospholipid-synthesizing enzymes in the yeast Saccharomyces cerevisiae. In this work, we examined the phosphorylation of Opi1p by protein kinase C. Using a purified maltose-binding protein-Opi1p fusion protein as a substrate, protein kinase C activity was time- and dose-dependent, and dependent on the concentrations of Opi1p and ATP. Protein kinase C phosphorylated Opi1p on a serine residue. The Opi1p synthetic peptide GVLKQSCRQK, which contained a protein kinase C sequence motif at Ser(26), was a substrate for protein kinase C. Phosphorylation of a purified S26A mutant maltose-binding protein-Opi1p fusion protein by the kinase was reduced when compared with the wild-type protein. A major phosphopeptide present in purified wild-type Opi1p was absent from the purified S26A mutant protein. In vivo labeling experiments showed that the phosphorylation of Opi1p was physiologically relevant, and that the extent of phosphorylation of the S26A mutant protein was reduced by 50% when compared with the wild-type protein. The physiological consequence of the phosphorylation of Opi1p at Ser(26) was examined by measuring the effect of the S26A mutation on the expression of the phospholipid synthesis gene INO1. The beta-galactosidase activity driven by an INO1-CYC-lacI'Z reporter gene in opi1Delta mutant cells expressing the S26A mutant Opi1p was about 50% lower than that of cells expressing the wild-type Opi1p protein. These data supported the conclusion that phosphorylation of Opi1p at Ser(26) mediated the attenuation of the negative regulatory function of Opi1p on the expression of the INO1 gene. (+info)
Enzymes that hydrolyze fungal cell wall polysaccharides. The carbonhydrate constitution of mycodextranse, an endo-alpha (1 yields 4)-D-glucanase from Pencillium melinii.
(75/1049)
The chemical constitution of the carbohydrate portion of mycodextranase, an exocellular endo-alpha(1 yields 4) D-glucanase of Penicillium melinii, has been investigated. At least 80% of the carbohydrate, consisting exclusively of mannose and glucose, is released from protein by treatment of the enzyme with 0.05 M potassium hydroxide plus 1 M sodium borohydride or 0.5 M sodium hydroxide at 50 degrees. There is concomitant destruction of 60% of the threonine and 15% of the serine of the treated enzyme and an increase in absorption, at 241 nm, of the treated protein's spectrum, indicative of an O-glycosidic beta-hydroxyamino acyl linkage between untreated protein and its associated carbohydrate. Mannose is the monosaccharide involved in this linkage. Smith degradation, methylation, and glycosidase digestions of the carbohydrate indicate that it is present in mycodextranase as side chains of mannose, glucosyl alpha(1 yields 2)-mannose, and mannosyl alpha(1 yields 2)-glucosyl alpha(1 yields 2)-mannose units with each enzyme molecule bearing a calculated average of 25 side chains. Separation of pronase glycopeptides by gel filtration on Sephadex G-25 revealed that 96% of the carbohydrate is present in the highest molecular weight fraction which contains 60% of the threonine of mycodextranase but only 3.5% of the aromatic acids judged by its absorbance at 280 nm. Further fractionation of this glycopeptide component on Sephadex G-75 indicates carbohydrate is restricted to two fractions, one containing 71% by weight of the threonine and serine of mycodextranase and 56% of its carbohydrate. These results suggest carbohydrate chains of mycodextranase are clustered in a few threonin-rich regions along the polypeptide chain rather than being separated from each other by nonglycosylated areas. (+info)
Susceptibility to mitomycin C and lecithinase activities of Clostridium oedematiens (C. novyi) type B and D.
(76/1049)
In tests with broth-culture products of Clostridium oedematiens, none of 15 type-B strains showed beta-toxin lecithinase activities exceeding 20 egg-units per ml, whereas 12 of 13 type-D strains consistently produced much greater amounts of the lecithinase. The types also differed in their susceptibility to lysis by mitomycin C (MC). Of 13 type-D strains tested, 12 were sensitive to MC at a concentration of 1 or 2 mug per ml, whilst 14 of 15 type-B strains were insensitive. Phage-like particles were observed in the MC-lysates of some type-D strains. No type-specific differences in the production of indole or the fermentation of maltose were demonstrated. (+info)
Concurrent maltodextrin and cellodextrin synthesis by Fibrobacter succinogenes S85 as identified by 2D NMR spectroscopy.
(77/1049)
1D and 2D NMR experiments were used to analyse the synthesis of various metabolites by resting cells of Fibrobacter succinogenes S85 when incubated with [1-(13)C]glucose, in both extracellular and cellular media. Besides the expected glycogen, succinate, acetate, glucose-1-P and glucose-6-P, maltodextrins and cellodextrins were detected. Maltodextrins were excreted into the external medium. They were found to have linear structures with a maximum degree of polymerization (DP) of about 6 or 7 units. Cellodextrins were located in the cells (cytoplasm and/or periplasm), and their DP was < or = 4. Both labelled (1-(13)C and 6-(13)C) and unlabelled maltodextrins and cellodextrins were detected, showing the contribution of carbohydrate cycling in F. succinogenes, including the reversal of glycolysis and the futile cycle of glycogen. The mechanisms of these oligosaccharide syntheses are discussed. (+info)
Purification and characterization of the heterologously expressed trehalose/maltose ABC transporter complex of the hyperthermophilic archaeon Thermococcus litoralis.
(78/1049)
We report the purification of the maltose/trehalose transporter complex MalFGK of the hyperthermophilic archaeon Thermococcus litoralis. The complex was expressed in Escherichia coli, solubilized in dodecyl maltoside and purified with the aid of a histidine tag on one of the membrane proteins. One hundred grams of cells yielded 3 mg of pure complex. The final product showed ATPase activity at 70 degrees C and was soluble at low detergent concentration. ATPase activity was not due to dissociation of the MalK subunit from the integral membrane proteins MalF and MalG but could not be further stimulated by trehalose/maltose binding protein (TMBP), be it the native protein as isolated from T. litoralis or the soluble engineered protein. The purified native TMBP was identified as a glycoprotein. (+info)
Carbohydrate mediation of boar sperm binding to oviductal epithelial cells in vitro.
(79/1049)
After mating, mammalian spermatozoa are transported to the lower oviductal isthmus. Spermatozoa are sequestered at the isthmus by attaching and interacting with oviductal epithelial cells, hence forming a sperm reservoir. In several mammalian species, specific carbohydrates mediate sperm-oviductal epithelial cell binding. A quantitative in vitro free cell bioassay was developed to investigate the involvement of carbohydrate recognition in pig sperm-oviductal epithelial cell interactions. This assay was validated. The sensitivity of the assay was such that it was possible to discriminate between different sperm concentrations and sperm-oviductal epithelial cell co-incubation periods, spermatozoa with damaged plasma membranes and epithelial cells of non-reproductive origin. Optimal conditions were used to incubate spermatozoa and oviductal epithelial cells in the presence of six hexose sugars at concentrations of 0, 2, 10 and 50 mmol l(-1). A significant (P < or = 0.05) reduction in the binding of spermatozoa to the oviductal epithelium was detected with 2, 10 and 50 mmol maltose l(-1), 50 mmol lactose l(-1) and 50 mmol mannose l(-1). These findings support the hypothesis that attachment of pig spermatozoa to oviductal epithelium before fertilization is mediated by carbohydrate recognition. (+info)
Conjugation of Arg-Gly-Asp (RGD) sequence in copolymer bearing sugar moiety for insulinoma cell line (MIN6) culture.
(80/1049)
Copolymers composed of an Arg-Gly-Asp (RGD) sequence for the adhesion molecule and sugar moieties were synthesized for an insulinoma cell (MIN6) culture. MIN6 cells attached on the poly(N-p-vinylbenzyl-D-maltonamide-co-6-(p-vinylbenzamido)-hexanoic acid-g-GRGDS) (p(VMA-co-VBGRGDS))-coated dishes were in a more aggregated form than other polymer-coated surfaces. P(VMA-co-VBGRGDS) also shows faster proliferation of MIN6 cells (about 18% higher) than with p(VLA-co-VBGRGDS). By interaction between cell and matrix, about 80% greater insulin secretion from MIN6 cells was produced with the p(VMA-co-VBGRGDS), and about 50% greater insulin secretion was produced with the poly(N-p-vinylbenzyl-D-lactonamide-co-6-(p-vinylbenzamido)-hexanoic acid-g-GRGDS) (p(VLA-co-VBGRGDS) as compared with unstimulated cells. Moreover, attachment of MIN6 cells treated with RGD monomer was suppressed approximately 50% for the p(VMA-co-VBGRGDS) surface. This result supported the idea that conjugation of adhesion molecules of RGD peptide in p(VMA-co-VBGRGDS) copolymer specifically interact with integrin families on MIN6 cell membrane. (+info)