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PubMed1999

Acquisition of species-specific O-linked carbohydrate chains from oviducal mucins in Rana arvalis. A case study.

The extracellular matrix surrounding amphibian eggs is composed of mucin-type glycoproteins, highly O-glycosylated and plays an important role in the fertilization process. Oligosaccharide-alditols were released from the oviducal mucins of the anuran Rana arvalis by alkali-borohydride treatment in reduced conditions. Neutral and acidic oligosaccharides were fractionated by ion-exchange chromatographies and purified by HPLC. Each compound was identified by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) spectrometry, NMR spectroscopy, electrospray ionization-tandem mass spectroscopy (ESI-MS/MS) and permethylation analyses. This paper reports on the structures of 19 oligosaccharide-alditols, 12 of which have novel structures. These structures range in size from disaccharide to octasaccharide. Some of them are acidic, containing either a glucuronic acid or, more frequently, a sulfate group, located either at the 6 position of GlcNAc or the 3 or 4 positions of Gal. This latter sulfation is novel and has only been characterized in the species R. arvalis. This structural analysis led to the establishment of several novel carbohydrate structures, demonstrating the structural diversity and species-specificity of amphibian glycoconjugates.

Animals↗
PubMed1996

Structures of five sulfated hexasaccharides prepared from porcine intestinal heparin using bacterial heparinase. Structural variants with apparent biosynthetic precursor-product relationships for the antithrombin III-binding site.

Porcine intestinal heparin was extensively digested with Flavobacterium heparinase and size-fractionated by gel chromatography. Subfractionation of the hexasaccharide fraction by anion exchange high pressure liquid chromatography yielded 10 fractions. Six contained oligosaccharides derived from the repeating disaccharide region, whereas four contained glycoserines from the glycosaminoglycan-protein linkage region. The latter structures were reported recently (Sugahara, K., Tsuda, H., Yoshida, K., Yamada, S., de Beer, T., and Vliegenthart, J.F.G. (1995) J. Biol. Chem. 270, 22914-22923). In this study, the structures of one tetra- and five hexasaccharides from the repeat region were determined by chemical and enzymatic analyses as well as 500-MHz 1H NMR spectroscopy. The tetrasaccharide has the hexasulfated structure typical of heparin. The five hexa- or heptasulfated hexasaccharides share the common core pentasulfated structure delta HexA(2S) alpha 1-4GlcN-(NS, 6S) alpha 1-4IdoA alpha/GlcA beta 1-4GlcN(6S) alpha 1-4GlcA beta 1-4GlcN (NS) with one or two additional sulfate groups (delta HexA, GlcN, IdoA, and GlcA represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid, D-glucosamine, L-iduronic acid, and D-glucuronic acid, whereas 2S, 6S and NS stand for 2-O-, 6-O-, and 2-N-sulfate, respectively). Three components have the following hitherto unreported structures: delta HexA(2S) alpha 1-4GlcN(NS, 6S) alpha 1-4GlcA beta 1-4GlcN(NS, 6S) alpha 1-4GlcA beta 1-4GlcN(NS,6S), delta HexA(2S) alpha 1-4GlcN(NS, 6S) alpha 1-4IdoA alpha 1-4GlcNAc(6S)-alpha 1-4GlcA beta 1-4GlcN(NS, 3S), and delta HexA(2S) alpha 1-4GlcN-(NS,6S) alpha 1-4IdoA (2S) alpha 1-4GlcNAc(6S) alpha 1-4GlcA beta 1-4GlcN(NS, 6S). Two of the five hexasaccharides are structural variants derived from the antithrombin III-binding sites containing 3-O-sulfated GlcN at the reducing termini with or without a 6-O-sulfate group on the reducing N,3-disulfated GlcN residue. Another contains the structure identical to that of the above heptasulfated antithrombin III-binding site fragment but lacks the 3-O-sulfate group and therefore is a pro-form for the binding site. Another has an extra sulfate group on the internal IdoA residue of this pro-form and therefore can be considered to have diverged from the binding site in the biosynthetic pathway. Thus, the isolated hexasacharides in this study include the three overlapping pairs of structural variants with an apparent biosynthetic precursor-product relationship, which may reflect biosynthetic regulatory mechanisms of the binding site.

Animals↗
PubMed2000

Regulation of urokinase/urokinase receptor interaction by heparin-like glycosaminoglycans.

We show here that the interaction between the urokinase-type plasminogen activator and its receptor, which plays a critical role in cell invasion, is regulated by heparan sulfate present on the cell surface and in the extracellular matrix. Heparan sulfate oligomers showing a composition close to the dimeric repeats of heparin (glucosamine-NSO(3)(6-OSO(3))-iduronic acid(2-OSO(3))) n = 5 and n > 5, where iduronic acid may alternate with glucuronic acid, exhibit affinity for urokinase plasminogen activator and confer specificity on urokinase/urokinase receptor interaction. Cell surface clearance of heparan sulfate reduces the affinity of such interaction with a parallel decrease of specific urokinase binding in the presence of an unaltered expression of receptor. Transfection of human urokinase plasminogen activator receptor in normal Chinese hamster ovary fibroblasts and in Chinese hamster ovary cells defective for the synthesis of sulfated glycosaminoglycans results in specific urokinase/receptor interaction only in nondefective cells. Heparan sulfate/urokinase and receptor/urokinase interactions exhibit similar K(d) values. We concluded that heparan sulfate functions as an adaptor molecule that confers specificity on urokinase/receptor binding.

Animals↗
PubMed1991

Isolation and characterization of hyodeoxycholic-acid: UDP-glucuronosyltransferase from human liver.

The enzyme hyodeoxycholic-acid: UDP-glucuronosyltransferase was purified about 230-fold from a solubilized human liver microsomal preparation utilizing anion-exchange chromatography, ampholyte-displacement chromatography and UDP-hexanolamine--Sepharose affinity chromatography. The homogeneity of the final enzyme preparation was judged by two criteria: the appearance of a single band of Mr 52000 in SDS/PAGE; the elution of a single peak in reversed-phase FPLC. The isolated enzyme catalyzed the glucuronidation of the 6 alpha-hydroxy bile acids hyodeoxycholic and hyocholic acids, and of the steroid hormone estriol, with a ratio of relative reaction rates of 13:1:2.7. UDP-glucuronosyltransferase activities toward the 3 alpha-hydroxy bile acid lithocholic acid, androsterone, testosterone, bilirubin and p-nitrophenol were not detectable in the pure enzyme preparation and were shown to be separated from enzyme activity toward hyodeoxycholic acid during ampholyte-displacement chromatography and/or UDP-hexanolamine--Sepharose affinity chromatography. Two-substrate kinetic analysis of hyodeoxycholic-acid-conjugating activity gave a sequential mechanism with apparent Km values of 12 microM and 4 microM for hyodeoxycholic acid and UDP-glucuronic acid, respectively. Phospholipids were required for reconstitution of maximal activity toward hyodeoxycholic acid. Phosphatidylcholine was the most effective activator of enzyme activity.

Bile Acids and Salts↗
PubMed2006

Galactosamine prevents ethinylestradiol-induced cholestasis.

Ethinylestradiol (EE) induces intrahepatic cholestasis in experimental animals being its derivative, ethinylestradiol 17beta-glucuronide, a presumed mediator of this effect. To test whether glucuronidation is a relevant step in the pathogenesis of cholestasis induced by EE (5 mg/kg b.wt. s.c. for 5 consecutive days), the effect of simultaneous administration of galactosamine (200 mg/kg b.wt. i.p.) on biliary secretory function was studied. A single injection of this same dose of galactosamine was able to decrease hepatic UDP-glucuronic acid (UDP-GA) levels by 85% and excretion of EE-17beta-glucuronide after administration of a tracer dose of [3H]EE by 40%. Uridine (0.9 g/kg b.wt. i.p.) coadministration reverted the effect of galactosamine on hepatic UDP-GA levels and restored the excretion of [3H]EE-17beta-glucuronide. When administered for 5 days, galactosamine itself did not alter any of the serum markers of liver injury studied (aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase) or biliary secretory function. When coadministered with EE, galactosamine partially prevented the impairment induced by this estrogen in total bile flow, the bile-salt-independent fraction of bile flow, basal bile salt secretion, and the secretory rate maximum of tauroursodeoxycholate. Uridine coadministration partially prevented galactosamine from exerting its anticholestatic effects. In conclusion, galactosamine administration partially prevented EE-induced cholestasis by a mechanism involving decreased UDP-GA availability for subsequent formation of EE 17beta-glucuronide. The evidence thus supports the hypothesis that EE 17beta-glucuronide is involved in the pathogenesis of EE cholestasis.

Animals↗
PubMed1990

Sugar-mediated induction of Agrobacterium tumefaciens virulence genes: structural specificity and activities of monosaccharides.

The virulence genes of Agrobacterium tumefaciens are induced by specific plant phenolic metabolites and sugars (G. A. Cangelosi, R. G. Ankenbauer, and E. W. Nester, Proc. Natl. Acad. Sci. USA, in press). In this report, monosaccharides, derivatives, and analogs which induce the vir regulon have been identified and the structural requirements for monosaccharide-mediated induction have been determined. Pyranose sugars with equatorial hydroxyls at C-1, C-2, and C-3 displayed strong vir gene-inducing activity; the C-4 hydroxyl could be epimeric and a wide variety of substitutions at C-5 were permissible. The acidic monosaccharide derivatives D-galacturonic acid and D-glucuronic acid were the strongest inducers among the monosaccharides tested. Eight of the 11 inducing compounds are known plant metabolites, and 7 are monomers of major plant cell wall polysaccharides. A role for monosaccharides and plant phenolic compounds as wound-specific plant metabolites which signal the ChvE/VirA/VirG regulatory system is proposed.

Bacterial Proteins↗
PubMed1993

Ultrastructure and chemical composition of the sheath of Leptothrix discophora SP-6.

Light microscopy and transmission electron microscopy of thin sections and metal-shadowed specimens showed that the sheath of Leptothrix discophora SP-6 (ATCC 51168) is a tube-like extracellular polymeric structure consisting of a condensed fabric of 6.5-nm-diameter fibrils underlying a more diffuse outer capsular layer. In thin sections, outer membrane bridges seen to contact the inner sheath layer suggested that the sheath fabric was attached to the outer layer of the gram-negative cell wall. The capsular polymers showed an affinity for cationic colloidal iron and polycationic ferritin, indicating that they carry a negative charge. Cell-free sheaths were isolated by treatment with a mixture of lysozyme, EDTA, and N-lauroylsarcosine (Sarkosyl) or sodium dodecyl sulfate (SDS). Both Sarkosyl- and SDS-isolated sheaths were indistinguishable in microscopic appearance. However, the Mn-oxidizing activity of Sarkosyl-isolated sheaths was more stable than that of SDS-isolated sheaths. The Sarkosyl-isolated sheaths also contained more 2-keto-3-deoxyoctanoic acid and more outer membrane protein than SDS-isolated sheaths. The oven-dried mass of detergent-isolated sheaths represented approximately 9% of the total oven-dried biomass of SP-6 cultures; the oven-dried sheaths contained 38% C, 6.9% N, 6% H, and 2.1% S and approximately 34 to 35% carbohydrate (polysaccharide), 23 to 25% protein, 8% lipid, and 4% inorganic ash. Gas-liquid chromatography showed that the polysaccharide was an approximately 1:1 mixture of uronic acids (glucuronic, galacturonic, and mannuronic acids and at least one other unidentified uronic acid) and an amino sugar (galactosamine). Neutral sugars were not detected. Amino acid analysis showed that sheath proteins were enriched in cysteine (6 mol%). The cysteine residues in the sheath proteins probably provide sulfhydryls for disulfide bonds that play an important role in maintaining the structural integrity of the sheath (D. Emerson and W.C. Ghiorse, J. Bacteriol. 175:7819-7827, 1993).

Amino Acids↗
PubMed1983

Hepatic metabolism of 3 alpha-hydroxy-5 beta-etianic acid (3 alpha-hydroxy-5 beta-androstan-17 beta-carboxylic acid) in the adult rat.

Normal human meconium has been shown to contain short-chain (C20-C22) bile acids and, recently, these compounds have been identified in sera of patients with cholestasis. This suggests that shortchain bile acids may be secreted in bile. We have examined this point by studying the hepatic metabolism and biliary secretion of one naturally occurring C20 bile acid, 3 alpha-hydroxy-5 beta-etianic acid (3 alpha-hydroxy-5 beta-androstan-17 beta-carboxylic acid). [3-3H]-3 alpha-hydroxy-5 beta-etianic acid was prepared and administered intravenously to rats prepared with an external biliary fistula. 85.5 +/- 1.2% of the administered dose was recovered in bile over 20 h with 71.5 +/- 1.3% appearing in the first hour. 11.9 +/- 1.6% of the dose was estimated to be distributed in body water and 0.6 +/- 0.2% was recovered as organic matter in urine. Total recovery of label was 98.0 +/- 2.6%. Administration of milligram quantities of 3 alpha-hydroxy-5 beta-etianic acid produced an increase in bile flow (58.9 +/- 7.1% over basal levels) within 20 min after injection of the steroid. The radiolabeled material in bile was shown by thin-layer chromatography (TLC) to be a polar conjugate which, after beta-glucuronidase hydrolysis, cochromatographed with authentic free 3 alpha-hydroxy-5 beta-etianic acid. After purification, and derivatization, the steroid moiety was proven by gas chromatography-mass spectrometry to be identical to 3 alpha-hydroxy-5 beta-etianic acid. Characterization of the conjugate by TLC and by 3 alpha-hydroxysteroid dehydrogenase assay, before and after beta-glucuronidase hydrolysis, indicated that the steroid was secreted in bile as the 3-O-beta-glucuronide. It is concluded that 3 alpha-hydroxy-5 beta-etianic acid is cleared from the plasma, conjugated with glucuronic acid, and secreted into bile rapidly and in high concentration. The choleretic properties of this shortchain bile acid contrast with the cholestatic effects of lithocholic acid, its C24 analog. Both the form of conjugation of etianic acid and its effect on bile flow suggest that the shortened side chain of this steroid markedly alters its hepatic metabolism and physiology.

Androstanols↗
PubMed1984

Metabolic fate of ethyl O-[N-(p-carboxyphenyl)-carbamoyl] mycophenolate (CAM), a new antitumor agent, in experimental animals.

Absorption, distribution, metabolism and excretion of ethyl O-[N-(p-carboxyphenyl)-carbamoyl] mycophenolate (CAM), a new antitumor agent, was studied in mice using 14C-labeled CAM and species difference of metabolism was studied in mice, rats, guinea pigs, rabbits and dogs. CAM was rapidly absorbed from the gastrointestinal tract of mice. Concentration of radioactivity in the plasma of mice showed two peaks reflecting enterohepatic circulation of metabolites, the first was 30 min and the second was 6 h after dosing. Extremely high concentration of radioactivity was observed in the bile as about 100 times of the radioactivity detected in the plasma. Wide distribution of radioactivity was observed in the whole body tissues of the tumor-bearing mice. Strong radioactivity was presented in the liver, kidney, bile and gastrointestinal content. Radioactivity in the tumor was higher than those in the liver and kidney 24 h after dosing. About 45% of the dose and 30% of the dose were excreted in the urine and feces, respectively, during 24 h after administration of 14C-CAM to mice. The metabolites of CAM were only mycophenolic acid (MPA) and its glucuronic acid conjugate (MPA-G) in all tested animals. Converting ratio of MPA to MPA-G differed greatly among animal species. In the plasma of rats and mice, MPA was presented as higher level than MPA-G at all times. While, in the plasma of rabbits, MPA-G was presented exclusively and MPA was observed only slightly.

Animals↗
PubMedDate not supplied

Beta-carotene-rich carotenoid-protein preparation and exopolysaccharide production by Rhodotorula rubra GED8 grown with a yogurt starter culture.

The underlying method for obtaining a beta-carotene-rich carotenoid-protein preparation and exopolysaccharides is the associated cultivation of the carotenoid-synthesizing lactose-negative yeast strain Rhodotorula rubra GED8 with the yogurt starter culture (Lactobacillus bulgaricus 2-11 + Streptococcus thermophilus 15HA) in whey ultrafiltrate (45 g lactose/l) with a maximum carotenoid yield of 13.37 mg/l culture fluid on the 4.5th day. The chemical composition of the carotenoid-protein preparation has been identified. The respective carotenoid and protein content is 497.4 microg/g dry cells and 50.3% per dry weight, respectively. An important characteristic of the carotenoid composition is the high percentage (51.1%) of beta-carotene (a carotenoid pigment with the highest provitamin A activity) as compared to 12.9% and 33.7%, respectively, for the other two individual pigments--torulene and torularhodin. Exopolysaccharides (12.8 g/l) synthesized by the yeast and lactic acid cultures, identified as acid biopolymers containing 7.2% glucuronic acid, were isolated in the cell-free supernatant. Mannose, produced exclusively by the yeast, predominated in the neutral carbohydrate biopolymer component (76%). The mixed cultivation of R. rubra GED8 with the yogurt starter (L. bulgaricus 2-11 + S. thermophilus 15HA) in ultrafiltrate under conditions of intracellular production of maximum amount of carotenoids and exopolysaccharides synthesis enables combined utilization of the culture fluid from the fermentation process.

Amino Acids↗
PubMedDate not supplied

Absorption and disposition of 2-[4-(2,2-dichlorocyclopropyl)phenoxy]-2-methylpropanoic acid, WIN 35,833, in rats, monkeys, and men.

2-[4-(2,2-Dichlorocyclopropyl)phenoxy]-2-methylpropanoic acid, Win 35,833, was readily absorbed after oral administration; in rats, rhesus monkeys, and human volunteers, peak concentrations of drug in plasma were attained within 2 hr of medication. The time-concentration curve of administered drug was biphasic in monkeys and men, while in rats the kinetics of a one-compartment model were observed. Distribution studies of 14C-labeled drug in the rat showed that most of the radioactivity was excreted in the feces and that significant quantities of 14C were sequestered by depot fat. Monkeys and human subjects both eliminated Win 35,833 primarily through the kidneys. The drug was excreted in rat bile and human urine, both as the free acid and conjugated with glucuronic acid. At physiological concentrations, Win 35,833 was extensively bound to rat, monkey, and human plasma proteins. A gas-chromatographic method for the analysis of drug in plasma, urine, or bile gave a linear relationship between peak height ratios and concentrations, in the range of 1-60 mug/ml.

Animals↗
PubMed1989

O-acetylation and de-O-acetylation of sialic acids. O-acetylation of sialic acids in the rat liver Golgi apparatus involves an acetyl intermediate and essential histidine and lysine residues--a transmembrane reaction?

Isolated intact rat liver Golgi vesicles utilize [acetyl-3H]coenzyme A to add 3H-O-acetyl esters to sialic acids of internally facing endogenous glycoproteins. During this reaction, [3H]acetate also accumulates in the vesicles, even though the vesicles are impermeant to free acetate. On the other hand, entry of intact AcCoA into the lumen of the vesicles could not be demonstrated, and permeabilization of the vesicles did not alter the reaction substantially (Diaz, S., Higa, H. H., Hayes, B. K., and Varki, A. (1989) J. Biol. Chem. 264, 19416-19426). When vesicles prelabeled with [acetyl-3H] coenzyme A are permeabilized with saponin, we can demonstrate a [3H]acetyl intermediate in the membrane that can transfer label to the 7- and 9-positions of exogenously added free N-acetylneuraminic acid but not to glucuronic acid or CMP-N-acetylneuraminic acid. This labeled acetyl intermediate represents a significant portion of the radioactivity incorporated into the membranes during the initial incubation and cannot be accounted for by nonspecifically "trapped" acetyl-CoA in the permeabilized vesicles. There was no evidence for involvement of acetylcarnitine or acetyl phosphate as an intermediate. The overall acetylation reaction appears to involve two steps. The first step (utilization of exogenous acetyl-CoA to form the acetyl intermediate) is inhibited by coenzyme A-SH (apparent Ki = 24-29 microM), whereas the second (transfer from the acetyl intermediate to sialic acid) is not affected by millimolar concentrations of the nucleotide. Studies with amino acid-modifying reagents indicate that 1 or more histidine residues are involved in the first step of the acetylation reaction. Diethylpyrocarbonate (which can react with both nonsubstituted and singly acetylated histidine residues) also blocks the second reaction, indicating that the acetyl intermediate on both sides of the membrane involves histidine residue(s). Taken together with data presented in the preceding paper, these results indicate that the acetylation of sialic acids in Golgi vesicles may occur by a transmembrane reaction, similar to that described for the acetylation of glucosamine in lysosomes (Bame, K. J., and Rome, L. H. (1985) J. Biol. Chem. 260, 11293-11299). However, several features of this Golgi reaction distinguish it from the lysosomal one, including the nature and kinetics of the reaction and the additional involvement of an essential lysine residue. The accumulation of free acetate in the lumen of the vesicles during the reaction may occur by abortive acetylation (viz. transfer of label from the acetyl intermediate to water). It is not clear if this is an artifact that occurs only in the in vitro reaction.

Acetates↗
PubMedDate not supplied

Impaired biliary elimination of beta-glucuronidase-resistant "glucuronides" of valproic acid after intravenous administration in the rat. Evidence for oxidative metabolism of the resistant isomers.

A major metabolite of valproic acid (VPA) is its glucuronic acid conjugate (VPA-G). The disposition of VPA-G was compared with that of its intramolecularly rearranged, beta-glucuronidase-resistant isomers (collectively called VPA-G-R) after iv bolus administration to pentobarbitone-anesthetized rats. VPA-G was eliminated from blood more rapidly than VPA-G-R. After administration of dose A (predominantly VPA-G) and dose B (predominantly VPA-G-R) to rats with catheterized bladders and bile ducts, total conjugated VPA in blood declined from 110 micrograms of VPA/ml at 2 min to 1.1 micrograms/ml at 1 and 3 hr, respectively. A role for systemic hydrolysis of VPA-G was demonstrated by blood concentrations of free VPA which increased until 30 min. A minor role for systemic hydrolysis of VPA-G-R may be possible but cannot be proved from the current data. Urinary excretion (57 and 56% of doses A and B, respectively, in 3 hr) was greater than biliary excretion (32 and 10% of the doses, respectively, in 3 hr). The lower biliary elimination of VPA-G-R may be caused in part by impaired transport from blood to hepatocytes and/or hepatocytes to bile, but a role for phase I metabolism of the VPA moiety of VPA-G-R was demonstrated by recovery of 4.4% of dose B as 4-hydroxy-VPA. This latter mechanism was less (or not) applicable to VPA-G since only 0.4% of dose A was recovered as 4-hydroxy-VPA. Other VPA metabolites measured were quantitatively less important. These results were consistent in rats where either or both of the urinary and biliary elimination routes were surgically blocked.

Animals↗
PubMed1983

Correlation between structure, fat-clearing and anticoagulant properties of heparins and heparan sulphates.

Heparins and heparan sulphates from different organs, of high purity by accepted criteria, were characterized by chemical and physical (including electrophoretic and 13C-NMR spectroscopic) methods. The fat-clearing activity of these preparations was shown to be correlated with their content of the trisulphated disaccharide units I2S-ANS,6S (L-iduronic acid 2-O-sulphate-N-sulphated D-glucosamino 6-O sulphate). Species with low anti-lipemic activity contained significant proportions (greater than or equal to 30%) of nonsulphated uronic acids (especially D-glucuronic acid), and of D-glucosamino residues undersulphated at C-6, these latter residues being partially N-acetylated. Heparins from beef lung and sheep mucosa, predominantly consisting of trisulphated disaccharide units, displayed consistently higher antilipemic activity than the more heterogeneous pig mucosal heparins. The anticoagulant activity (as determined by the U.S.P., APTT and anti-Xa tests) was not a simple function of the measured physicochemical parameters. Trends were confirmed for pig mucosal heparins being more anticoagulant than beef lung preparations, and low molecular weight (usually undersulphated) species being more active in the anti-Xa test than in the U.S.P. and APTT tests.

Amino Acids↗
PubMed1978

Increased D-glucaric acid excretion by jaundiced patients.

The urinary excretion of D-glucaric acid, a catabolite of glucuronic acid, is considered to be a reliable index of the state of hepatic microsomal enzyme activity. Because enzyme activity may be altered in liver disease, we examined the effect of liver disease on the excretion of this metabolite and its correlation with liver function tests. We studied 89 patients with nonhemolytic jaundice, 39 with viral hepatitis, 33 with obstructive jaundice, six with cirrhosis, and 11 patients with jaundice of mixed etiology. Glucaric acid excretion was significantly increased in all these patients as compared to controls, most pronounced in the obstructive jaundice group. No correlation was found between glucaric acid excretion and concentrations of bilirubin, albumin, globulin, aspartate aminotransferase, alkaline phosphatase, cholesterol, or gamma-glutamyltransferase in serum, even though the concentrations of these analytes did vary with the type of liver disease. We suggest that this increase in glucaric acid excretion is an indication of normal or even increased glucuronidation (UDP-glucuronosyltransferase activity), which occurs in liver disease.

Adult↗
PubMed1993

A method for the determination of UDP-glucuronosyltransferase activity toward arylcarboxylic acids.

A fast and reliable method for the assay of UDP-glucuronosyltransferase (UGT) activity toward aglycones containing a carboxylic acid function is described. The procedure involves incubation with UDP-[U-14C]-glucuronic acid, the common substrate for the reaction, solid-phase separation of the radiolabeled acylglucuronides and unreacted cofactor, and quantification by liquid scintillation counting. The technique was validated for each of the seven substrates tested by reversed-phase HPLC, and was then applied successfully to the determination of optimal conditions for the activation of the carboxylic acid-UGT, and the estimation of kinetic constants for the glucuronidation of clofibric acid, 2-naphthylacetic acid, naproxen, and 4,4,4-triphenylbutanoic acid in rat liver microsomes. From the results obtained, we believe that this is an assay which, with only minor modification, could be applied to a wide range of carboxylic acid substrates for which, until now, specific and sensitive assays have been largely unavailable.

Animals↗
PubMed2004

A two-component covRS regulatory system regulates expression of fructosyltransferase and a novel extracellular carbohydrate in Streptococcus mutans.

The expression of fructosyltransferase (FTF), the enzyme that synthesizes fructan from sucrose, is regulated in the cariogenic bacterium Streptococcus mutans. However, the exact mechanism of FTF regulation is unknown. In this study, the role of a two-component regulatory system (covRS) in FTF expression was investigated. A CovR-defective mutant of S. mutans NG8 was constructed by homologous recombination. By use of immunoblotting, the mutant was shown to overexpress FTF in the absence of sucrose, while the wild type and a covRS-complemented mutant showed sucrose-inducible FTF expression. Reverse transcription-PCR showed that the ftf transcript levels were increased in the covR mutant, suggesting regulation at the transcriptional level. The covR mutant was also found to overproduce extracellular carbohydrate, and this phenotype was reversed by covRS complementation. Paper chromatographic studies and chemical tests showed that the extracellular carbohydrate contained glucose and glucuronic acid but not fructose. These results suggest that the extracellular carbohydrate was not fructan. The production of a glucose- and glucuronic acid-containing extracellular carbohydrate has not been reported for S. mutans and may be considered novel. In conclusion, the results indicate that the expression of FTF and a glucose- and glucuronic acid-containing carbohydrate was negatively regulated by the covRS two-component regulatory system in S. mutans.

Bacterial Proteins↗