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At least 937 records · Page 52Linked to original sources
PubMed2004

A sensitive enzymatic assay for the determination of sucrose in serum and urine.

BACKGROUND: Sucrose permeability has been suggested as a simple and non-invasive marker of gastric mucosal damage. We here report on a sensitive enzymatic assay using four sequential enzyme reactions coupled with reduced thio-NADPH. METHODS: Sucrose is phosphorylated by sucrose phosphorylase (EC2.4.1.7). The subsequent reaction in the presence of phosphoglucomutase (EC5.4.2.2) and glucose-1,6-diphosphate forms glucose-6-phosphate. Sucrose of the monad forms the dyad thio-NADPH. The reaction is monitored by changes in absorbance at 405 nm. RESULTS: The lower limit of detection (3SD method) was 2.8 micromol/l for serum and 7.0 micromol/l for urine. The precision of the method was <4.0%, and has sufficient analytical range. CONCLUSIONS: The assay was sensitive enough to monitor serum sucrose concentrations during the sucrose permeability test and an automated assay may be useful in a large number of subjects.

Gastric Mucosa↗
PubMedDate not supplied

Molecular aspects of rapid, reversible, Ca2+-dependent de-phosphorylation of pp63/parafusin during stimulated exo-endocytosis in Paramecium cells.

Ca2+ signalling governs stimulated exocytosis and exocytosis-coupled endocytosis also in Paramecium cells. Upon stimulation, the < or =10(3) dense-core exocytotic organelles (trichocysts) can be synchronously (80 ms) released, followed by endocytotic membrane resealing (350 ms) and retrieval. Paramecium is the most synchronous dense-core exocytotic system known, allowing to dissect rapidly reversible Ca2+-dependent phenomena. This holds for the reversible de-/re-phosphorylation cycle of a 63 kD phosphoprotein, pp63/parafusin (pf), which we have cloned, immuno-localised, and characterised as phosphoglucomutase, the enzyme funneling glucose into the glycolytic pathway. It was isolated ex vivo, followed by MALDI analysis, while X-ray structure analysis was performed after heterologous expression. We found multiple phosphorylation of superficial Ser/Thr residues. Although present also in exo(-) mutants, pp63/pf is selectively de-phosphorylated only in exo(+) strains during synchronous exocytosis (80 ms) and re-phosphorylated within approximately 20 s, i.e., the time required to re-establish [Ca2+] homeostasis. We have isolated relevant protein phosphatases and kinases and probed their activity on pp63/pf in vitro. We consider Ca2+/calmodulin-activated PP2B (calcineurin, whose subunits have been cloned) relevant for de-phosphorylation. Re-phosphorylation can be achieved by two protein kinases that also have been cloned. One is activated by cGMP (PKG) which in turn is formed by Ca2+-activated guanylate cyclase. Another kinase, casein kinase 2, is inhibited by Ca2+ and, hence, activated with some delay in parallel to decreasing [Ca2+] after exocytosis. In total, several Ca2+-sensitive cycles cooperate whose protein components have been localised to the cell cortex. Regulation of the phosphorylation degree of pp63/pf may affect structure binding on a microscale and/or its enzymatic activity. All this may serve fueling substrate into glycolysis with increased ATP re-formation (compromised in exo(-) mutants) and NADH formation, with effects on Ca2+ signalling including mobilisation from cortical stores (alveolar sacs) and overall effects on ATP and Ca2+ dynamics during synchronous exo- and endocytosis.

Animals↗
PubMed2004

Characterization of the Azotobacter vinelandii algC gene involved in alginate and lipopolysaccharide production.

Azotobacter vinelandii is a soil gamma-proteobacteria that fixes nitrogen and forms desiccation-resistant cysts. The exopolysaccharide alginate is an integral part of the layers surrounding the cysts. Here, we reported the cloning of A. vinelandii algC, encoding the enzyme catalyzing the second step of alginate pathway. We showed that AlgC is involved not only in alginate production, but also in lipopolysaccharide (LPS) synthesis and that it seems to have both phosphomannomutase and phosphoglucomutase activities. The transcriptional analysis of the A. vinelandii algC gene showed that it contained two start sites, one of which was dependent on the alternative sigma factor AlgU/AlgT. This finding explains why alginate biosynthesis is dependent on AlgU activity, since all other alginate biosynthetic genes have been characterized previously and algC is the only alginate structural gene that is directly transcribed by this sigma factor.

Alginates↗
PubMed2006

Altered nucleotide sugar metabolism in Streptococcus thermophilus interferes with nitrogen metabolism.

Exopolysaccharide (EPS)-producing Streptococcus thermophilus strains have attracted interest recently, since the EPSs act as natural viscosifiers and texture enhancers of fermented foods. We have previously reported that the low level of EPS production by S. thermophilus LY03 could be improved by altering the activities of enzymes in the central carbon metabolism involved in the nucleotide sugar metabolism. In this study, we observed a reduced growth in milk for the strains with increased UDP-glucose pyrophosphorylase (GalU) activity together with either enhanced phosphoglucomutase activity, and/or enhanced activity of the Leloir enzymes. Rapid growth of these mutants in milk could be restored by the addition of four specific amino acids, i.e. Glu, His, Met, and Val. This amino acid requirement was confirmed in a defined medium. Furthermore, the 31P NMR spectra showed higher levels of the GalU reactants pyrophosphate (PPi) and UDP-glucose in the engineered strain, TMB 6013, compared to the parent strain, LY03. These products plus Glu and the GalU reactant UTP are known to be involved in the nitrogen regulatory system in many bacteria. Thus, these results suggest that the reaction catalyzed by GalU is connected to the nitrogen demand of these engineered strains.

Animals↗
PubMed2004

First record of Trichinella pseudospiralis in the Slovak Republic found in domestic focus.

Infection of Trichinella spp. is widespread among wildlife in Slovakia and the red fox (Vulpes vulpes) is the main reservoir of Trichinella britovi. Trichinella spiralis has been rarely documented in sylvatic and domestic animals of this country. During routine examination of domestic pigs at the slaughter, Trichinella larvae were detected by artificial digestion in a domestic pig of a large-scale breeding farm in Eastern Slovakia. The parasite has been identified by molecular (PCR) and biochemical (allozymes) analyses and by the morphology of the nurse cell as the non-encapsulated species Trichinella pseudospiralis infecting both mammals and birds. The epidemiological investigation carried out at the farm level revealed the presence of the same parasite species in other three pigs of 192 examined (2.1%), in 3 of 14 (21.4%) examined synanthropic rats (Rattus norvegicus) and in a domestic cat. The farm was characterized by inadequate sanitary conditions, insufficient nutrition, cannibalism and the presence of rat population. A different profile has been observed at the phosphoglucomutase locus in T. pseudospiralis isolates from Slovakia in comparison with the T. pseudospiralis reference isolate from the Palearctic region. This is the first documented focus of T. pseudospiralis from Central Europe. The detection in domestic pigs of a non-encapsulated parasite infecting both mammals and birds stresses the need to avoid the use of trichinelloscopy to detect this infection at the slaughterhouse.

Adenylate Kinase↗
PubMed1984

Fasting-induced impairment of glucose-1,6-bisphosphate synthesis in pancreatic islets.

In pancreatic islets removed from rats fasted for 48 hours, the insulin secretory response to glucose is decreased. Although the activity of phosphoglucomutase is unaffected by fasting, the decrease in glucose-stimulated insulin release coincides with a suppression of the glucose-induced increment in both glucose-1,6-P2 content and lactate or pyruvate output. These findings are compatible with a regulatory role of glucose-1,6-P2 in the control of glycolysis in pancreatic islets.

Animals↗
PubMed2000

A novel enzymic determination of maltose.

A novel enzymic determination of maltose with four enzymes (a new enzyme, maltose 1-epimerase [EC 5.1.3.-], maltose phosphorylase [EC 2.4.1.8], beta-phosphoglucomutase [EC 5.4.2.6], and glucose-6-phosphate dehydrogenase [EC 1.1.1.49]) is described. Maltose was rapidly and quantitatively determined within about 2 min by means of maltose 1-epimerase. The standard curve was linear up to 1.5 micromol/mL. The within-run and between-run studies gave precision (CV) values of < 2.0% and < 3.0%, respectively. No significant interferences by mono- and disaccharides were observed with the proposed method under this study. There was a good correlation (r = 0.997) between the results obtained by the enzymic and HPLC methods. This method fulfills the need for an accurate, specific and simple assay of maltose, and it is less time consuming than HPLC and enzymic methods previously reported.

Carbohydrate Epimerases↗
PubMed2001

High yielding one-pot enzyme-catalyzed synthesis of UDP-glucose in gram scales.

Uridine diphosphoglucose is an important cofactor of glucosylating enzymes. A simple and high yielding one-pot enzymatic synthesis of UDPG on a gram scale from glucose via hexokinase, phosphoglucomutase and UDPG pyrophosphorylase (UGPase) is described. Repetitive addition of substrate was used to avoid inhibition of UGPase. The approach allows recovery of active enzymes and their re-use. The synthesis of UDP-[4-(13)C]-glucose on a 0.5 g scale resulted in a final yield of 70% and a purity of >95% after chromatographic purification.

Carbon Radioisotopes↗
PubMedDate not supplied

The evaluation of five electrophoretic phenotyping systems for routine screening of bloodstains.

The performance of the polymorphic marker systems group-specific component (GC), phosphoglucomutase-1 (PGM), alpha-2-HS-glycoprotein (A2HS), haptoglobin (Hp), and erythrocyte acid phosphatase (EAP) was evaluated on control bloodstains. The major factors considered were: sensitivity of the test system; stability of the marker; laboratory economics of each test; and distinguishing power (Dp) of the system. GC was considered to be the most suitable marker for routine screening because of its high stability and Dp, and the sensitivity of the immunoblotting detection method. PGM and A2HS were the next most valuable markers followed by Hp. EAP could only be considered useful when large amounts of relatively fresh bloodstain were available.

Acid Phosphatase↗
PubMedDate not supplied

Typing of human red cell and serum mixtures in three electrophoretic systems.

The isoenzymes erythrocyte acid phosphatase and phosphoglucomutase were typed in mixed red cell samples which had been derived from two individuals; the protein group specific component was typed in mixed serum samples. Typing was performed by isoelectric focusing on ultrathin polyacrylamide gels. Depending upon the mixture, from 2 to 20% (but typically 5-10%) by volume of a second blood or serum needed to be present in a mixture before it could be detected. In the majority of cases when there was significant mixing, samples were readily identified as a mixture when the results consisted of unusual band patterns or unusual band intensities. There would be a few instances when blood or serum could not be identified as a mixture when masking effects occurred or when the mixture produced a combined, apparently normal, pattern.

Acid Phosphatase↗
PubMedDate not supplied

Genetic responses to metal contamination in two clams: Ruditapes decussatus and Ruditapes philippinarum.

Coastal ecosystems are subjected to a wide variety of disturbances, including those due to xenobiotics of agricultural and industrial origin. These pollutants as heavy metals can modify the genetic diversity of populations by favouring or counter-selecting certain alleles or genotypes by differential mortality. In the present study, two genetic markers (phosphoglucomutase and glucosephosphate isomerase) and a protein marker (metallothionein) were monitored in order to determine the impact of heavy metals in different clam populations. Analysis of the genetic structure of the clam populations examined reveals that those inhabiting environments contaminated by heavy metals exhibit a higher allelic diversity and possess alleles at PGM loci that could be selected by the presence of heavy metals. The evaluation of metallothionein levels using a specific polyclonal antibody developed in the Pacific oyster (Crassostrea gigas) demonstrated the existence of a relationship between metallothionein concentrations and the level of metal pollution for clam populations sampled from different sites. An inter-specific difference was also detected between Ruditapes decussatus and Ruditapes philippinarum living in sympatry at the same site, suggesting a differential response of these two species upon exposure to an identical heavy metal concentration.

Adaptation, Physiological↗
PubMed2002

Effect of medium composition and temperature and pH changes on exopolysaccharide yields and stability during Streptococcus thermophilus LY03 fermentations.

To increase the exopolysaccharide (EPS) yields from Streptococcus thermophilus LY03 and to unravel the nature of the EPS degradation process, fermentation experiments were carried out with this strain in a customized MRS medium, using different additional carbohydrates or amino acids possibly related to growth and EPS production. No significant increase of the EPS yields or activities of the enzymes alpha-phosphoglucomutase, UDP-glucose pyrophosphorylase and UDP-galactose 4-epimerase that are correlated with EPS production, or of the activity of dTDP-glucose pyrophosphorylase involved in the rhamnose synthetic branch of EPS biosynthesis, was observed. The EPS monomer composition remained unchanged for all experiments. Fermentations with a sudden temperature increase or lowered pH were carried out as well to try to avoid EPS degradation upon prolonged fermentation. It was demonstrated that EPS degradation took place enzymatically. Incubations of purified high-molecular-mass EPS with cell-free culture supernatant or cell extracts showed its degradation by enzymes with an endo-activity. This glycohydrolytic activity probably encompasses several enzymes having a molecular mass lower than 50,000 and 10,000 Da, and seems to be rather stable at high temperature and low pH. These results contribute to a better understanding of the physiological and chemical factors influencing EPS production and degradation.

Carbohydrate Metabolism↗
PubMed1977

[Presence of a pathway for biosynthesis of UDP-glucose in the Golgi apparatus of rat hepatocytes].

Glucokinase, phosphoglucomutase and glucose-1-phosphate uridylyltransferase are the three enzymes involved in a microsomic pathway for the synthesis of UDP glucose. Evidence is given, in this paper, for the localization of these three enzymes in a Golgi-rich fraction of rat liver. This fraction is prepared, from smooth microsomes, by the means of a discontinuous four-step sucrose gradient. Three of the lighter fractions (d = 1.08-1.13) are enriched in the Golgi markers (galactosyltransferase, sialytransferase and thiamin pyrophosphatase), especially the one with density 1.13. The three enzymes we are interested in are enriched in the two upper hands (d 1.08-1.11), which display an activity for the biosynthesis of UDP-glucose from glucose equivalent to the one obtained in a crude microsomic preparation, and which are not contaminated by other subcellular components.

Animals↗
PubMed1989

Age differences between distributions of genotypes among pregnant women: evidence of fertility selection.

The number of children produced by a modern woman is usually below her total reproductive capacity and is determined by circumstances other than natural selection. It is, therefore, practically impossible to detect differences in natural fertilities associated with different types (e.g. phenotypes, genotypes) of women. This does not mean, however, that natural selection at the reproductive level cannot at all be detected today. If women of a particular type have high natural fertility, this usually means that they reproduce (become pregnant) at a higher rate than women of a type with lower natural fertility. Hence, when there is a limit on the number of children, women of the first type will reach the limit at an earlier age than women of the second type. As a result, types that have a higher natural fertility should be overrepresented among pregnant women of younger ages and, consequently, underrepresented among older ones, as compared to types with a lower natural fertility. Based on this notion, a model of age-related differences between distributions of types among pregnant women is suggested. The model is applied to data on MNSs-blood group and PGM1 (phosphoglucomutase) types in a sample of pregnant women and an evidence of natural selection at the reproduction level associated with these genetic markers is obtained.

Adolescent↗
PubMed1983

Isozyme patterns of Schistosoma japonicum and S. mansoni.

Isozyme patterns of six enzymes, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, hexokinase, malate dehydrogenase, 6-phosphate dehydrogenase and phosphoglucomutase were examined in electrophoresed homogenates of adult male worms of Schistosoma japonicum and S. mansoni. In general, enzyme patterns obtained from the parasite homogenates differed from that of host (mouse) blood and muscle, indicating that electrophoretic patterns from parasite extracts are most probably of parasite origin. Adult male and female S. mansoni worms yielded identical patterns. However, all six enzyme patterns showed distinct differences between S. japonicum and S. mansoni. These results suggest that S. japonicum is clearly distinguishable from S. mansoni at the molecular level.

Animals↗
PubMed1989

Enzyme polymorphism in Schistosoma mattheei from cattle in the Eastern Transvaal Lowveld.

Enzyme electrophoresis was conducted on 10 Schistosoma mattheei adult worm samples, comprising 270 individuals, collected from cattle in the Eastern Transvaal Lowveld. Glucose-6-phosphate dehydrogenase (G6PDH) was studied in all the samples and phosphoglucomutase (PGM) and malate dehydrogenase (MDH) in five populations each. Only one population was polymorphic for G6PDH. In this population, in addition to the allele found in all the other samples, a second allele occurred with a similar Rf value to S. haematobium. The two alleles were in Hardy-Weinberg equilibrium. MDH-1 exhibited two alleles. However, these alleles were not in equilibrium. In certain populations, heterozygotes occurred together with homozygotes of one of the alleles only. PGM was monomorphic in all the populations studied.

Alleles↗
PubMed1997

Comparison of human and murine isolates of Schistosoma mansoni from Richard-Toll, Senegal, by isoelectric focusing.

Studies on human and murine isolates of Schistosoma mansoni, from Richard-Toll, Senegal, were carried out by isoelectric focusing in polyacrylamide gels. Seven enzyme systems; lactate dehydrogenase (LDH), malate dehydrogenase (MDH), glucose-6-phosphate dehydrogenase (G6PD), acid phosphatase (AcP), hexokinase (HK), glucose phosphate isomerase (GPI), and phosphoglucomutase (PGM), were used to compare the two isolates. All systems tested, apart from LDH, were found to be polymorphic for both isolates. Interestingly, one phenotype is more frequent than the remainder. The results show that there is no significant genetic variation between the S. mansoni isolates from man and the rodents, Arvicanthis niloticus and Mastomys huberti.

Acid Phosphatase↗