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Biomedical subjects

R H Glew

Publications and source records attributed to R H Glew.

At least 19 recordsLinked to original sources
PubMed1995

Effects of oligosaccharide binding on glycogen debranching enzyme activity and conformation.

Glycogen debranching enzyme contains two catalytic activities (4-alpha-glucanotransferase and amylo-1,6-glucosidase) on its single polypeptide chain, and they are affected differently by the binding of oligosaccharides. Glucose, maltose, and maltotriose are competitive inhibitors of the amylo-1,6-glucosidase activity measured by the hydrolysis of alpha-glucosyl fluoride, whereas saccharides with four or more glucose units are activators of the same activity, showing apparent "uncompetitive" kinetics. This suggests that they do not bind until the alpha-glucosyl fluoride is bound. In either case the potency of the effect increases with the length of the oligosaccharide chain. On the other hand, all oligosaccharides tested (maltose to maltohexaose, alpha-cyclodextrin, and beta-cyclodextrin) are competitive inhibitors of the transferase activity and also cause a decrease in the intrinsic fluorescence, both functions again increased by chain length, thus indicating that these saccharides do bind to the free enzyme. These interesting results can be reconciled if the extended main chain resulting from the transferase reaction has to be reoriented into a different binding mode in order to position the alpha-1,6-linked side-chain glucose into the correct position for the glucosidase reaction. Therefore, activating oligosaccharides behave kinetically as if they had not been previously bound. It is concluded that the main chain of the natural limit dextrin substrate has a different mode of binding for the two catalytic reactions in order to position properly first the maltotetraosyl side chain in the transferase catalytic site and then the glucosyl side chain in the glucosidase catalytic site.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Serum levels of the pyridinoline crosslinked carboxyterminal telopeptide of type I collagen (ICTP) and osteocalcin in rachitic children in Nigeria.

OBJECTIVES: We measured the levels of the pyridinoline crosslinked carboxyterminal telopeptide of type I collagen (ICTP) and osteocalcin (OC) in the serum of 12 rachitic and 27 healthy Nigerian children, and compared the performance of these relatively new markers of bone metabolism with established laboratory parameters of skeletal disease. DESIGN AND METHODS: Active rickets was diagnosed on the basis of clinical and biochemical criteria. Serum calcium and phosphorus concentration and alkaline phosphatase activity were determined using clinically accepted methods. Radioimmunoassay was performed to quantify parathyroid hormone, 1-,25-dihydroxyvitamin D, OC, and ICTP. RESULTS: The rachitic children had statistically significant serum elevations of ICTP and osteocalcin as compared with age- and sex-matched controls. Serum levels of ICTP correlated with alkaline phosphate activity. CONCLUSIONS: As a marker of abnormal bone metabolism, ICTP performs at least as well as alkaline phosphate. ICTP and OC are valuable additions to the growing repertoire of bone markers.

Age Factors↗
PubMed1995

Decreased urinary excretion of beta-glucuronidase in sickle cell anemia in Nigeria.

The activities of five lysosomal hydrolases--namely beta-glucuronidase, beta-hexosaminidase, beta-galactosidase, alpha-galactosidase, and alpha-mannosidase--were measured in the plasma and urine of children (ages, 7 to 15 years) with sickle cell anemia (n = 11) and controls (n = 11) from Jos, Nigeria. The presence of SS hemoglobin was confirmed by electrophoresis of red cell hemolysates. Albuminuria was absent in all of the patients with sickle cell anemia. The creatinine-indexed urinary activity level (units of enzyme activity/milligrams creatinine) and the fractional enzyme excretion (FEE) value, which is defined as the ratio of enzyme clearance to creatinine clearance, were determined for each of the five lysosomal enzymes and compared between the two groups. The mean FEE values for beta-glucuronidase and alpha-galactosidase in the sickle cell patients were 10- and 3.5-fold lower, respectively, than the corresponding control values, and these differences were statistically significant (p < .03) for both enzymes; however, beta-hexosaminidase, beta-galactosidase, and alpha-mannosidase levels in urine were not different between the two groups. When indexed to creatinine, a comparison of the urinary enzyme levels of control and sickle cell patients showed significant differences for beta-glucuronidase (p < .01) and alpha-galactosidase (p < .05) but not for the other three enzymes. Differences in level of plasma enzyme activity between control and sickle cell patients were not significant, except for alpha-galactosidase (p < .05), which was increased slightly (25%) in the sickle cell group. These data indicate that there may be abnormalities in the metabolism of lysosomal enzymes in the kidneys of patients with sickle cell anemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1994

Human glucocerebrosidase catalyses transglucosylation between glucocerebroside and retinol.

The basal activity of human placental glucocerebrosidase is elevated 16-fold by n-pentanol when assayed using p-nitrophenyl beta-D-glucopyranoside (pNPGlc) as the beta-glucosidase substrate. This enhancement of activity is the result of the formation of a transglucosylation product, n-pentyl beta-D-glucoside, in rate-determining competition with the hydrolytic reaction. The transglucosylation product accounts for approximately 80% of the reaction product generated in the presence of n-pentanol (0.18 M) when either glucocerebroside or pNPGlc was used as the substrate. This stimulatory effect can be increased an additional 3-fold by the inclusion of phosphatidylserine (20 micrograms/ml) or sodium taurodeoxycholate (0.3%, w/v) in the incubation medium. In the presence of retinol, glucocerebrosidase also catalyses the synthesis of a novel lipid glucoside, retinyl glucoside, when either glucocerebroside or pNPGlc serves as the substrate. The reaction product was identified as retinyl beta-D-glucoside, based on its susceptibility to hydrolysis by almond beta-D-glucosidase and the subsequent release of equimolar amounts of retinol and glucose. The rate of retinyl-beta-glucoside formation is dependent on the concentration of retinol in the incubation medium, reaching saturation at approximately 0.3 mM retinol. Retinyl beta-D-glucoside is a substrate for two broad-specificity mammalian beta-glucosidases, namely the cytosolic and membrane-associated beta-glucosidases of guinea pig liver. However, retinyl beta-D-glucoside is not hydrolysed by placental glucocerebrosidase. These data indicate that the glucocerebrosidase-catalysed transfer of glucose from glucocerebroside to natural endogenous lipid alcohols, followed by the action of a broad-specificity beta-glucosidase on the transglucosylation product, could provide mammals with an alternative pathway for the breakdown of glucocerebroside to glucose and ceramide.

Animals↗
PubMed1994

Why renal cysts grow.

The growth of renal cysts leads to morbidity, contributes to mortality, and is a lesson in applied physiology. The fact that chemical, electrical, osmotic, and hydrostatic driving forces determine cyst volumes is inferred from observations that cyst fluids vary in their concentrations of osmotically active substances. Most have concentrations of sodium-based salts that are lower and nonsodium-based salts that are higher than those present in normal plasma. Nonsodium solutes include osmolytes that normally are present in higher concentrations inside of cells, including potassium, amino acids, and so-called idiogenic osmoles. The basic process of cyst growth therefore involves 1) the osmotic equilibration of water across cysts walls that have variable permeability characteristics and 2) the replacement of sodium salts with other osmotically active solutes in cyst fluids. Cyst volume is governed by the amounts and kinds of osmolytes that enter and become entrapped in lumina. Proliferation and necrosis of mural cells are events that are fundamental to the growth of renal cysts.

Animals↗
PubMed1993

Variable expression of leukocyte cytosolic broad-specificity beta-glucosidase activity.

The cytosolic beta-glucosidase activity that is found in a variety of mammalian tissues has no clearly defined function. In vitro assay conditions under which the broad-specificity beta-glucosidase hydrolyzes glucocerebroside at a significant rate have not been described. Nonetheless, it has been suggested that this enzyme might play an accessory role with lysosomal glucocerebrosidase in catalyzing the hydrolysis of glucosylceramide. However, this hypothesis would require that activity of both enzymes be low in severe cases of Gaucher disease in which there are pathological accumulations of glucosylceramide in one or more of the affected organs, i.e. spleen, liver and bone marrow. Information is lacking regarding the normal range of cytosolic beta-glucosidase activity in humans. p-Nitrophenyl-beta-D-mannoside was found to be a potent inhibitor (Ki = 0.068 mM) of cytosolic beta-glucosidase. In parallel studies, the activity of glucocerebrosidase was found to be minimally affected by p-nitrophenyl-beta-D-mannoside at concentrations as high as 2.5 mM. This information was used to design an assay system that would allow us to estimate glucocerebrosidase and cytosolic beta-glucosidase activities in extracts of human leukocytes. Average cytosolic beta-glucosidase activity with 4-heptyl-umbelliferyl-beta-D-glucoside as a substrate was 8.8 nmol/h/mg protein in leukocytes from 356 subjects (range, 0.2-28). Average leukocyte glucocerebrosidase specific activity was 16 nmol/h/mg protein (range 5.3-45.7). No correlation was observed between cytosolic beta-glucosidase and glucocerebrosidase activity for control and Gaucher heterozygote populations (r = 0.19 and 0.25, respectively). The wide range of leukocyte cytosolic beta-glucosidase activity in individuals tested in this study indicates that a substantial proportion of the population may lack sufficient cytosolic beta-glucosidase activity to assist a defective lysosomal glucocerebrosidase in hydrolyzing glucosylceramide.

Chromatography, High Pressure Liquid↗
PubMed1993

Decreased renal excretion of beta-hexosaminidase in adults with insulin-dependent diabetes mellitus and normal renal function.

The activities of three lysosomal hydrolases and creatinine levels were measured in the plasma and urine of 11 adults (mean age, 28.1 years) with insulin-dependent diabetes mellitus and 14 non-diabetic controls (mean age, 27.9 years). All of the patients were free of diabetic complications and non exhibited microalbuminuria. Fractional enzyme excretion (FEE) values between the two groups of subjects were calculated and compared for the following enzymes: beta-hexosaminidase (N-acetyl-glucosaminidase), beta-glucuronidase and alpha-galactosidase. The FEE value was calculated as the ratio of enzyme clearance to creatinine clearance. Relative to the non-diabetic control group, the FEE value for beta-hexosaminidase was approximately 2-fold lower (P = 0.02) in the diabetic subjects (means, 0.424 vs. 0.242, respectively). The FEE values for beta-glucuronidase and alpha-galactosidase were not significantly different (P > 0.4) between the diabetic and control groups. These easily measured biochemical parameters in blood and urine and the resultant FEE value for beta-hexosaminidase may provide a means of assessing subtle deteriorative changes in renal function which occur in the early stage of diabetes before the onset of clinically evident complications.

Adult↗
PubMed1993

Altered urinary excretion of lysosomal hydrolases in pregnancy.

Creatinine concentrations and the activities of five lysosomal hydrolases were measured in the serum and urine of 14 healthy nonpregnant control women and 19 healthy pregnant women. Fractional enzyme excretion (FEE) values for beta-glucuronidase, beta-hexosaminidase, alpha-galactosidase, beta-galactosidase, and alpha-mannosidase were calculated and compared between the two groups of subjects. Fractional enzyme excretion was calculated as the ratio of enzyme clearance to creatinine clearance. The FEE values for beta-galactosidase and alpha-mannosidase between the nonpregnant and pregnant populations were not statistically different; however, relative to the nonpregnant control group, the median FEE values for beta-glucuronidase (P < 0.03), beta-hexosaminidase (P < 0.06), and alpha-galactosidase (P < 0.02) were decreased approximately 1.5-, 1.8-, and 2.7-fold, respectively, in the pregnant population. The median urinary beta-galactosidase activity for the pregnant population, when expressed on the basis of creatinine, was twofold higher than that of the control group (P < 0.0005). These data indicate that with pregnancy there are marked changes in the urinary excretion of selected lysosomal enzymes, particularly alpha-galactosidase and beta-glucuronidase. When the molecular weights of these five hydrolases were compared between kidney homogenate and control urine, a correlation of 0.96 was observed, while the correlation between control serum and control urine was 0.69. This suggests that the FEE value differences between the pregnant and control groups are most likely due to changes in tubule cell metabolism, either decreased secretion or increased reabsorption. These biochemical changes may provide a means of assessing changes in renal function during pregnancy.

Adolescent↗
PubMed1993

Acid phosphatase activity in Coxiella burnetii: a possible virulence factor.

High-speed supernatant fluids derived from sonicated Coxiella burnetii contained considerable acid phosphatase activity when assayed by using 4-methylumbelliferylphosphate; they also contained a factor that blocked superoxide anion production by human neutrophils stimulated with formyl-Met-Leu-Phe. The pH optimum of the enzyme was approximately 5.0. The level of phosphatase activity detected in several isolates of C. burnetii implicated in acute (Nine Mile) and chronic (S Q217, PRS Q177, K Q154) Q fever was 25 to 60 times greater than that reported in other microorganisms, including Leishmania and Legionella spp. The enzyme was found in rickettsiae grown in different hosts (L929 cells and embryonated eggs) and, in the case of L929 cells, for both short periods (less than a month) and the long term (years). Cytochemical techniques coupled with electron microscopy localized the phosphatase activity to the periplasmic gap in the parasite. Ion-exchange chromatography revealed a major species of the enzyme and showed that the enzyme of the parasite was distinct from that of the host cell (L929 fibroblasts); its apparent molecular weight was 74,000. Phosphatase inhibitors (i.e., molybdate heteropolyanions) had differential effects on the phosphatases of the parasite and host cell. C. burnetii supernatant fluid inhibited superoxide anion production by formyl-Met-Leu-Phe-stimulated human neutrophils; molybdate inhibitors reversed the inhibition. Treatment of C. burnetii-infected L929 cells with one of the molybdate compounds (complex B') significantly reduced the level of infection and did not affect the viability or growth of the host cell. These data suggest that the acid phosphatase of the parasite may be a major virulence determinant, allowing the agent to avoid being killed during uptake by phagocytes and subsequently in the phagolysosome.

Acid Phosphatase↗
PubMed1992

Comparison of urinary excretion of four lysosomal hydrolases in healthy elderly and young adults.

The activities of four lysosomal enzymes and creatinine levels were measured in the plasma and urine of 17 healthy elderly and 7 young adults. Fractional enzyme excretion (FE ENZ) values for beta-hexosaminidase (N-acetylglucosaminidase), alpha-galactosidase, beta-galactosidase and beta-glucuronidase were calculated and compared between the two groups of subjects. FE ENZ was calculated as the ratio of enzyme clearance to creatinine clearance. The FE ENZ values for alpha-galactosidase, beta-galactosidase and beta-glucuronidase between the elderly and young populations were not statistically different; however, relative to the young control group, the FE ENZ value for beta-hexosaminidase was elevated approximately 2-fold in the elderly population (P = 0.06). The mean urinary alpha-galactosidase activity for the elderly population, when expressed on the basis of creatinine, was 50% lower than that of the control group (P = 0.03), whereas the mean urinary beta-hexosaminidase activity for the elderly was significantly higher compared to the control group (P = 0.008). When data for all subjects was analyzed, no correlation was observed between the urinary excretion of beta-hexosaminidase or alpha-galactosidase and glomerular filtration rate. These data indicate that with advancing age there are changes in the tubular secretion or reabsorption of selective lysosomal enzymes, particularly beta-hexosaminidase and alpha-galactosidase. These biochemical changes may provide a means of assessing subtle progressive deterioration of renal function.

Adult↗
PubMed1992

Exolytic hydrolysis of toxic plant glucosides by guinea pig liver cytosolic beta-glucosidase.

We demonstrate that although the guinea pig liver cytosolic beta-glucosidase does not catalyze the hydrolysis of gentiobiose, it does hydrolyze, disaccharide-containing glycosides such as p-nitrophenyl-beta-D-gentiobioside (Glc beta 1----6Glc beta-pNP) and mandelonitrile-beta-D-gentiobioside (amygdalin). Furthermore, we establish that the enzyme attacks disaccharide glycosides exolytically; specifically, we document the exolytic deglucosylation of amygdalin and the generation of the intermediate monosaccharide glycoside mandelonitrile-beta-D-glucoside prior to the formation of the aglycone (mandelonitrile). We also show that the cytosolic beta-glucosidase catalyzes the hydrolysis of various phenolic (e.g. arbutin and salicin) and cyanogenic plant glucosides (e.g. prunasin). Using the everted gut-sack technique, we demonstrate that the plant glucosides, amygdalin, prunasin, and vicine, are transported across the small intestine of the guinea pig efficiently and without being hydrolyzed. Based on these data we speculate that the cytosolic beta-glucosidase may participate in biotransformation of toxic plant glucosides.

Amygdalin↗
PubMed1992

Transglucosylation as a probe of the mechanism of action of mammalian cytosolic beta-glucosidase.

This study establishes that guinea pig liver cytosolic beta-glucosidase generates a common glucosyl-enzyme intermediate from a variety of aryl beta-D-glucoside substrates and that the intermediate can react with various acceptors to form distinct products at rates which are dependent on the structure, nucleophilicity, and concentration of the acceptor. Specifically, we demonstrate that water and linear alkanols will react with the glucosyl-enzyme intermediate to form D-glucose and alkyl-beta-D-glucoside (e.g. octyl-beta-D-glucoside), respectively. The rate of alcoholysis is 24-fold greater than the rate of hydrolysis of the glucosyl-enzyme intermediate and accounts for the increase in steady-state rate of substrate disappearance in the presence of alcohols. In addition, the substrate molecule itself (e.g. p-nitrophenyl-beta-D-galactoside (pNP-Gal)) can serve as an acceptor in the transglycosylation reaction, thereby enabling the enzyme to synthesize disaccharide glycosides (e.g. pNP-beta-Gal(6----1)beta-Gal). The transglycosylation data point to the presence of two hydrophobic subsites in the active site of the cytosolic beta-glucosidase. These data support a model in which the cytosolic beta-glucosidase binds an acceptor and a glycosyl donor simultaneously within its catalytic center and efficiently catalyzes the transfer of a sugar residue from the donor to the acceptor.

1-Butanol↗
PubMed1992

Heparin enhances the interaction of infective Leishmania donovani promastigotes with mouse peritoneal macrophages. A fluorescence flow cytometric analysis.

Human visceral leishmaniasis results from the infection of macrophages by the protozoan parasite Leishmania donovani. Both forms of the parasite, the extracellular promastigote and the obligate intracellular amastigote, require cell surface molecules to ensure their recognition and uptake by the host cell, the macrophage. We have proposed previously that the heparin-binding protein on the surface of promastigotes is an adhesion molecule. The present report provides experimental evidence to support this hypothesis. Fluorescence flow cytometry using FITC-heparin was employed to study the heparin-binding protein of L. donovani promastigotes and amastigotes. We demonstrate the presence of the heparin-binding protein on the surface of amastigotes and document the heparin specificity of the binding protein for both forms of the parasite. Two-color fluorescence analysis was performed to compare R-PNA reactivity and FITC-heparin binding during the parasite's 7-day growth curve. Using this strategy we show that the expression of heparin binding activity coincides with the differentiation of the noninfective promastigote into the infective metacyclic from of the parasite. Macrophages that were challenged for 30 min with heparin-treated, FITC-labeled parasites became 2.82-fold more fluorescent than their counterparts which were exposed to non-heparin-treated FITC-labeled promastigotes. Finally, using Kolmogorov-Smirnov analysis we show that the adhesion of promastigotes to mouse peritoneal macrophages is significantly enhanced in the presence of 3.3 microM heparin. The experiments described in the present report provide evidence for the hypothesis that L. donovani's heparin-binding protein is a virulence factor that functions as an adhesion molecule in the parasite-macrophage interaction.

Animals↗
PubMed1992

Continuous spectrophotometric assays for beta-glucosidases acting on the plant glucosides L-picein and prunasin.

The neutral pH optimum beta-glucosidases of mammalian liver and almonds are each capable of hydrolyzing a number of plant glucosides, including L-picein (p-hydroxyacetophenone-beta-D-glucoside) and prunasin (D-mandelonitrile-beta-D-glucoside). Taking advantage of the marked differences in the spectra of the substrate/product pairs of L-picein/p-hydroxyacetophenone and prunasin/mandelonitrile, we have devised spectrophotometric assays that permit the continuous monitoring at pH 7.0 of p-hydroxyacetophenone (piceol) release from L-picein by guinea pig hepatic cytosolic beta-glucosidase and mandelonitrile from prunasin by almond beta-glucosidase. When L-picein hydrolysis was monitored at 320 nm and prunasin at 282 nm, the molar absorption coefficients determined for their products, namely piceol and mandelonitrile, were 3200 and 1360 M-1 cm-1, respectively. The kinetic parameter Km and Vmax values obtained using these spectrophotometric procedures for the guinea pig liver cytosolic beta-glucosidase acting on L-picein were 0.88 mM and 5.29 x 10(5) units/mg protein and for the almond beta-glucosidase acting on prunasin, Km 1.1 mM and Vmax 5.24 x 10(6) units/mg protein. These values agreed well with previously reported values obtained using less convenient, discontinuous assay procedures.

Animals↗
PubMed1992

Virulence factors of the family Legionellaceae.

Whereas bacteria in the genus Legionella have emerged as relatively frequent causes of pneumonia, the mechanisms underlying their pathogenicity are obscure. The legionellae are facultative intracellular pathogens which multiply within the phagosome of mononuclear phagocytes and are not killed efficiently by polymorphonuclear leukocytes. The functional defects that might permit the intracellular survival of the legionellae have remained an enigma until recently. Phagosome-lysosome fusion is inhibited by a single strain (Philadelphia 1) of Legionella pneumophila serogroup 1, but not by other strains of L. pneumophila or other species. It has been found that following the ingestion of Legionella organisms, the subsequent activation of neutrophils and monocytes in response to both soluble and particulate stimuli is profoundly impaired and the bactericidal activity of these cells is attenuated, suggesting that Legionella bacterial cell-associated factors have an inhibitory effect on phagocyte activation. Two factors elaborated by the legionellae which inhibit phagocyte activation have been described. First, the Legionella (cyto)toxin blocks neutrophil oxidative metabolism in response to various agonists by an unknown mechanism. Second, L. micdadei bacterial cells contain a phosphatase which blocks superoxide anion production by stimulated neutrophils. The Legionella phosphatase disrupts the formation of critical intracellular second messengers in neutrophils. In addition to the toxin and phosphatase, several other moieties that may serve as virulence factors by promoting cell invasion or intracellular survival and multiplication are elaborated by the legionellae. Molecular biological studies show that a cell surface protein named Mip is necessary for the efficient invasion of monocytes. A possible role for a Legionella phospholipase C as a virulence factor is still largely theoretical. L. micdadei contains an unusual protein kinase which catalyzes the phosphorylation of eukaryotic substrates, including phosphatidylinositol and tubulin. Since the phosphorylation of either phosphatidylinositol or tubulin might compromise phagocyte activation and bactericidal functions, this enzyme may well be a virulence factor. Administration of the L. pneumophila exoprotease induces lesions resembling those of Legionella pneumonia and kills guinea pigs, suggesting that this protein plays a role in the pathogenesis of legionellosis. However, recent work with a genetically engineered strain has convincingly shown that the protease is not necessary for intracellular survival or virulence. As might be expected with a complex process like intracellular parasitism, it appears that the capability of Legionella strains to invade and multiply in host phagocytes is multifactorial and that no single moiety which is responsible for the virulence phenotype will be found.

Humans↗