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PubMed1996

M-glycogenin, the protein moiety of Neurospora crassa proteoglycogen, is an auto- and transglucosylating enzyme.

Neurospora crassa proteoglycogen was purified and its protein moiety, M-glycogenin, was released by amylolytic treatment. The released protein was capable of autoglucosylation from UDP-glucose forming glucosyl-alpha 1,4-glucosyl linkage. The kinetics of autoglucosylation suggested an intramolecular mechanism of reaction. M-glycogenin was also able to glucosylate dodecyl-beta-maltoside and autoglucosylate, simultaneously and independently. Both auto- and transglucosylation reactions were dependent on Mn2+. Thus, M-glycogenin, which has also been described as the constituent of Escherichia coli proteoglycogen (A. Goldraij and J. A. Curtino. 1993, Biochem. Mol. Biol. Int. 30, 453-458), is a glucosyltransferase that bears similar catalytic properties with mammalian glycogenin. This is the first report on the enzymatic character of the protein constituent of proteoglycogen in primitive organisms, which suggest that the mechanism for the de novo biosynthesis of glycogen was conserved over a very long period of evolution.

Glucose↗

PubMed2000

Modulation of EGF receptor activity by changes in the GM3 content in a human epidermoid carcinoma cell line, A431.

Gangliosides have been described as modulators of growth factor receptors. For example, GM3 addition in cell culture medium inhibits epidermal growth factor (EGF)-stimulated receptor autophosphorylation. Furthermore, depletion of ganglioside by sialidase gene transfection appeared to increase EGF receptor (EGFR) autophosphorylation. These data suggested that changes in GM3 content may result in different responses to EGF. In this study, the ceramide analog d-threo-1-phenyl-2-decannoylamino-3-morpholino-1-propanol ([D]-PDMP), which inhibits UDP-glucose-ceramide glucosyltransferase, and addition of GM3 to the culture medium were used to study the effects of GM3 on the EGFR. Addition of 10 microM [D]-PDMP to A431 cells resulted in significant GM3 depletion. Additionally, EGFR autophosphorylation was increased after EGF stimulation. When exogenous GM3 was added in combination with [D]-PDMP, the enhanced EGFR autophosphorylation was returned to control levels. [D]-PDMP also increased EGF-induced cell proliferation, consistent with its effect on autophosphorylation. Once again, the addition of GM3 in combination with [D]-PDMP reversed these effects. These results indicate that growth factor receptor functions can be modulated by the level of ganglioside expression in cell lines. Addition of GM3 inhibits EGFR activity and decrease of GM3 levels using [D]-PDMP treatment enhances EGFR activity. Modulation of growth factor receptor function may provide an explanation for how transformation-dependent ganglioside changes contribute to the transformed phenotype.

Carcinoma, Squamous Cell↗

PubMed2000

Biological activity of SeMNPV, AcMNPV, and three AcMNPV deletion mutants against Spodoptera exigua larvae (Lepidoptera: noctuidae).

Virulence and speed of action, as related to dose, are important effectiveness-determining properties of insect-pathogenic biocontrol agents. We used the droplet-feeding bioassay to compare dose responses between two wild-type baculoviruses, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV), and three deletion mutants of AcMNPV in S. exigua larvae. In each mutant one gene was deleted by genetic engineering: pp34, coding for the polyhedral membrane; egt, coding for ecdysteroid UDP-glucosyltransferase; or p10, coding for fibrillar structures in infected insect cells. SeMNPV had the lowest median lethal dose (LD(50)) as well as the highest speed of action (LT(50)) of all viruses investigated. In our comparative bioassays the only significant effect of gene deletions in AcMNPV was a slightly lower speed of action for the p10 deletion mutant. Otherwise, wild-type and recombinant AcMNPVs had similar biological activities. Our results suggest, in contrast to what is generally assumed, that gene deletions in AcMNPV for improved insecticidal activity should be critically assessed in each host system prior to further implementation as a control agent. Insertion of foreign genes coding for entomotoxins is less questionable and more promising in this respect.

Animals↗

PubMed1995

Automatic analysis of protein conformational changes by multiple linkage clustering.

An automatic algorithm is presented for analyzing protein conformational changes such as those occurring upon substrate binding or in different crystal forms of the same protein. Using, as sole information, the atomic coordinates of a pair of protein structures, the procedure first generates structure alignments, which optimize the root-mean-square deviation of the backbone atoms. To this end, equivalent secondary structures and/or loops from both proteins are combined by a multiple linkage hierarchic clustering algorithm, which generates several intertwined clustering trees. Automatic analysis of these clustering trees is used to dissect the mechanism of the conformational change. It allows the identification of the static core, representing the collection of secondary structures which undergo no structural changes, as well as other entities which move like rigid bodies. It also permits the description of the movement of secondary structures or loops relative to this core or entities. USing this information, it can be inferred whether a particular conformational change involves shear or hinge motion, or components of both. The algorithm is applied to the analysis of the conformational changes of citrate synthase, lactate dehydrogenase, lactoferrin and beta-glucosyltransferase, representing typical examples of shear- and hinge-type mechanisms, and a varied range in movement size. The results are shown to be in excellent agreement with previous analyses, and to provide additional information which gives a more complete and objective picture of the conformational change. Using our automatic algorithm, we find that any conformational change may be viewed as having components of both shear- and hinge-type motion. Determining which of these is most appropriate requires the combination of the information provided by our procedure with detailed knowledge of the protein tertiary structures.

Algorithms↗

PubMed2000

Analysis of multiple mutations in the hALG6 gene in a patient with congenital disorder of glycosylation Ic.

Congenital disorder of glycosylation Ic is caused by mutations in the hALG6 gene that encodes an alpha-1,3 glucosyltransferase. This enzyme is required for the addition of the first glucose residue to the lipid-linked oligosaccharide precursor for N-linked glycosylation. Here we describe the biochemical and molecular analysis of a patient with three mutations in the hALG6 gene. The maternal allele has an intronic G --> A mutation resulting in skipping of exon3 (IVS3 + 5G > A). This produces a nonfunctional enzyme as shown by its inability to restore normal glycosylation in a Saccharomyces cerevisiae strain lacking a functional ALG6. The paternal allele has two mutations. One is a deletion of three bases (895-897delATA) leading to an in-frame deletion of isoleucine 299 (delI299) located in a transmembrane domain. The second mutation on the same allele 911T > C causes a F304S change. When expressed in the ALG6 deficient yeast strain, this allele restores glycosylation but the mRNA is unstable or inefficiently transcribed, contributing to the impaired glycosylation in the patient.

Alleles↗

PubMed2002

Sequential NO production by mitochondria and endoplasmic reticulum during induced apoptosis.

Early stages of rat thymocyte apoptosis measured as annexin-V positive events and induced by methylprednisolone (MPS), etoposide, and thapsigargin, showed a sequential increase in nitric oxide (NO) production by mitochondrial and endoplasmic reticulum membranes. Thapsigargin induced the highest NO production, a sevenfold increase as compared with untreated thymocytes, in mitochondrial and microsomal membranes. MPS and etoposide were equally effective in increasing NO production by mitochondrial membranes by a factor of 4-5, with only a slight increase in NO production by endoplasmic reticulum membranes. Western blot analysis of both types of membrane indicated that a nitric oxide synthase (NOS) isoenzyme is present in mitochondrial membranes and reacts with antibodies to i-NOS (type II), while reactivity to antibodies to e-NOS (type III) was restricted to endoplasmic reticulum. The participation of endoplasmic reticulum during apoptosis was further determined by alterations in UDP-Glucosyltransferase (UDP-GT) and NADPH cytochrome P450 reductase. Increased UDP-GT activity was observed after thapsigargin treatment, and no changes were found after treatment with etoposide or MPS. NADPH cytochrome P450 reductase activity markedly decreased during apoptosis, being stronger after thapsigargin treatment. The latest stage of the apoptotic process was measured by caspase activities. Caspase 3 activity was markedly increased by the three apoptosis inducers; caspase 6 was only activated by MPS and etoposide, while caspase 8 was not activated by any of these inducers. It is clear that mitochondria and endoplasmic reticulum are involved in thapsigargin induced thymocyte apoptosis. Meanwhile, other thymocyte apoptotic pathways, such as those induced by MPS or etoposide, seem to centrally involve mitochondria but not endoplasmic reticulum.

Animals↗

PubMed1995

Development of a heterodimer plasmid system for the introduction of heterologous genes into streptococci.

We have previously constructed a model secretion system for oral streptococci using the secretory domain of the Streptococcus mutans GS-5 gtfB gene. As an initial step in developing systems for secreting protein fusions containing a glucan-binding polypeptide from streptococci, a DNA fragment corresponding to the glucan-binding domain (GBD) of the glucosyltransferase-S enzyme from strain GS-5 was fused to the gtfB secretory domain. However, it was not possible to clone the hybrid gene into Escherichia coli-streptococcal shuttle plasmids using E. coli as host cells. Integration of the hybrid GBD gene into the Streptococcus gordonii chromosome was directly accomplished utilizing a strategy involving a novel heterodimeric plasmid system. The heterodimer was constructed by ligating together two plasmids each containing DNA fragments homologous with a corresponding region of the S. gordonii chromosome flanking the hybrid GBD gene following a double crossover recombination event. However, this single-copy integrant secreted limited quantities of the GBD protein. In order to achieve high-level expression of the protein, the hybrid GBD gene was integrated into resident plasmids in S. gordonii following construction of intermediate heterodimeric plasmids. These plasmids contained the GBD gene flanked by sequences homologous to regions of the resident plasmids. The presence of the GBD protein in culture fluids from transformants harboring the multicopy plasmids demonstrated the secretion of the functional GBD protein. The strategy successfully developed for secreting the GBD in the streptococci should be adaptable for other organisms for which gene transfer systems are available. In addition, these systems will allow the direct introduction of genetic constructs when the constructs cannot be stably maintained in shuttle vectors within intermediate hosts such as E. coli.

Binding Sites↗

PubMed1978

Preliminary results in the immunization of Irus monkeys against dental caries.

Twelve Irus monkeys were divided into four groups which were immunized with placebo, two cell wall preparations and one vaccine containing a glucosyltransferase preparation of S. mutans. After 310 days of challenge by a cariogenic diet, the caries score in all the immunized animals was below that of the controls. Furthermore, the protected animals were characterized by a high specific serum titer against the antigens used for immunization and the extension of plaque seemed to be smaller when compared with the controls.

Animals↗

PubMed1980

Modification of the glomerular basement membrane in sucrose-fed and streptozotocin-diabetic rats.

Rats were fed on diets containing either sucrose or starch as the carbohydrate component (55%) for eight months. Diabetes was induced in animals of both groups by injecting streptozotocin (50 mg/kg body weight). Diabetic rats failed to gain weight, had enlarged kidneys, polyuria and elevated blood glucose levels. Starch and sucrose fed rats gained weight normally and had normal blood glucose levels. Sucrose fed rats had enlarged kidneys. Regional thickening of the glomerular basement membrane was present in sucrose-fed and diabetic rats but not in starch-fed controls. Glomerular basement membrane isolated from pooled kidney cortices from rats in the different experimental groups were analysed for amino acid, disaccharide and hexosamine content. Hydroxylysine (9 to 20%), hydroxyproline (21 to 24%), disaccharide (27%) and hexosamine (26%) were increased in membranes insolated from the three experimental groups, compared with starch-fed non-diabetic controls. An increase in low molecular weight components of the glomerular basement membrane of sucrose-fed and diabetic rats was observed using electrophoresis in sodium dodecyl sulphate. Significantly higher (p < 0.001) glucosyltransferase activity was present in kidney supernatants prepared from sucrose-fed (1050 +/- 60 nmol/2h/kidney) compared to starch-fed rats (510 +/- 40 nmol/2h/kidney). Sucrose feeding induces changes similar to those found in diabetes and the induction of diabetes made little difference over the feeding of sucrose alone.

Amino Acids↗

PubMed1984

Glycogen synthesis by cell-free extracts from the dimorphic yeast Candida albicans.

A particulate glucosyltransferase prepared from budding and filamentous cultures of Candida albicans used uridine diphosphate glucose as sole glucosyl donor in a reaction (measured by following the incorporation of [14C]-glucose from UDP [14C]-glucose into polymer) stimulated by glucose-6-phosphate and inhibited by adenosine triphosphate and guanosine triphosphate. The radiolabelled reaction product was solubilized by alpha-amylase, and, on oxidation with periodate followed by reduction with borohydride and acid hydrolysis, yielded erythritol and glycerol in the ratio of 4 to 1. The radiolabelled glucosyl residues were attached to an endogenous acceptor of high molecular weight.

Adenosine Triphosphate↗

PubMed1989

Age- and training-related changes in the collagen metabolism of rat skeletal muscle.

The effects of ageing and life-long endurance training on the collagen metabolism of skeletal muscle were evaluated in a longitudinal study. Wistar rats performed treadmill running 5 days a week for 2 years. The activities of collagen biosynthesis enzymes, prolyl-4-hydroxylase and galactosylhydroxylysyl glucosyltransferase, were highest in the muscles of the youngest animals, decreased up to the age of 2 months and from then on remained virtually unchanged. The enzyme activity in young animals was higher in the slow collagenous soleus muscle than in the rectus femoris muscle. The enzyme activity in the soleus muscle was higher for older trained rats than older untrained rats. The relative proportion of type I collagen increased and that of type III collagen decreased with age, suggesting a more marked contribution by type I collagen to the age-related accumulation of total muscular collagen. The results show that collagen biosynthesis decreases with maturation and that life-long endurance training maintains a higher level of biosynthesis in slow muscles.

Aging↗

PubMed1996

Studies of the action of ceramide-like substances (D- and L-PDMP) on sphingolipid glycosyltransferases and purified lactosylceramide synthase.

We have studied the effects of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) and its L-enantiomer on glycosphingolipids in cultured normal human kidney proximal tubular cells. We found that D-PDMP exerted a concentration-dependent reduction in the metabolic labelling and cellular levels of glucosylceramide (GlcCer), lactosylceramide (LacCer), and the globo-series glycosphingolipids, GbOSe3Cer and GbOse4Cer. It also directly inhibited the activity of UDP-glucose:ceramide beta 1--> 4-glucosyltransferase (GlcT-1) and UDP-galactose: GlcCer beta 1-->4 galactosyltransferase (GalT-2). In contrast, L-PDMP had opposite effects on the metabolic labelling of GlcCer, LacCer, and GbOse3Cer. The levels of GlcCer and LacCer were increased, while the labelling and level of GbOse4Cer were strongly reduced. Purified GalT-2 from human kidney was inhibited by D-PDMP and stimulated by L-PDMP. It appears likely that the different glycosphingolipid glycosyltransferases possess similar binding sites for the ceramide moiety, which are blocked by binding to D-PDMP and, in the case of GbOse4Cer synthase, by L-PDMP as well. The stimulatory effects of L-PDMP on GlcCer and LacCer synthases may be the result of binding to a modulatory site on the glycosyltransferases; in intact cells, the enzyme-analog complex may afford protection against the normal catabolic inactivation of the enzymes.

Carbohydrate Conformation↗

PubMed1990

Effect of local deposition of antigen on salivary immune responses and reaccumulation of mutans streptococci.

In this study glucosyltransferase (GTF) from Streptococcus sobrinus was topically administered onto the lower lips of young adults. The effect of this route of antigenic exposure on labial and parotid gland and serum antibody levels to GTF and on the reaccumulation of indigenous mutans streptococci after a dental prophylaxis was then measured. Young adults between 18 and 42 years of age were screened for levels of antibody activity to GTF in parotid and labial gland salivas and levels of mutans streptococci in their whole saliva. Prior to antigen administration, two groups were formed which had similar distributions of mutans streptococci in their whole saliva. Antigen (GTF) or placebo, each combined with aluminum phosphate (AP), was then administered to the lower lip daily for 5 days. Immediately prior to topical application of GTF or placebo to the labial salivary glands, all subjects were given a dental prophylaxis. Statistically significant differences in anti-GTF IgA antibody activity in parotid or labial salivas were not observed between the GTF-administered and the placebo groups during the 6 weeks following topical application. However, the proportions of indigenous mutans streptococci/total streptococcal flora, or total cultivable flora, were always lower in the whole salivas of the GTF, compared with the placebo group. These differences were statistically significant on days 13, 20, 34, and 41 after initial topical application. Delays in reaccumulation were significantly associated (P less than 0.025) with elevations in parotid saliva IgA antibody levels of all subjects. Seven of 10 of the subjects demonstrating this association were in the group to which GTF was topically administered.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Topical↗

PubMed1982

Distribution of lipid synthesizing enzymes, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and myelin proteins in rat forebrain subfractions during development.

The distribution of UDP-galactose:ceramide galactosyltransferase (CGalT) was studied in subcellular fractions of rat forebrain during development using zonal centrifugation on linear gradients. Specialized subfractions: SN 1, a microsomal fraction, SN 4, a myelin-related fraction, and purified myelin were also used for this study. For comparison, two microsomal lipid synthesizing enzymes, a myelin-specific enzyme, 2',3'-cyclic nucleotide 3'-phosphodiesterase and myelin proteins were measured in the same subfractions. UDP-glucose:ceramide glucosyltransferase and cerebroside sulfotransferase were confined to microsomes. CGalT was localized in microsomes, but also in myelin and myelin-related fractions. The developmental change in distribution of CGalT in adult animals toward myelin containing fractions could indicate that the replacement of galactosylceramide in compact myelin could be carried out in close proximity to compact myelin (mesaxon, paranodal loops) rather than in the distant oligodendrocyte perikaryon.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1994

Microbial transformation of immunosuppressive compounds. III. Glucosylation of immunomycin (FR 900520) and FK 506 by Bacillus subtilis ATCC 55060.

The regiospecific glucosylation of FK 506 and immunomycin (FR 900520) at the 24-hydroxy position was performed using resting cells of Bacillus subtilis ATCC 55060. 24-Glucopyranosyl FK 506 and 24-glucopyranosyl immunomycin were isolated by methylene chloride extraction and purification using reverse phase HPLC. The metabolite structures were established using spectroscopic techniques including MS and NMR. The glucose conjugate was further confirmed by chemical degradation. Enzymatic glucosylation was demonstrated using cell-free extracts derived from Bacillus subtilis ATCC 55060. The 24-glucosyltransferase, which appears UDP-glucose dependent, was solubilized from cell membranes by treatment with 0.1% Nonidet P-40 detergent. The optimal conditions for assay of the enzyme have been determined.

Bacillus subtilis↗

PubMed1983

Increased prolyl 4-hydroxylase activity in the myocardium of endurance-trained mice.

Endurance training over 3, 10 or 20 days increased the activity of prolyl 4-hydroxylase (PH) in the left ventricle of mice. No increase was observed in the weight of the left ventricle, in galactosylhydroxylysyl glucosyltransferase activity or in hydroxyproline concentration. The increase in PH suggests that the synthesis of collagen increases during physiological adaptation of the heart to endurance exercise without changes in the ventricle weight or its total collagen content.

Animals↗

PubMed1981

A 2-phase liquid scintillation assay for glycolipid synthetases.

Glycolipid synthetases can be assayed conveniently by incubating the lipid substrate with the radiosugar-labeled nucleotide in a small plastic scintillation vial. At the end of the incubation period, water and perchloric acid are added, then n-butanol, then a toluene-based scintillation cocktail. The radioactive lipid partitions into the scintillation fluid, leaving excess sugar nucleotide in the aqueous phase. Only a small fraction of the total radioactivity in the aqueous layer is detectable. This method is illustrated for ceramide:UDP-glucose glucosyltransferase. The approach should be applicable to other lipid synthetases that can be assayed with radioactive hydrophilic substrate.

Animals↗

PubMed1985

Induction of rat liver bilirubin-conjugating enzymes and glutathione S-transferase by rifampicin.

After oral administration of rifampicin and 25-desacetylrifampicin, which is a major metabolite of rifampicin in man but not in rat, to male Wister rats for 7 days, hepatic microsomal cytochrome P450, cytochrome b5, and activities of aniline hydroxylase, aminopyrine demethylase, bilirubin-conjugating enzymes and supernatant glutathione S-transferase were measured. Rifampicin induced bilirubin UDP-glucuronyltransferase, bilirubin UDP-glucosyltransferase, bilirubin UDP-xylosyltransferase and glutathione S-transferase activities, but did not induce mixed function oxidase activities. No inductive effect of desacetylrifampicin on any enzymes was observed. Serum bilirubin increased till the third day, and decreased after 7 days of rifampicin treatment. Plasma clearances of indocyanine green and sulfobromophthalein showed a marked delay after 1 day and 7 days of rifampicin treatment. Induction of bilirubin-conjugating enzymes and glutathione S-transferase by rifampicin in rats was different from that in humans, in which selective induction of mixed function oxidase is reported to occur. This species difference does not seem to be derived from the species difference of rifampicin metabolism, because no effect of desacetylrifampicin was observed. These results suggested that in rats rifampicin directly inhibits the hepatic excretion of bilirubin, whereas it enhances bilirubin conjugation due to enzyme induction.

Animals↗