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PubMed1988

Effects of joint loading on articular cartilage collagen metabolism: assay of procollagen prolyl 4-hydroxylase and galactosylhydroxylysyl glucosyltransferase.

The degree of joint loading on the femoral head cartilage was experimentally altered in dogs by splinting of the knee joint for 11 weeks (n = 9), and making another group (n = 6) to run on a treadmill with 15 degrees uphill inclination during a period of 15 weeks (4 km/day, 5 days a week). A third group (n = 9) served as controls. The influence of these altered loading conditions on articular cartilage collagen synthesis was measured by assaying the activity of procollagen prolyl 4-hydroxylase (PPH) and galactosylhydroxylysyl glucosyltransferase (GGT) in chondrocytes. The average activity of PPH was 11-13% elevated in the runner and contralateral (more loaded) cartilages, while the splinted (unloaded) cartilages showed a significant (53%) increase of PPH. In some samples of the runner, splinted and contralateral cartilages the activity of GGT was also high, but the differences did not reach statistical significance. The increase of the activity of PPH, combined with unaltered total content of collagen, indicates that the synthesis of collagen and probably also its turnover, are enhanced in the cartilage atrophied due to reduced weight-bearing, but only slightly, if at all in the cartilages subjected to moderately elevated loading.

Animals↗
PubMed1993

DNA sequence of the glucosyltransferase gene of serotype d Streptococcus sobrinus.

A glucosyltransferase (GTF) gene was cloned into Escherichia coli from serotype d Streptococcus sobrinus OMZ176. Transformed E. coli strain MI expressed water-insoluble glucan synthesizing activity. Restriction enzyme map of pGT31 extracted from MI shows that the enzyme gene exists in the 6.4-KB PstI-fragment inserted into pBR322 vector. DNA sequence analysis indicates that a single ORF (530-5,300) is located in the PstI-fragment. The putative amino-acid composition (1,590 residues) resembles those of other GTF-I enzymes obtained from serotype g S. sobrinus and serotype h Streptococcus downei. However, at the same positions of the sequence, 18 and 142 amino-acid residues are different between serotype d and g, and serotype d and h GTF-I enzymes, respectively. The differences between serotype d and h GTF-Is are distributed in N and C-terminal regions.

Amino Acid Sequence↗
PubMed1997

Nucleotide sequence analysis of the Streptococcus gordonii glucosyltransferase gene, gtfG.

Streptococcus gordonii has an extracellular glucosyltransferase (GTF) that polymerizes the glucose moiety of sucrose to form both water-soluble and water-insoluble glucans. Whereas multiple gtf genes have been identified in strains of mutans streptococci and Streptococcus salivarius, a single gene, designated gtfG, encodes the GTF of S. gordonii Challis. gtfG is also unique among the characterized gtfs in that it has a described regulatory determinant, rgg. Furthermore, the GTF activity in S. gordonii undergoes reversible phase variation between high and low levels. In order to gain insight into this novel GTF system, the nucleotide sequence of gtfG was determined and found to consist of a 4,734 base pair open reading frame encoding a protein with a deduced molecular weight of ca. 174,000. gtfG was similar to other sequenced gtfs with a conserved signal sequence followed by a ca. 600-bp region distinctive for gtfG, a conserved region encoding a putative catalytic active site and a series of six direct repeats in the carboxyl terminal region implicated in glucan binding. Although comparison of gtfG to other gtfs did not show a basis for the primer-independence of the encoded enzyme or the nature of the glucan products, the gtfG sequence data provide an important basis for further studies of these enzymes.

Amino Acid Sequence↗
PubMed1996

Kinetic properties of glucosyltransferase adsorbed onto saliva-coated hydroxyapatite.

Results from previous studies have shown that several properties of glucosyltransferase (GTF) adsorbed onto saliva-coated hydroxyapatite beads differ from those of GTF in solution. For example: thermostability, pH-activity dependency, sensitivity to inhibitors. The aim of this study was to compare the kinetics of the adsorbed GTF with its kinetic properties in solution. Hydroxyapatite beads were coated with human parotid saliva (sHA). Following washes, cell-free GTF enzyme from Streptococcus sobrinus 6715 (S. sobrinus 6715) or Streptococcus mutans GS-5 (S. mutans GS-5) was adsorbed onto sHA. The GTF-coated sHA were then incubated with radiolabeled sucrose for intervals of 5-360 minutes and the amount of glucans synthesized in situ by the adsorbed GTF was determined and compared with that produced in solution. The adsorbed GTF (from S. sobrinus 6715) exhibited a sharp increase in glucan production within the first 5 minutes of incubation while surface-bound GTF of S. mutans GS-5 displayed an initial burst of activity within the first 15 minutes of incubation. During the next 6 hours (duration of experiment) the amount of glucan on the beads did not increase with either enzyme. In contrast, the kinetic profile of the two GTFs in solution demonstrated a linear increase in the amount of glucans formed, with no initial burst effect. The results indicate that the rapid formation of glucans by GTF adsorbed onto sHA could have implications for colonization by oral microorganisms on tooth surfaces. The accelerated synthesis of glucan on tooth surfaces may affect the microbiology of the dental plaque, and might also influence the movement of substances, such as acids and antiplaque agents, across the acquired pellicle and dental plaque.

Adsorption↗
PubMed1988

Characteristics of M-GTFI, a new inhibitor of Streptococcus mutans glucosyltransferase.

M-GTFI, originally screened as an inhibitor of Streptococcus mutans glucosyltransferase, strongly inhibited alpha-glucosidase, in a non-competitive manner especially when the synthetic substrate p-nitrophenyl-alpha-D-glucopyranoside was used. It also inhibited beta-glucosidase, beta-amylase and, to a lesser extent, beta-glucuronidase. The inhibitor was stable in neutral and alkaline pH ranges and dependency of the inhibition on pH and temperature was not observed. Some proteinases and polysaccharides-hydrolyzing enzymes as well as human saliva did not inactivate the inhibitor. There was a correlation between the release of sulfate anions from the inhibitor molecule on incubation with HCl (0.2 N) at 100 degrees C and loss of inhibitory properties of the molecule. It is suggested that the presence of sulfate ester linkages in the inhibitor molecule play an important role in the inhibition process.

Enzyme Inhibitors↗
PubMed1989

The structure of M-GTFI, an inhibitor of glucosyltransferase from S. mutans, and its inhibitory relationship with other sulfate ester-containing inhibitors.

M-GTFI, an inhibitor of glucosyltransferase from S. mutans was produced by Micromonospora narashinoensis strain No. 731. The isolation procedure for M-GTFI was improved and established for spectroscopic analyses, and some properties of the inhibitor were investigated. The structure of M-GTFI was shown to be trisodium [2-sulphonato-(E)-9-undecenyll-oxacyclotriacont-(E)-3- en-2-one, 16, 18-bis sulphonate. The chemical structure of M-GTFI was therefore similar to that of izumenolide which is a beta-lactamase inhibitor containing sulfate ester groups in its molecule. The inhibitory characteristics of M-GTFI were parallel to that of other inhibitory compounds containing sulphate esters but the spectrum of activity was wider.

Alkanesulfonates↗
PubMed1997

[Ultrastructure of adherence of Streptococcus mutans MT8148 and its glucosyltransferase deficient mutants].

The ultrastructure of adherence of Streptococcus mutans MT8148 and its glucosyltransferase deficient mutants were compared by Scanning Electronic Microscope. Four patterns of adherence of tested strains in the presence of sucrose were observed. These results suggest that the cell-associated GTase is responsible for firm aggregation of cells. However, cell free GTase is also important in aggregation between cells.

Bacterial Adhesion↗
PubMed1999

Characterization of the ecdysteroid UDP-glucosyltransferase gene of Spodoptera litura multinucleocapsid nucleopolyhedrovirus.

The ecdysteroid UDP-glucosyltransferase (EGT) gene of Spodoptera litura multinucleocapsid nucleopolyhedrovirus (SpltMNPV), Guangzhouisolate was sequenced. The open reading frame of the gene is 1530 nucleotides long, encoding a putative protein of 509 amino acids with Mr of 58,500. The 5' noncoding region has a TATA box. A polyadenylation signal, AATAAA, is contained downstream of the translation stop codon. A putative signal peptide is present at the N-terminus of the protein. Homology comparison with other baculovirus egt genes showed that these genes are highly conserved. The SpltMNPV egt gene is closely related to that of Spodoptera littoralis nucleopolyhedrovirus (SliNPV) with the identities of 84% in nucleotide and 91% in amino acid, respectively. In this study, an alignment of amino acid sequences has been made and a tentative phylogenetic tree of twelve baculovirus egts is presented.

Amino Acid Sequence↗
PubMedDate not supplied

Subsite specificity of divalent metal ions to glucosyltransferase.

Glucosyltransferase from oral bacteria Streptococcus mutans is the most significant virulent factor in causing dental caries. The enzyme has two subsites. The binding specificity of divalent metal ions to glucosyl or fructosyl subsite was examined using multiple inhibition kinetics. The interaction factor "alpha" identifies whether the two subsites are exclusive or non-exclusive.

Binding Sites↗
PubMed1975

Intracellular localization and some properties of UDPG: sterol glucosyltransferase from Calendula officinalis.

UDPG: sterol glucosyltransferase is localized in the 2-week-old C. officinalis seedling in the membrane structures, separated from chloroplasts and mitochondria, and consisting probably of fragments of the Golgi apparatus. A minor part of the enzyme activity is associated with the microsomal fraction. A number of synthetic detergents stimulate the activity of the membrane-bound enzyme causing its solubilization. The enzyme preparation purified about 70-fold is strongly inhibited by HgCl2 and p-chloromercuribenzoate; it is markedly stimulated by mercaptoethanol and dithiothreitol, and to a lesser extent by Mg2+ and Ca2+ as well as by some chelating and reducing agents. UMP stimulates and UDP and UTP markedly inhibit the enzyme activity. The enzyme does not act on 4-methylsterols although it utilizes a number of 4-demethylsterols. It seems that the presence of a double bond in ring B enhances the affinity of the substrate for the enzyme. Delta-5-Sterols are utilized at a higher rate than delta-7-sterols. Saturated sterols and delta-25-sterols are poor substrates.

Cell Membrane↗
PubMed2000

Effect of lactoperoxidase system on glucosyltransferase D of Streptococcus mutans.

OBJECTIVE: To investigate the effects of the peroxidase system (LP, H2O2, SCN) and its components on the activity of glucosyltransferase D (GtfD) originating from Streptococcus mutans (S. mutans). METHODS: GtfD was incubated in buffered-KCl assay mixture that contained 20 microM dextran and 100 mM sucrose including 14C-glucose. The activity of GtfD was measured with a scintillation counter. The effects of lactoperoxidase system and its components on GtfD activity were examined by incubating different components separately and together with GtfD. RESULTS: The lactoperoxidase system-generated hypothiocyanite (OSCN) had no effect on the activity of streptococcal GtfD, while the lactoperoxidase enzyme inhibited GtfD. The ratio between the 2 enzymes, LP and GtfD, was important for the inhibition. However, if LP was combined with its substrates, SCN and/or a high concentration of H2O2, it enhanced the activity of GtfD. CONCLUSION: Peroxidase-generated antimicrobial agent HOSCN/OSCN is not inhibitory against GtfD, whereas low concentrations of the LP-enzyme inhibit GtfD.

Anti-Bacterial Agents↗
PubMed2001

Developmental patterns of ceramide glucosyltransferase (GlcT-1) expression in the mouse: in situ hybridization using DIG-labeled RNA probes.

Glycosphingolipids (GSLs) play significant roles in a variety of cell membrane events, including cellular interactions, signaling, and trafficking. Ceramide glucosyltransferase (glucosylceramide synthase, GlcT-1, EC 2.4.1.80) catalyzes the initial step in GSL synthesis, the transfer of glucose from UDP-glucose to ceramide. The reaction product of glucosylceramide serves as a core structure for over 400 species of GSLs. The enzyme is a key regulatory factor controlling intracellular levels of ceramide and GSLs. Appearance and differential distribution of GlcT-1 mRNA during mice postimplantation embryogenesis [embryonic (E) days; E9, E11, E13, E15] were investigated by in situ hybridization with digoxigenin-labeled RNA probes, coupled with alkaline phosphatase detection. On E9, tissues of the mesencephalon, myelecephalon, diencephalons, and telencephalon expressed GlcT-1. On E11, it was expressed to a detectable extent in various tissues including mesencephalon, myelecephalon, diencephalon, telencephalon, nose, lung, liver, vertebra, tail, spinal cord, and tongue. The expression patterns of E13 were similar to those of E11, except that the heart and stomach became positive. On E15, a specific signal for GlcT-1 was detected in all organs of the embryo. These results provide the first evidence that GlcT-1 is differentially expressed during postimplantation embryogenesis.

Animals↗
PubMed2000

[The effect of immune bovine whey on cell-associated glucosyltransferase activity of Streptococcus mutans].

OBJECTIVE: To study the effect of immune bovine whey on cell-associated glucosyltransferase (GTF) activity of S. mutans MT8148. METHODS: The immune milk was collected from cows immunized with cell-associated GTF overexpression strain B-29-33 of S. mutans MT8148. The control milk was from non-immunized cows. The immune absorbed whey was gotten from immune bovine whey which was absorbed with lyophilized Formalin-killed B-29 whole cells. Three kinds of whey were subdivided into three groups: 50 microliters, 70 microliters, 90 microliters. The content of insoluble glucan was estimated colorimetrically by anthrone method. RESULTS: The control bovine whey had an enhancing GTF activity (407.00%-485.62%). The immune absorbed whey inhibited partly the enhancing GTF activity (208.74%-273.00%). The immune whey inhibited significantly the GTF activity (70.24%-38.62%) and the inhibition showed a tendency to depend on doses. CONCLUSIONS: The immune bovine whey inhibits significantly the cell-associated GTF activity of S. mutans MT8148.

Animals↗
PubMed2002

Differential and quantitative analyses of mRNA expression of glucosyltransferases from Streptococcus mutans MT8148.

Streptococcus mutans produces three glucosyltransferases, coded by gtfB, gtfC, and gtfD, whose cooperative action is essential for sucrose-dependent cellular adhesion. This cellular adhesion plays an important role in the formation of dental plaque and the initiation of dental caries. Since they bear genetic similarities and are large in size, differentiation of their gene expression has been difficult, and little is known about the dynamic process of gtf expression. Using a real-time reverse-transcription/polymerase chain-reaction, we determined the expression of each gtf. Under various conditions, the relative levels of transcription were gtfB > gtfD > gtfC. Sucrose enhanced gtfD expression, whereas it reduced that of gtfB and gtfC, suggesting the presence of independent promoters. Quantitative analyses demonstrated coincidence between the ratio of expression of each gtf and the ratio previously identified as optimal for sucrose-dependent adhesion in vitro, suggesting that S. mutans produces GTF at an optimal ratio to adhere to the tooth surface.

Analysis of Variance↗
PubMed2002

Increased proliferative response of peripheral blood mononuclear cells and T cells to Streptococcus mutans and glucosyltransferase D antigens in the exacerbation stage of recurrent aphthous ulcerations.

BACKGROUND AND PURPOSE: Cytotoxic T lymphocyte-induced lysis of virus-infected oral epithelial cells has been shown to be a cause of early ulcer formation in recurrent aphthous ulcerations (RAU). To test whether bacteria and their associated antigens are involved in the disease process of RAU, the proliferative response (PR) to different streptococcal species in peripheral blood mononuclear cells (PBMC) and T cells isolated from RAU patients at the exacerbation stage was determined. METHODS: PBMC and T cells were isolated from 39 patients with RAU, 21 patients with erosive oral lichen plaus (EOLP, disease control group), and 22 healthy subjects (normal control group). Streptococcus mutans, Streptococcus sanguis, Streptococcus oralis, Streptococcus gordonii, Streptococcus mitis and their associated antigen, glucosyltransferase D (GtfD), were used to stimulate isolated PBMC and T cells in in vitro proliferation assays. RESULTS: PBMC and T cells isolated from RAU patients at the exacerbation stage showed a significantly higher PR to S. mutans antigen and GtfD than those isolated from EOLP patients or healthy control subjects (p < 0.05). GtfD was a more potent stimulation antigen than S. mutans. However, elevated PRs to S. mutans antigen and GtfD were transient and present only in the exacerbation stage of RAU. These elevated PRs declined to normal levels in the postexacerbation and convalescence stages of RAU. Furthermore, the GtfD-stimulated PR in PBMC and T cells was correlated with the severity of RAU. CONCLUSION: In addition to viral infections, streptococci and their associated antigen, GtfD, may be involved in the disease process of RAU, especially in the exacerbation stage.

Adolescent↗
PubMed2001

[Construction of eukaryotic expression plasmid pcDNA3-gtfB expressing glucosyltransferase B of Streptococcus mutans].

OBJECTIVE: Glucosyltransferase (GTF) of Streptococcus mutans is considered as a cariogenic virulence factor due to its ability to synthesize glucan, which facilitate sucrose-depended adherence and cell-to-cell accumulation of bacteria. In this study, gtfB, the target gene fragment which encodes multiple catalytic sites and antigen epitopes of GTF, was recombined into eukaryotic expression vector pcDNA3. The feasibility of the recombination plasmid pcDNA3-gtfB used as gene vaccine will be investigated in further study. METHODS: The target gene fragment gtfB (904-4578 bp) was obtained by standard PCR amplification while genome DNA of streptococcus mutans GS-5 was used as template. Then the PCR products were extracted and purified from low-melting temperature agarose. The gtfB and plasmid pcDNA3 were cut by Kpn I, Xho I, and the digested products were extracted and purified again for recombination. The purified gtfB and plasmid pcDNA3 were recombined by T4 DNA ligase, ligation products were transformed into competent cell, Escherichia coli JM109. Transformed colonies were screened by Ampr LB plate, then recombined plasmids were isolated and identified by restricted endonuclease cutting and Sanger dideoxy DNA sequencing. RESULTS: Identified by agarose gel electrophoresis, the target gene-gtfB obtained PCR amplification had the same molecular size (36 kb) as predicted. It was indicated that recombined plasmids contained inserted gtfB gene fragment by restricted endonuclease cut analysis, the sequencing data also indicated that inserted gtfB gene had correct DNA sequence and orientation according to DNA sequence of Streptococcus mutans GS-5 (gene bank M17361). CONCLUSION: Inserted gene-gtfB of recombined plasmid pcDNA3-gtfB encoded multiple catalytic sites and epitopes. It was proved that these epitopes had high immune antigenicity and that antiserum could significantly inhibit the synthesis of water-insoluble glucans and water-soluble glucan. In vitro adherence experiment also indicated that it could inhibit streptococcus mutans adherence to saliva-coated hydroxyapatite. Vector pcDNA3 was high expressing eukaryotic vector, and could stimulate antigen-representing cell. It was suggested that recombined plasmid pcDNA3-gtfB had high immune antigenicity and immune responsiveness, and this supported its use as gene vaccine candidates in the development of anti-caries vaccines.

Dental Caries↗
PubMed2002

[Immunogenicity of a synthetic peptide vaccine from glucosyltransferase].

OBJECTIVE: To measure the immunogenicity of a synthetic peptide of glucosyltransferase (GTF) for designing synthetic peptide-based vaccine of dental caries. METHODS: A fusion 27-mer peptide, containing the conserved regions within catalytic and glucan-binding domains of GTF, was synthesized. Serum antibodies to the synthetic peptide were determined by ELISA method. Inhibitions of both GTF activity and S. mutans adherence were detected for the functions of antisera. RESULTS: The sequence of fusion peptide vaccine was ANDVDNSNPVVQAEQLYFRANGVQVKG. The spleen weights of immunized mice were heavier than the control ones. Specific antibodies were effectually elicited. The immune sera not only inhibited GTF enzymatic activity but also inhibited the vitro adherence of S. mutans. CONCLUSIONS: The peptide vaccine which involves antibody-mediated inhibition of the catalytic and the glucan-binding activities of GTF may be valuable for controlling the dental caries.

Amino Acid Sequence↗