首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
A gene encoding an ATP-dependent fructokinase from Streptococcus mutans GS-5 was identified within a 2 kb DNA fragment immediately downstream from the scrA gene. The gene cloned in Escherichia coli also expressed mannokinase activity. Insertional inactivation of this gene in S. mutans markedly decreased both fructokinase and mannokinase activities. Nucleotide sequence analysis of the 2 kb fragment revealed an ORF starting 199 bp downstream from the scrA gene, preceded by potential ribosome-binding (Shine-Dalgarno) and promoter-like sequences. This ORF specified a putative protein of 293 amino acids with a calculated M(r) of 31,681. The deduced amino acid sequence of the fructokinase gene, scrK, from S. mutans exhibited no significant similarity to fructokinase genes from Klebsiella pneumoniae, E. coli plasmid pUR400 or Vibrio alginolyticus, but was similar to a comparable gene from Zymomonas mobilis.  相似文献   

2.
Glutamine:fructose-6-phosphate amidotransferase (glucosamine-6-phosphate synthase) catalyzes the first step of the hexosamine pathway required for the biosynthesis of cell wall precursors. The Candida albicans GFA1 gene was cloned by complementing a gfa1 mutation of Saccharomyces cerevisiae (previously known as gcn1-1; W. L. Whelan and C. E. Ballou, J. Bacteriol. 124:1545-1557, 1975). GFA1 encodes a predicted protein of 713 amino acids and is homologous to the corresponding gene from S. cerevisiae (72% identity at the nucleotide sequence level) as well as to the genes encoding glucosamine-6-phosphate synthases in bacteria and vertebrates. In cell extracts, the C. albicans enzyme was 4-fold more sensitive than the S. cerevisiae enzyme to UDP-N-acetylglucosamine (an inhibitor of the mammalian enzyme) and 2.5-fold more sensitive to N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (a glutamine analog and specific inhibitor of glucosamine-6-phosphate synthase). Cell extracts from the S. cerevisiae gfa1 strain transformed with the C. albicans GFA1 gene exhibited sensitivities to glucosamine-6-phosphate synthase inhibitors that were similar to those shown by the C. albicans enzyme. Southern hybridization indicated that a single GFA1 locus exists in the C. albicans genome. Quantitative Northern (RNA) analysis showed that the expression of GFA1 in C. albicans is regulated during growth: maximum mRNA levels were detected during early log phase. GFA1 mRNA levels increased following induction of the yeast-to-hyphal-form transition, but this was a response to fresh medium rather than to the morphological change.  相似文献   

3.
Streptococcus mutans constitutively expresses three glucosyltransferases, i.e., GtfB, GtfC, and GtfD; which synthesize glucan polymers from sucrose. To obtain individual GTF without complexing with one another, a purification strategy was developed to recover recombinant GTF expressed from Escherichia coli. The recombinant GtfC was aggregated and associated with the insoluble fraction in E. coli homogenates. GtfC was solublized with the 8M urea, renatured to its biologically active form by serial dialysis against sodium phosphate buffer, and subsequently purified to homogeneity by DEAE-Sephacel and hydroxylapatite column chromatography. The GtfC enzyme preparation was purified 16.3-fold and the molecular weight was estimated to be 140 kDa. GtfC synthesized water insoluble glucan in a primer independent manner and its enzymatic activities could be enhanced by dextran. Purified GtfC had a pH optimum of 6.5, a K(m) of 9.26 mM for sucrose and a pI of 5.5. Distinct from the previous reports, results from this study offers an alternative for the purification of the recombinant GTFs free from any detergent contamination to make it more suitable for utilization in vivo.  相似文献   

4.
An Arabidopsis thaliana cDNA clone, AtTPS1, that encodes a trehalose-6-phosphate synthase was isolated. The identity of this protein is supported by both structural and functional evidence. On one hand, the predicted sequence of the protein encoded by AtTPS1 showed a high degree of similarity with trehalose-6-phosphate synthases of different organisms. On the other hand, expression of the AtTPS1 cDNA in the yeast tps1 mutant restored its ability to synthesize trehalose and suppressed its growth defect related to the lack of trehalose-6-phosphate. Genomic organization and expression analyses suggest that AtTPS1 is a single-copy gene and is expressed constitutively at very low levels.  相似文献   

5.
Glutamine:fructose-6-phosphate amidotransferase (GFA) is the rate-limiting enzyme in hexosamine biosynthesis, an important pathway for cellular glucose sensing. Human GFA has two potential sites for phosphorylation by cAMP-dependent protein kinase A (PKA). To test whether GFA activity is regulated by cAMP-dependent phosphorylation, rat aortic smooth muscle cells were treated in vivo with cAMP-elevating agents, 10 micromol/l forskolin, 1 mmol/l 8-Br-cAMP, or 3-isobutyl-1-methylxanthine. All treatments resulted in rapid and significant increases (2- to 2.4-fold) in GFA activity assayed in cytosolic extracts. Maximal effects of forskolin were observed at 10 micromol/l and 60 min. Preincubation of cells with cycloheximide did not abolish the effect of forskolin. Incubation of cytosolic extracts at 37 degrees C for 10 min in a buffer without phosphatase inhibitors led to a 79% decrease of GFA activity. This loss of activity was inhibited by the addition of phosphatase inhibitors (5 mmol/l sodium orthovanadate, 50 mmol/l sodium fluoride, or 5 mmol/l EDTA, but not 100 nmol/l okadaic acid), suggesting that GFA undergoes rapid dephosphorylation by endogenous phosphatases. Purified GFA is phosphorylated in vitro by purified PKA, resulting in a 1.7-fold increase in GFA activity. Treatment of GFA with purified protein kinase C had no effect. We conclude that GFA activity may be modulated by cAMP-dependent phosphorylation.  相似文献   

6.
OBJECTIVES: To investigate the degree of association between tactile and optical criteria as used to assess the carious status of the enamel-dentine junction (EDJ) during cavity preparation, assessment with a caries detector dye and detection of Streptococcus mutans using culture and polymerase chain reaction (PCR) techniques. METHODS: Twenty-nine teeth, extracted within the previous 30 min, and 15 teeth prepared under rubber dam in vivo, were clinically assessed at the EDJ after the removal of evident carious tissue. Demineralisation was then assessed using a caries detector dye (1% acid red in propylene glycol; Cavex). A rosehead bur was used to remove tissue at the EDJ for culture and PCR analysis. Culture was carried out on a tryptone yeast cystine sucrose bacitracin selective medium, and PCR used to amplify a sequence (192 bp) of the spaP gene, which encodes the surface protein antigen I/II of S. mutans. RESULTS: Demineralised tissue at the EDJ, as shown using the dye, was found in 52% of teeth. Removed tissue was culture and PCR positive for S. mutans in 2 and 47% of teeth, respectively. A highly significant association (77% of cases; P < 0.001) was shown between dye and PCR assessment methods. No association was found between any other combination of assessment methods. CONCLUSIONS: Culture methods may underestimate the presence of S. mutans. Removal of sufficient dye-stained tissue is therefore recommended to prevent further carious assault from residual S. mutans.  相似文献   

7.
Endothelial cells contain leukotriene (LT) A4 hydrolase (LTA-H) as detected by Northern and Western blotting, but several studies have been unable to detect the activity of this enzyme. Since LTA-H could play a key role in determining what biologically active lipids are generated by activated endothelium during the inflammatory process, we studied possible mechanisms by which this enzyme may be regulated. We find that LTA-H is phosphorylated under basal conditions in human endothelial cells and in this state does not exhibit epoxide hydrolase activity (i.e. conversion of LTA4 to LTB4). LTA-H purified from endothelial cells is efficiently dephosphorylated by incubation with protein phosphatase-1 in the presence of an LTA-H peptide substrate and not at all in the absence of substrate. Under conditions that lead to dephosphorylation, protein phosphatase-1 activates the epoxide hydrolase activity of LTA-H. Using peptide mapping and site-directed mutagenesis, we have identified serine 415 as the site of phosphorylation of LTA-H by a kinase found in endothelial cell cytosol. In parallel, we have studied a human lung carcinoma cell line that expresses active LTA-H. Although these cells have cytosolic kinases that phosphorylate recombinant LTA-H, they do not target serine 415 and thus do not inhibit LTA-H activity. We believe that LTA-H is regulated in intact cells by a kinase/phosphatase cycle and further that the kinase in endothelial cells specifically recognizes and phosphorylates a regulatory site in the LTA-H.  相似文献   

8.
P element-mediated transformation has been used to investigate the regulation of expression of the sn-glycerol-3-phosphate dehydrogenase gene of Drosophila melanogaster. A 13-kb construct containing the eight exons and associated introns, 5 kb of the 5' region, and 3 kb downstream from the structural gene produced normal levels of enzyme activity and rescued the poor viability of flies lacking the enzyme. All the regulatory elements essential for normal enzyme expression were located in a fragment that included the exons and introns and 1-kb upstream noncoding sequence. Deletions of the 1.6-kb second intron reduced activity to 25%. Transformants with fusion constructs between the sn-glycerol-3-phosphate dehydrogenase gene and the beta-galactosidase gene from E. coli revealed three elements that affected expression. A (CT)9 repeat element at the 5' end of the second intron increased expression in both larvae and adults, particularly at emergence. A second regulatory element, which includes a (CT)7 repeat, was located 5' to the TATA box and had similar effects on the gene's expression. A third, undefined, enhancer was located in the second intron, between 0.5 and 1.8 kb downstream of the translation initiation codon. This element increases enzyme activity to a similar extent in larvae and adults but has little effect when the enhancer at the 5' end of the intron is present.  相似文献   

9.
A comparison of the amino acid sequences of the glucosyltransferases (GTFs) of mutans streptococci with those from the alpha-amylase family of enzymes revealed a number of conserved amino acid positions which have been implicated as essential in catalysis. Utilizing a site-directed mutagenesis approach with the GTF-I enzyme of Streptococcus mutans GS-5, we identified three of these conserved amino acid positions, Asp413, Trp491, and His561, as being important in enzymatic activity. Mutagenesis of Asp413 to Thr resulted in a GTF which expressed only about 12% of the wild-type activity. In contrast, mutagenesis of Asp411 did not inhibit enzyme activity. In addition, the D413T mutant was less stable than was the parental enzyme when expressed in Escherichia coli. Moreover, conversion of Trp491 or His561 to either Gly or Ala resulted in enzymes devoid of GTF activity, indicating the essential nature of these two amino acids for activity. Furthermore, mutagenesis of the four Tyr residues present at positions 169 to 172 which are part of a subdomain with homology to the direct repeating sequences present in the glucan-binding domain of the GTFs had little overall effect on enzymatic activity, although the glucan products appeared to be less adhesive. These results are discussed relative to the mechanisms of catalysis proposed for the GTFs and related enzymes.  相似文献   

10.
We have isolated dextran-aggregation-negative mutants of Streptococcus mutans following random mutagenesis with plasmid pVA891 clone banks. A chromosomal region responsible for this phenotype was characterized in one of the mutants. A 2.2-kb fragment from the region was cloned in Escherichia coli and sequenced. A gene specifying a putative protein of 583 amino acid residues with a calculated molecular weight of 63,478 was identified. The amino acid sequence deduced from the gene exhibited no similarity to the previously identified S. mutans 74-kDa glucan-binding protein or to glucan-binding domains of glucosyltransferases but exhibited similarity to surface protein antigen (Spa)-family proteins from streptococci. Extract from an E. coli clone of the gene exhibited glucan-binding activity. Therefore, the gene encoded a novel glucan-binding protein.  相似文献   

11.
An enzyme that deacetylates N-acetylglucosamine to glucosamine from Vibrio cholerae non-O1 was purified to homogeneity by sequential procedures. The native enzyme had a molecular mass of 190,000 Da and was predicted to be composed of four identical subunits with molecular masses of 45,000 Da. The purified enzyme hydrolyzed N-acetylglucosamine, N-acetylglucosamine 6-phosphate, and N-acetylglucosamine 6-sulfate, but not chitin oligosaccharides, and N-acetylgalactosamine. The deacetylase activity was completely abolished by N-ethylmaleimide, p-chloromercuribenzoate, EDTA, and Cu2+. On the other hand, the activity was activated by Co2+. The amino-terminal amino acids of the purified enzyme were sequenced. Among the 22 N-terminal amino acid residues, 12 residues of Vibrio deacetylase were identical with that of Escherichia coli GlcNAc 6-phosphate deacetylase.  相似文献   

12.
The net stimulation of dextransucrase EC 2.4.1.5) activity from Streptococcus mutans HS6 by dextran, secretory immunoglobulin A, or secretory component was investigated. Approximately equal stimulation resulted from treatment with these three components.  相似文献   

13.
The intracellular redox potential, which is determined by the level of oxidants and reductants, has been shown to play an important role in the regulation of cell growth. The principal intracellular reductant is NADPH, which is mainly produced by the pentose phosphate pathway through the actions of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, and by 6-phosphogluconate dehydrogenase. Previous research has suggested that an increase in G6PD activity is important for cell growth. In this article, we suggest that G6PD activity plays a critical role in cell growth by providing NADPH for redox regulation. The results show the following: 1) inhibition of G6PD activity abrogated growth factor stimulation of [3H]thymidine incorporation in all cell lines tested; 2) overexpression of G6PD stimulated cell growth, as measured by an increase in [3H]thymidine incorporations as compared with cells transfected with vector alone; 3) inhibition of G6PD caused cells to be more susceptible to the growth inhibitory effects of H2O2; 4) inhibition of G6PD led to a 30-40% decrease in the NADPH/NADP ratio; and 5) inhibition of G6PD inhibited cell anchorage and significantly decreased the growth-related stimulation of tyrosine phosphorylation.  相似文献   

14.
Plasmin, the primary fibrinolytic enzyme, has a broad substrate spectrum and participates in other biological processes dependent upon proteolytic activity. Consequently, plasmin activity is tightly regulated by plasminogen activators and protease inhibitors. In this study, we examined whether regulation of plasminogen gene expression also might provide a new mechanism for controlling this system. We examined the effects of recombinant human interleukin-6 (rhIL-6), a pleiotropic cytokine, on plasminogen mRNA expression in primary murine hepatocytes and Hep3B human hepatoma cells. In primary hepatocytes, rhIL-6 and hydrocortisone separately increased plasminogen mRNA expression, but hydrocortisone did not markedly enhance the response to rhIL-6. Hep3B hepatoma cells exhibited more modest responses to rhIL-6. We used the polymerase chain reaction to amplify a 1,067-bp fragment of the human plasminogen promoter/5' flanking region. This fragment was cloned upstream of a luciferase reporter gene. Hep3B cells transiently transfected with this construct provided approximately 100-fold higher luciferase activity compared to cells transfected with control plasmids, and luciferase activity was increased approximately 4.5-fold when these cells were treated with rhIL-6. Furthermore, mice injected with rhIL-6 exhibited increases in hepatic plasminogen mRNA. Circulating plasminogen levels were significantly higher in the mice injected with rhIL-6 compared to mice injected with saline. Mice injected with lipopolysaccharide (an inducer of IL-6 in vivo) also showed increased hepatic plasminogen mRNA. Thus, plasminogen gene expression can be modulated by rhIL-6, suggesting a new mechanism for regulating biological systems that use plasmin.  相似文献   

15.
A new type of nylon oligomer degradation enzyme (EIII) was purified from an Escherichia coli clone harboring the EIII gene (nylC). This enzyme hydrolyzed the linear trimer, tetramer, and pentamer of 6-aminohexanoate by an endo-type reaction, and this specificity is different from that of the EI (nylA gene product) and EII (nylB gene product). Amino acid sequencing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified EIII demonstrated that the enzyme is made of two polypeptide chains arising from an internal cleavage between amino acid residues 266 and 267.  相似文献   

16.
The physiological role of the catecholamines (CA), adrenaline and noradrenaline in fish has been frequently reviewed, but the metabolic consequences of these hormones have received less attention. The purpose of this review is to examine the recent literature dealing with CA actions on whole fish and tissue metabolism. The CA increase glucose production both in vivo and in vitro, at least in isolated hepatocytes. Although the data are less clear, lipid mobilization is also a consequence of elevated circulating CA. The difficulty with using the whole fish for such studies is that CA may alter other circulating hormone levels, CA turnover in the circulation quickly, and it is difficult to define precisely the tissue being affected. Much of our understanding is derived, therefore, from the study of isolated tissues, and especially the hepatocyte. Catecholamines stimulate both glycogenolysis and gluconeogenesis in hepatocytes isolated from a large number of fish species. This review examines the steps involved in the signal transduction system, from the binding of CA to alpha- and beta-adrenoceptors to the ultimate effects of specific enzyme phosphorylation. Recent literature demonstrates that the complexity of the adrenoceptor system noted for mammals, also is expressed in fish. Adrenoceptor subtypes are specific to species, to tissues and to function of the tissues, and these issues are discussed especially as they are related to external and to internal stressors. Future research will pursue better definitions of the adrenoceptor systems, molecular biology of the components of these receptor systems and development of alternative cell models. There still remains a poor explanation of the reason for the diversity of adrenoceptor systems, and there are a number of fish systems that may provide unique opportunities to understand this question.  相似文献   

17.
18.
19.
Human salivary immunoglobulin A (IgA) and serum IgG antibodies to the Streptococcus mutans glucosyltransferases (Gtfs) and to a synthetic peptide of 19 amino acids from a conserved region in the Gtfs (residues 435 to 453) were determined in young adults by enzyme-linked immunosorbent assay. Varying levels of antibody to Gtfs were detected in saliva or serum, with significantly higher levels of antibody to GtfD than to GtfB/C or GtfC. Anti-Gtf IgA levels in saliva did not correlate with those of IgG in serum. Caries-free (CF) volunteers exhibited significantly higher salivary IgA antibody levels to the peptide and to GtfB/C or GtfC than did the caries-active (CA) subjects. Preincubation of CF saliva and serum with the peptide inhibited the antibodies to the Gtfs in a dose-dependent manner, whereas preincubation of the samples from the CA group resulted in only partial inhibition. Our results indicated that this 19-amino-acid peptide includes one of the major B-cell epitopes of Gtfs and that CF individuals have higher titers of antibodies than CA subjects.  相似文献   

20.
The proinflammatory leukotrienes (LT) play important roles in host defense and disease states. However, no endogenous mechanisms to downregulate 5-lipoxygenase (5-LO), the enzyme catalyzing LT synthesis, have been described. We observed that the cytosolic fraction of rat alveolar macrophages (AMs) and peritoneal macrophages (PMs), and of peripheral blood monocytes (PBMs) contain substantial amounts of 5-LO protein, but little detectable 5-LO activity. We therefore examined these mononuclear phagocyte (MNP) cytosolic fractions for inhibitory activity against 5-LO. MNP cytosol dose-dependently reduced the 5-LO activity in neutrophil (PMN) cytosol and AM membrane. Furthermore, MNP cytosol dose-dependently prolonged the lag phase of soybean lipoxygenase (LO) without affecting the rate of product formation. This effect was overcome by subsequent addition of 13(S)-hydroperoxy-9-cis-11-trans-octadecadienoic acid (13-HpOD), suggesting that the active factor scavenges hydroperoxides. Inactivation by boiling and roteinase K suggest that is a protein. We speculate that this cytosolic factor(s) may serve as an endogenous means for the down-regulation of 5-LO in macrophages.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号