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ABSTRACT: A gene (amyA) encoding an extracellular alpha-amylase from a marine bacterium Pseudoalteromnonas sp. MY-1 was cloned and expressed in Escherichia coli. It comprised an open-reading-frame of 2,007 base pairs and encoded a protein of 669 amino acids with a predicted molecular weight of 73,770 daltons and a pI of 5.15. The entire amino acid sequence of amyA gene showed 86% similarity to the alpha-amylase preproprotein from Pseudoalteromonas haloplanktis. It consisted of a signal peptide, alpha-amylase catalytic domain and an amy C domain. The recombinant amylase was purified to homogeneity and biochemically characterized. The enzyme revealed maximum activity at pH 7.0 and 40 degrees C. The enzyme hydrolyzed soluble starch and some maltooligosaccharides to several oligosaccharides, and maltose was the common product from different substrates.
Indian journal of biochemistry & biophysics 11/2008; 45(5):305-9. · 1.14 Impact Factor
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ABSTRACT: To investigate whether valproic acid (VPA) modulates human GM3 synthase (hST3Gal V) mRNA expression, as a part of ganglioside GM3 biosynthesis, in human neuroblastoma cells.
Using RT-PCR and immunofluorescent confocal microscopy, we examined hST3Gal V mRNA and GM3 levels during VPA-induced differentiation of human neuroblastoma SK-N-BE(2)-C cells. We characterized the VPA-inducible promoter region within the hST3- Gal V gene using luciferase constructs carrying 5'-deletions of the hST3Gal V promoter.
RT-PCR indicated that VPA-mediated hST3Gal V induction is transcriptionally regulated. Functional analysis of the 5'-flanking region of the hST3Gal V gene demonstrated that the -177 to -83 region, which contains a cAMP-responsive element (CRE) at -143, functions as the VPA-inducible promoter by actively binding CRE binding protein (CREB). In addition, sitedirected mutagenesis and electrophoretic mobility shift assay indicated that the CRE at -143 is crucial for the VPA-induced expression of hST3Gal V in SK-NBE( 2)-C cells.
Our results isolated the core promoter region in the hST3Gal V promoter, a CRE at -143, and demonstrated that it is essential for transcriptional activation of hST3Gal V in VPA-induced SK-N-BE(2)-C cells. Subsequent CREB binding to this CRE mediates VPA-dependent upregulation of hST3Gal V gene expression.
Acta Pharmacologica Sinica 10/2008; 29(9):999-1005. · 1.95 Impact Factor
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ABSTRACT: In this study, we have shown the transcriptional regulation of the human GD3 synthase (hST8Sia I) induced by valproic acid (VPA) in human neuroblastoma SK-N-BE(2)-C cells. To elucidate the mechanism underlying the regulation of hST8Sia I gene expression in VPA-stimulated SK-N-BE(2)-C cells, we characterized the promoter region of the hST8Sia I gene. Functional analysis of the 5’-flanking region of the hST8Sia I gene by the transient expression method showed that the -1146 to -646 region, which contains putative binding sites for transcription factors c-Ets-1, CREB, AP-1 and NF-kappa B, functions as the VPA-inducible promoter of hST8Sia I in SK-N-BE(2)-C cells. Site-directed mutagenesis and electrophoretic mobility shift assay indicated that the NF-kappa B binding site at -731 to -722 was crucial for the VPA-induced expression of hST8Sia I in SK-N-BE(2)-C cells. In addition, the transcriptional activity of hST8Sia I induced by VPA in SK-N-BE(2)-C cells was strongly inhibited by SP600125, which is a c-Jun N-terminal kinase (JNK) inhibitor, and GO6976, which is a protein kinase C (PKC) inhibitor, as determined by RT-PCR (reverse transcription-polymerase chain reaction) and luciferase assays. These results suggest that VPA markedly modulated transcriptional regulation of hST8Sia I gene expression through PKC/JNK signal pathways in SK-N-BE(2)-C cells.
Molecules and Cells 10/2008; 27(1). · 2.18 Impact Factor
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ABSTRACT: Triphenylmethane reductase (TMR) catalyzes the NADH-dependent reduction of triphenylmethane dyes. Sequence alignment revealed a region with a conserved GXXGXXG motif near its N-terminus, which corresponds to a conserved structural motif of known dinucleotide-binding proteins. To verify whether some of these glycine residues are important for the enzyme catalysis, these three glycine residues (Gly-7, Gly-10 and Gly-13) were individually replaced by alanine using site-directed mutagenesis. The secondary structures of these mutants, as measured by circular dichroism spectroscopy, did not show remarkable differences as compared with the wild type. The V(max)/K(m) values of mutants G7A and G13A for both Basic fuchsin and NADH were increased about three and twofold over that of the wild type, respectively, whereas the V(max)/K(m) value of mutant G10A were decreased about sixfold. These results suggest that these three glycine residues are involved in the interaction with both substrate and cofactor for the catalytic activity of TMR.
FEMS Microbiology Letters 07/2007; 271(1):78-82. · 2.04 Impact Factor
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Web and Wireless Geographical Information Systems, 7th International Symposium, W2GIS 2007, Cardiff, UK, November 28-29, 2007. Proceedings; 01/2007
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ABSTRACT: An extracellular β-amylase, which was easily adsorbable onto raw corn starch, was purified 22.5-fold from a new isolate of Bacillus polymyxa No 26–1 with a Mr of 53 kDa and pI of 9.1. The optimum temperature was 45°C and pH 5.5 for raw corn starch. Thermal stability at 40°C and pH stability at 5.0–8.5 were shown. The degradation ofraw starch by β-amylase was greatly stimulated by pullulanase addition. Scanning electron micrographs revealed that starch granule degradation by the enzyme alone occured at the equatorial grooves of lecticular granules. Corn starch granules hydrolyzed by β-amylase had large holes on granule surfaces.
Journal of Food Science 08/2006; 61(1):230 - 234. · 1.66 Impact Factor
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Advances in Information Systems, 4th International Conference, ADVIS 2006, Izmir, Turkey, October 18-20, 2006, Proceedings; 01/2006
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ABSTRACT: N-Acetylglucosaminyltransferase (GnT)-III catalyzes the attachment of an N-acetylglucosamine (GlcNAc) residue to mannose in beta(1-4) configuration in the region of N-glycans and forms a bisecting GlcNAc. To investigate the pathophysiological role of dysregulated glycosylation mediated by aberrantly expressed GnT-III, we generated transgenic mice hyperexpressing the human GnT-III in the liver by introducing human GnT-III cDNA under the control of mouse albumin enhancer/promoter. Total five transgenic founder mice (pGnTSVTpA-10, -14, -20, -25, and -51) expressed the human GnT-III in their livers and were characterized by molecular genetic means. The copy number of transgene integrated into the genome of these mice ranged between 1 and 3 copies per haploid genome. Northern and Western blot analyses showed that the transgene is specifically expressed in the liver but not in any other tissues tested. The triglyceride level in GnT-III transgenic mice was significantly decreased, however, no significant differences in the levels of glucose, cholesterol, or albumin were observed between transgenic and nontransgenic mice. Although glutamate oxaloacetic transaminase and glutamic pyruvic transaminase activities of transgenic mice were also higher than those of nontransgenic mice, no differences in total bililubin and total protein were observed between the two animal lines. Large amounts of apolipoprotein (Apo) A-I and Apo B were specifically detected in the intracellular liver of transgenic mice. The accumulation of Apo A-I in hepatocytes may be due to aberrant glycosylation, since glycosylated Apo A-I was not observed in transgenic mice. However, the accumulated Apo B was severely glycosylated. Therefore, it is suggested that highly expressed transgenic GnT-III allowed unknown target proteins to be glycosylated in large amounts, and the resulting target protein(s) disrupted in assembly formation of Apo A-I in the hepatocytes and cause a decrease in the release of lipoproteins and accumulations of Apo A-I and Apo B in the liver. The transgenic mice showed aberrant glycosylation by GnT-III, resulting in numerous lipid droplets in liver tissues and the obesity. These mice showed microvesicular fatty changes with abnormal lipid accumulation in the hepatocytes. Our study provides the basis for future analysis of the role of glycosylation in hepatic pathogenesis. In the transgenic mice, Apo A-I and Apo B were significantly increased compared with levels in nontransgenic liver tissues.
Archives of Biochemistry and Biophysics 07/2004; 426(1):18-31. · 2.93 Impact Factor
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ABSTRACT: We studied the transcriptional regulation of human GM3 synthase (hST3Gal V) during megakaryocytic differentiation of K562 cells induced by PMA. Northern blot and reverse transcription polymerase chain reaction (RT-PCR) indicated that the induction of hST3Gal V by phorbol 12-myristate 13-acetate (PMA) is regulated at transcriptional level. To elucidate the mechanism underlying the regulation of the hST3Gal V gene expression during the differentiation of K562 cells induced by PMA, we characterized the promoter region of the hST3Gal V gene. Functional analysis of the 5(')-flanking region of the hST3Gal V gene by transient expression method showed that the -177 to -83 region, which contains a CREB binding site at -143, functions as the PMA-inducible promoter in K562 cells. In addition, gel shift assay and site-directed mutagenesis indicated that the CREB binding site at -143 is crucial for the PMA-induced expression of the hST3Gal V in K562 cells.
Biochemical and Biophysical Research Communications 02/2004; 313(1):142-7. · 2.48 Impact Factor
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ABSTRACT: Fast updates in spatial databases become a crucial issue in several application domains, such as LBS (location-based service). In particular, moving objects with frequent updates on their positions require fast update mechanisms in spatiotemporal database systems. In this paper, we propose a new method for the indexing of the current positions of moving objects on road network. Our method significantly improves not only the update cost but also the performance of query processing. The only constraint of our method is that the moving objects should be on roads, but we believe that most applications dealing moving objects imply this constraint. We provide a performance analysis on our model and by this we suggest further improvements on performance of our method. By experimental studies, we show that our method takes about 40% less in disk accesses compared to TPR-tree for updating and at maximum 4 times faster than TPR-tree for processing timestamp range queries.
Web Information Systems Engineering Workshops, 2003. Proceedings. Fourth International Conference on; 01/2004
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ABSTRACT: We studied the transcriptional regulation of human GM3 synthase (hST3Gal V) during monocytic differentiation of HL-60 cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Northern blot and reverse transcription polymerase chain reaction indicated that the induction of hST3Gal V by TPA is regulated at the transcriptional level. To elucidate the mechanism underlying the regulation of hST3Gal V gene expression during the differentiation of HL-60 cells induced by TPA, we characterized the promoter region of the hST3Gal V gene. Functional analysis of the 5'-flanking region of the hST3Gal V gene by the transient expression method showed that the -177 to -83 region, which contains a cAMP responsive element binding protein (CREB) binding site at -143, functions as the TPA-inducible promoter in HL-60 cells. In addition, gel shift assays and site-directed mutagenesis indicated that the CREB binding site at -143 is crucial for the TPA-induced expression of the hST3Gal V in HL-60 cells.
FEBS Letters 01/2004; 555(2):204-8. · 3.54 Impact Factor
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ABSTRACT: In order to prevent hyperacute rejection in pig-to-human xenotransplantation, it would be very useful to be able to down-regulate the Gal alpha1-3 Galbeta 1-4 GlcNAc-R (alpha-Gal epitope) in mouse and swine tissues. When the beta-D-mannoside beta-1,4-N-acetylglucosaminyl-transferase III (GnT-III) gene was introduced into mouse aorta endothelial cells (MEC) their susceptibility to complement-mediated cell lysis by normal human serum (NHS) was reduced. Expression of GnT-III also suppressed the antigenicity of MEC to human natural antibodies as shown by binding of Griffonia simplicifolia 1 isolectin (GS1B4 lectin) to the alpha-Gal epitope. Western blot analysis indicated that the reactivity of the glycoproteins of the transfectants to NHS and GSIB4 lectin was reduced to approximately the same extent. Thus GnT-III, a key enzyme involved in the formation of branched N-linked sugars, reduces the expression of xenoantigens, suggesting that this approach may be of value in clinical xenotransplantation.
Molecules and Cells 01/2004; 16(3):368-76. · 2.18 Impact Factor
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Web and Wireless Geographical Information Systems, 4th InternationalWorkshop, W2GIS 2004, Goyang, Korea, November 2004, Revised Selected Papers; 01/2004
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ABSTRACT: Geiji-Bokryung-Hwan (GBH), consisting of herbes of Cinnamomi ramulus (Geiji), Poria cocos (Bokryun), Mountan cortex radicis (Mokdanpi), Paeoniae radix (Jakyak), and Persicae semen (Doin), on antiplatelet activity in human platelet suspensions was studied. The mechanism involved in the antiplatelet activity of GBH in human platelet suspensions was investigated. GBH did not significantly affect the thromboxane synthetase activity of aspirin-treated platelet microsomes and GBH (15 and 30 microg/ml) significantly inhibited [3H]arachidonic acid released in collagen-activated platelets but not in unactivated-platelets. Nitric oxide (NO) production in human platelets was measured by a chemiluminesence detection method in this study. GBH did not significantly affect nitrate production in collagen (10 microg/ml)-induced human platelet aggregation. Various concentrations of GBH (0, 5, 10, 15, and 30 microg/ml) dose-dependently inhibited [3H]inositol monophosphate formation stimulated by collagen (10 microg/ml) in [3H]myoinositol-loaded platelets at different incubation times (1, 2, 3, and 5 min). These results indicated that the antiplatelet activity of GBH may possibly be due to the inhibition of phospholipase C (PLC) activity, leading to reduce phosphoinositide breakdown, followed by the inhibition of thromboxane A(2) formation, and then inhibition of [Ca(2+)](i) mobilization of platelet aggregation stimulated by agonists. In conclusion, GBH suppressed PLC in a dose-dependent manner, and may have pharmaceutical applications. These data suggest that GBH extracts merit investigation as a potential anti-atherosclerogenic agent in humans.
International Immunopharmacology 08/2003; 3(7):971-8. · 2.38 Impact Factor
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ABSTRACT: We have cloned the genomic DNA encoding the human NeuAc alpha2,3Gal beta1,3GalNAc alpha2,6-sialyltransferase (hST6GalNAc IV) and analysed its structure. The hST6GalNAc IV gene was found to span about 9 kb and to be composed of six exons. The 5'-RACE (rapid amplification of cDNA ends) results indicated that mRNA isoform of the hST6GalNAc IV was generated by alternative splicing in the 5'-untranslated region. The expression of this gene was highly restricted in human fetal tissues. The potential transcriptional start site was determined by CapSite hunting. Sequence analysis of the 5'-flanking region of this gene lacked canonical TATA and CAAT boxes, but contained several putative binding sites for transcription factors SP1, MZF1, GATA1, LMO2COM, NFAT, HFH8 and USF, etc. Functional analysis of the 5'-flanking region by transient expression method revealed a high transcriptional activity in both HepG2 cells and Molt4 cells in a cell type-dependent manner, but not in SK-N-MC cells. These results suggest cell type-specific regulation of the basal hST6GalNAc IV promoter activity.
Gene 03/2003; 305(1):113-20. · 2.34 Impact Factor
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ABSTRACT: GM3 synthase, which transfers CMP-NeuAc with an alpha2,3-linkage to a galactose residue of lactosylceramide, plays a key role in the biosynthesis of all complex gangliosides. The expression of this gene is highly restricted in both human fetal and adult tissues. To elucidate the mechanisms that regulate the tissue-specific expression of the human GM3 synthase (hST3Gal V) gene, we have isolated and characterized its 5'-flanking region. Potential transcriptional start site was determined by CapSite hunting. Sequence analysis of the 5'-flanking region revealed that hST3Gal V promoter lacked canonical TATA and CAAT boxes but contained several putative binding sites for transcription factors AP4, MZF1, SP1, ATF/CREB, NFY, IK2 and LYF1, etc. Functional analysis of the 5'-flanking region of the hST3Gal V gene by transient expression method revealed that the -177 to -83 region is important for transcriptional activity of the hST3Gal V gene in SK-N-MC and HepG2 cells. The present results also suggested that both positive and negative regulatory elements are present in this TATA-less promoter of the hST3Gal V gene.
Biochimica et Biophysica Acta 11/2002; 1578(1-3):84-9. · 4.66 Impact Factor
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ABSTRACT: Sialic acids are key determinants for biological processes, such as cell-cell interaction and differentiation. sialyltransferases
contribute to the diversity in carbohydrate structure through their attachment of sialic acid in various terminal positions
on glycolipid and glycoprotein (N-linked and O-linked) carbohydrate groups. Galß 1,3(4)GlcNAc α2,3-sialyltransferase (ST3Gal
III) is involved in the biosynthesis of sLex and sLea known as selectin ligands and tumor-associated carbohydrate structures. The appearance and differential distribution of ST3Gal
III mRNA during mice embryogenesis [embryonic (E) days; E9, E11, E13, E15] were investigated byin situ hybridization with digoxigenin-labeled RNA probes coupled with alkaline phosphatase detection. On E9, all tissues were positive
for ST3Gal III mRNA expression, whereas ST3Gal III mRNA on E11 was not detected throughout all tissues. On E13, ST3Gal III
mRNA was expressed in different manner in various tissues. In this stage, ST3Gal III mRNA was positive only in the liver,
pancreas and bladder. On E15, specific signal for ST3Gall III was detected in the liver, lung and forebrain. These results
indicate that ST3GAl III is differently expressed at developmental stages of mice embryo, and this may be importantly related
with regulation of organogenesis in mice.
Archives of Pharmacal Research 04/1999; 22(3):243-248. · 1.59 Impact Factor
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ABSTRACT: To elucidate the mechanism underlying the regulation of human GD3 synthase gene expression in human melanoma SK-MEL-2 cells, we identified the promoter region of the human GD3 synthase gene. The 5′-rapid amplification of cDNA end (5′-RACE) using mRNA prepared from SK-MEL-2 cells revealed the presence of multiple transcription start sites of human GD3 synthase gene. Promoter analyses of the 5′-flanking region of the human GD3 synthase gene using luciferase gene reporter system showed the strong promoter activity in SK-MEL-2 cells. Deletion study revealed that the region as the core promoter from − 1146 to − 646 (A of the translational start ATG as position + 1) was indispensable for endogenous expression of human GD3 synthase gene. This region lacks apparent TATA and CAAT boxes but contains putative binding sites for transcription factors c-Ets-1, CREB, AP-1 and NF-κB. Electrophoretic mobility shift assays using specific competitors, chromatin immunoprecipitation assay and site-directed mutagenesis demonstrated that only NF-κB element in this region is required for the promoter activity in SK-MEL-2 cells. These results indicate that NF-κB plays an essential role in the transcriptional activity of human GD3 synthase gene essential for GD3 synthesis in SK-MEL-2 cells.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression.
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ABSTRACT: The cDNA encoding human Sia-α2,3-Gal-β1,4-GlcNAc-R:α2,8-sialyltransferase, hST8Sia III, was isolated by screening of a human brain cDNA library with polymerase chain reaction-amplified DNA probe generated from the sequence of mouse ST8Sia III (mST8Sia III) and by 5′ rapid amplification of cDNA ends of mRNA isolated from human brain tissues. Comparative analysis of the predicted protein-coding region between our cloned hST8Sia III and mST8Sia III showed 92 and 96% identities in the nucleotide and the amino acid sequence, respectively. The soluble hST8Sia III protein expressed in COS-7 showed an extremely high catalytic activity of transferring sialic acid through α2,8-linkage to intact fetuin glycoprotein, whereas the transferring activity was completely undetectable toward either α2,6-sialylated glycoprotein or desialylated glycoprotein acceptors. Northern analysis of hST8Sia III showed that the transcript corresponding to 11 kb was expressed in both human fetal and adult brain, while the expression of the 5.5-kb transcript was restricted to fetal liver, indicating that the expression of hST8Sia III is developmentally and tissue-specifically regulated.
Archives of Biochemistry and Biophysics.
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ABSTRACT: A cDNA of human Galβ1,3GalNAc α2,3-sialyltransferase (hST3Gal II), which has been known to exhibit much more acceptor substrate preference for glycolipid than for O-linked oligosaccharides of glycoproteins, was isolated from the human liver cDNA library by plaque hybridization using the cDNA of mouse ST3Gal II (mST3Gal II) cloned previously as a probe. Comparative analysis of this cDNA with mST3Gal II indicates 89 and 94% homologies in the nucleotide and amino acid levels, respectively, between the two sequences in the predicted coding region. Northern analysis indicated that the expression of hST3Gal II mRNA is tissue-specific, it being prominent in skeletal muscle and heart, while that in lung and kidney is very low. This enzyme expressed in COS cells showed a similiar activity with that of mST3Gal II.
Biochemical and Biophysical Research Communications.
