Triphenylmethane were reductase (TMR) catalyzes the NADH-dependent reduction of triphenylmethane dyes. Sequence alignment revealed a region with a conserved GXXGXXG motif near catalytic its N-terminus, which corresponds to a conserved structural motif of known dinucleotide-binding proteins. To verify whether some of these glycine fuchsin residues are important for the enzyme catalysis, these three glycine residues (Gly-7, Gly-10 and Gly-13) were individually replaced by alanine catalysis, using site-directed mutagenesis. The secondary structures of these mutants, as measured by circular dichroism spectroscopy, did not show remarkable differences of as compared with the wild type. The V(max)/K(m) values of mutants G7A and G13A for both Basic fuchsin and NADH GXXGXXG were increased about three and twofold over that of the wild type, respectively, whereas the V(max)/K(m) value of mutant G10A the were decreased about sixfold. These results suggest that these three glycine residues are involved in the interaction with both substrate of and cofactor for the catalytic activity of TMR.

To and identify genes involved in the decolorization of malachite green, random mutants generated by transposon insertion in the malachite green-decolorizing bacterium,decolorization Citrobacter sp. were isolated. The resulting mutant bank yielded 24 mutants with complete defects in their abilities to decolorize malachite protein; green. Southern hybridization with a Tn5 fragment as a probe showed a single hybridized band in 7 mutants, which appeared single to have insertions at different sites of the chromosome. The Tn5-inserted genes were isolated and the DNA sequence flanking Tn5 malachite was determined. Based on a sequence database, the putative protein products encoded by the mg genes were identified as follows.bacterium, mg3, an ABC transporter homolog; mg6, a LysR-type regulatory protein; m11, an oxidoreductase; mg17, a MalG protein in the maltose probe transport system; and mg21, a sugar kinase. The deduced sequences from two mg genes (mg7 and mg18) showed no significant mg3, similarity to any protein with a known function, suggesting that these two mg genes encode unidentified proteins that are responsible mg6, for the decolorization of malachite green.

Angiogenesis GM3 is associated with growth, invasion and metastasis of human solid tumor and the aberrant activation of endothelial cells and induction a of microvascular permeability by vascular endothelial growth factor (VEGF)/VEGF receptor-2 (VEGFR-2)-mediated signaling pathway is observed in pathological angiogenesis including tumor,other wound healing, arthritis, psoriasis, diabetic retinopathy and others. Here, we show that GM3 regulated the activity of various downstream signal and pathways and biological events through the inhibition of VEGF-stimulated VEGFR-2 activation in vascular endothelial cells in vitro. Furthermore, GM3 strongly human blocked VEGF-induced neovascularization for in vivo models, such as the chick chorioallantoic membrane and matrigel plug assay. Interestingly, GM3 suppressed by VEGF-induced VEGFR-2 activation by blocking VEGF receptor-2 dimerization and the binding between VEGF and VEGFR-2 through GM3-specific interaction with extracellular downstream domain of VEGFR-2, but not VEGF. In other in vivo experiments, the growth of tumor in mice with primary tumors interaction was obviously inhibited by subcutaneous injection of GM3. Immunohistochemical analysis showed inhibition of angiogenesis and tumor cell proliferation. GM3 also but resulted in the suppression of VEGF-stimulated microvessel permeability in mouse skin capillaries. These results suggest that GM3 clearly inhibits VEGF/VEGFR-2-mediated in vascular endothelial cell function and angiogenesis, and might be a therapeutic avenue for anti-angiogenesis.

Ganglioside also GM3 inhibits growth of several cancer cells and induces cell cycle arrest by regulating cellular signal pathways. Our previous results GM3-stimulated have shown that GM3 suppresses tumor suppressor PTEN-mediated cancer cell proliferation. However, the precise molecular mechanism(s) for the transcriptional regulation obviously of PTEN gene induced by GM3 remains unclear. Here, we show, for the first time, that GM3 induces transcription factor and AP-2alpha-mediated PTEN expression in colon cancer cells. The enhanced expression of PTEN by GM3 in both HCT116 and p53-null HCT116 arrest cells has been shown to be not associated with p53 function. Thus, to further determine the mechanism underlying the regulation cell of PTEN gene expression by GM3, we characterized the promoter region of the PTEN gene. Promoter analysis of the 5'-flanking GM3 region of the PTEN gene showed that region between -1175 and -1077 from translational initiation site, which contains AP-2alpha binding colon site, functions as the GM3-inducible promoter in colon cancer cells. Furthermore, gel shift assays, site-directed mutagenesis and chromatin immunoprecipitation assay mutagenesis obviously indicated that the AP-2alpha is essential for the expression of PTEN in GM3-stimulated colon cancer cells. Moreover, siRNA against Additionally, AP-2alpha diminished the enhancement of AP-2alpha and PTEN expression in GM3-induced colon cancer cells. The transient expression of AP-2alpha also cells. results in the induction of PTEN transcription in AP-2alpha-negative colon cancer cells. Additionally, GM3 induced AP-2alpha-mediated PTEN expression through the The inhibition of autocrine-ligand-mediated EGFR activation. These results suggest that the AP-2alpha transcription factor is required for the ganglioside GM3-stimulated transcriptional AP-2alpha regulation of PTEN gene.

To region elucidate the mechanism underlying the regulation of human GD3 synthase gene expression in human melanoma SK-MEL-2 cells, we identified the GD3 promoter region of the human GD3 synthase gene. The 5'-rapid amplification of cDNA end (5'-RACE) using mRNA prepared from SK-MEL-2 binding cells revealed the presence of multiple transcription start sites of human GD3 synthase gene. Promoter analyses of the 5'-flanking region region of the human GD3 synthase gene using luciferase gene reporter system showed the strong promoter activity in SK-MEL-2 cells. Deletion GD3 study revealed that the region as the core promoter from -1146 to -646 (A of the translational start ATG as of position +1) was indispensable for endogenous expression of human GD3 synthase gene. This region lacks apparent TATA and CAAT boxes of but contains putative binding sites for transcription factors c-Ets-1, CREB, AP-1 and NF-kappaB. Electrophoretic mobility shift assays using specific competitors,region chromatin immunoprecipitation assay and site-directed mutagenesis demonstrated that only NF-kappaB element in this region is required for the promoter activity boxes in SK-MEL-2 cells. These results indicate that NF-kappaB plays an essential role in the transcriptional activity of human GD3 synthase These gene essential for GD3 synthesis in SK-MEL-2 cells.

In inhibitor, this study, we have shown the transcriptional regulation of the human Sia-alpha2,3-Gal-beta1,4-GlcNAc-R:alpha2,8-sialyltransferase (hST8Sia III) induced by retinoic acid (RA), a signal potent neuronal cell regulator in glioblastoma cell line (U-87MG). The induction of hST8Sia III by RA is regulated at the for transcriptional level in a dose- and time-dependent manner, as evidenced by reverse transcription-polymerase chain reaction (RT-PCR). To elucidate the mechanism expression underlying the regulation of hST8Sia III gene expression in RA-stimulated U-87MG cells, we characterized the promoter region of the hST8Sia the III gene. Functional analysis of the 5'-flanking region of the hST8Sia III gene by the transient expression method showed that glioblastoma the -1194 to -816 region, which contains a retinoic acid nucleic receptor (RAR) at -1000 to -982, functions as the of RA-inducible promoter in U-87MG cells. Site-directed mutagenesis indicated that the RA binding site at -996 to -991 is crucial for that the RA-induced expression of the hST8Sia III in U-87MG cells. In addition, the transcriptional activity of hST8Sia III induced by -996 RA in U-87MG cells was strongly inhibited by SP600125, c-Jun N-terminal Kinase (JNK) inhibitor, as determined by RT-PCR and luciferase promoter assay of hST8Sia III promoter containing the -1194 to -816 regions. These results suggest that RA markedly modulates transcriptional regulation by of hST8Sia III gene expression through JNK signal pathway in U-87MG cells.

The oligosaccharides chitinase A (ChiA)-coding gene of Pseudomonas sp. BK1, which was isolated from a marine red alga Porphyra dentata, was cloned bacterial and expressed in Escherichia coli. The structural gene consists of 1602 bp encoding a protein of 534 amino acids, with at a predicted molecular weight of 55,370 Da. The deduced amino acid sequence of ChiA showed low identity (less than 32%)ChiA with other bacterial chitinases. The ChiA was composed of multiple domains, unlike the arrangement of domains in other bacterial chitinases.BK1, Recombinant ChiA overproduced as inclusion bodies was solubilized in the presence of 8 M urea, purified in a urea-denatured form and and re-folded by removing urea. The purified enzyme showed maximum activity at pH 5. and 40 degrees C. It exhibited acid high activity towards glycol chitosan and glycol chitin, and lower activity towards colloidal chitin. The enzyme hydrolyzed the oligosaccharides from by (GlcNAc)4 to (GlcNAc)6, but not GlcNAc to (GlcNAc)3. The results suggest that the ChiA is a novel enzyme, with different showed domain structure and action mode from bacterial family 18 chitinases.

In the the present study, we have shown that KCl known as an inducer for differentiation of neuronal cells increases the human increasing Sia-alpha2,3-Gal-beta1,4-GlcNAc-R:alpha2,8-sialyltransferase (hST8Sia III) gene transcription via phosphoinositide 3 kinase (PI-3K) in glioblastoma U-87MG cells. The induction of hST8Sia III by signal KCl is regulated at the transcriptional level in a dose- and time-dependent manner as evidenced by reverse transcription-polymerase chain reaction regulation (RT-PCR). To elucidate the mechanism underlying the regulation of hST8Sia III gene expression in U-87MG cells induced by KCl, we as characterized the promoter region of the hST8Sia III gene. Functional analysis of the 5'-flanking region of the hST8Sia III gene via by the transient expression method showed that the -1194 to -816 region functions as the KCl-inducible promoter in U-87MG cells.mechanism Furthermore, as evidenced by Western blot analysis and RT-PCR, KCl-induced expression of hST8Sia III gene was dependent on the PI-3K KCl-induced signal transduction pathway during the neuronal differentiation of U-87 cells, as an increase in beta-tubulin III known as a neuronal dependent differentiation marker was observed. In KCl-depolarization on U-87 cells, the PI-3K-dependent promoter activation at the -1194 to -816 region up-regulated results expression of hST8Sia III gene. These results suggest that the expression of hST8Sia III gene via the PI-3K signaling pathway the is enhanced during KCl-induced differentiation of U-87 cells by increasing expression of beta-tubulin III.

To bg identify genes involved in the decolorization of brilliant green, we isolated random mutants generated by transposon insertion in brilliant green-decolorizing the bacterium, Citrobacter sp. The resulting mutant bank yielded 19 mutants with a complete defect in terms of the brilliant green protein color removing ability. Southern hybridization with a Tn5 fragment as a probe showed a single hybridized band in 7 mutants hybridized and these mutants appeared to have insertions at different sites of the chromosome. Tn5-inserted genes were isolated and the DNA brilliant sequence flanking Tn5 was determined. By comparing these with a sequence database, putative protein products encoded by bg genes were Citrobacter identified as follows: bg 3 as a LysR-type regulatory protein; bg 11 as a MalG protein in the maltose transport showed system; bg 14 as an oxidoreductase; and bg 17 as an ABC transporter. The sequences deduced from the three bg a genes, bg 2, bg 7 and bg 16, showed no significant similarity to any protein with a known function, suggesting 11 that these three bg genes may encode unidentified proteins responsible for the decolorization of brilliant green.

A to gene encoding glucansucrase was identified in Leuconostoc lactis EG001 isolated from lactic acid bacteria (LAB) in Kimchi, a traditional Korean antioxidant fermented food. The L. lactis EG001 glucansucrase gene consists of 4503bp open reading frame (ORF) and encodes an enzyme of properties 1500 amino acids with an apparent molecular mass of 165kDa. The deduced amino-acid sequence showed the highest amino-acid sequence identity with (75%) to that of dextransucrase of L. mesenteroides. The gene was cloned and over-expressed in Escherichia coli strain. The recombinant in enzyme was purified via Ni-NTA affinity chromatography and its enzymatic properties were characterized. The enzyme exhibited optimum activity at 30 Kimchi, degrees C and pH 5. . In addition, the enzyme was able to catalyze the glycosylation of l-ascorbic acid to l-ascorbic 1500 acid 2-glucoside. The glycosylated product via EG001 glucansucrase has the potential as an antioxidant in industrial applications.

Sphingomyelinase calculated C (SMC) was purified to homogeneity from the culture supernatant of Streptomyces griseocarneus NBRC13471. The purified enzyme appeared as a acid single band of 38 kDa by using an electropherogram trace. The molecular mass of the enzyme as determined by MALDI-TOF and MS was 32,102 Da, indicating that SMC is monomeric in nature. Under experimental conditions, the highest enzyme activity was found 5 at pH 9. and 50-55 degrees C, and the enzyme was stable from pH 5 to 10 and up to of 37 degrees C. The SMC activity requires Mg(2+) or Mn(2+) and the order of potency to enhance the activity was electropherogram Zn(2+)> or =Mn(2+)>Cu(2+)> or =Fe(2+). Phenylmethylsulfonyl fluoride and EDTA inhibited the enzyme activity, showing that SMC belongs to a group was of metalloenzymes and a class of serine hydrolases. The enzyme activity was inhibited by DTT, but not by mercaptoethanol and inhibited iodoacetamide. SDS inhibited the enzyme activity; by contrast, Triton X-100 stimulated the activity. The N-terminal and internal amino-acid sequences were stimulated determined as H(2)N-APAAATPSLK, AREIAAAGFFQGND, and NTVVQETSAP. The gene encoding SMC consisted of 1020 bp encoding a signal peptide of 42 enzymes amino acids and a mature protein of 297 amino acids with a calculated molecular mass of 32,125 Da. The conserved of region of DNase I-like family enzymes and the amino acid residues that are highly conserved in the active center of 297 other bacterial SMCs were also found in the deduced amino acid sequence of S. griseocarneus SMC.

A was new deoC gene encoding deoxyribose 5-phosphate aldolase (DERA) was identified in Yersinia sp. EA015 isolated from soil. The DERA gene mM) had an open reading frame (ORF) of 672 base pairs encoding 223 amino acids to yield a protein of molecular The mass 24.8 kDa. The amino acid sequence was 94 % identical to that of DERA from Yersinia intermedia ATCC 29909.to DERA was over-expressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The specific activity was 137 mumol/min/mg. The Michaelis deoxyribose constant (Km value) of DERA was 9.1 mM. DERA was optimally active at pH 6. and 50 degrees C. DERA sp. was tolerant to a high concentration (300 mM) of acetaldehyde.

The and release of malachite green, a commonly used triphenylmethane dye, into the environment is causing increasing concern due to its toxicity,by mutagenicity, and carcinogenicity. A bacterial strain that could degrade malachite green was isolated from the water of an aquatic hatchery.other It was identified as a Pseudomonas sp. based on the morphological, physiological, and biochemical characteristics, as well as the analysis It of 16S rRNA gene sequence and designated as MDB-1. This strain was capable of degrading both malachite green and leucomalachite commonly green, as well as other triphenylmethane dyes including Crystal Violet and Basic Fuchsin. The gene tmr2, encoding the triphenylmethane reductase concern from MDB-1, was cloned, sequenced and effectively expressed in E. coli. These results highlight the potential of this bacterium for aquatic the bioremediation of aquatic environments contaminated by malachite green.

Citrobacter oxygen sp. strain KCTC 18061P was found to be able to decolorize textile plant effluent containing different types of reactive dyes.7 Effects of physico-chemical parameters, such as aeration, nitrogen source, glucose and effluent concentrations on the color removal of real dye effluent effluent by this strain were investigated. The observed changes in the visible spectra indicated color removal by the absorption of this dye to cells during incubation with the strain. This strain showed higher decolorization ability under aerobic than static culture conditions.found With 1% glucose, this strain removed 70% of effluent color within 5 days. Decolorization was not significantly dependent on the different nitrogen sources tested. Chemical oxygen demand (COD) and biological oxygen demand (BOD) were decreased in proportion to incubation times, and dye their removal rates were about 35% and 50%, respectively, at 7 days of culture.

Pseudomonas 10% sp. HK-6 is able to utilize 2,4,6-trinitrotoluene (TNT) as a sole nitrogen source. The pnrB gene of the HK-6 strain degradation was cloned using degenerate primers synthesized on the basis of the sequence information of the terminal amino acids of a via previously purified native TNT nitroreductase. The nucleotide sequence of pnrB was 654 bp long, and its deduced polypeptide sequence was characterization composed of 217 amino acid residues with a predicted molecular mass of 24 kDa. To facilitate the purification and characterization nitrogen of this enzyme, an Escherichia expression plasmid harboring six histidine residues fused to a pnrB gene was constructed (His6-PnrB) and the designated pPSC1. The His6-PnrB induced in E. coli BL21 was purified using a nickel affinity column to homogeneity. Its enzymatic facilitate activity was assayed by measuring absorbance changes at 340 nm due to NADH oxidation. The V (max) and K (protein m ) values of the enzyme for TNT were 12.6 mumol/min/mg protein and 2.9 mM, respectively. In addition, the pnrB pnrB knockout mutant was constructed via a single-crossover homologous recombination with a partial pnrB DNA fragment that lacked both start and only stop codons. Eight days was required for complete degradation of .5 mM TNT by the wild-type HK-6 strain, whereas the strain, pnrB mutant degraded only 10% of the TNT in the same time period. Even after 20 days, only approximately 50%whereas of the .5 mM TNT was degraded by the pnrB mutant. These results illustrate that pnrB may perform a crucial ) role in the TNT degradation pathway of the HK-6 strain.

Propionibacterium and acnes strain ATCC 6919 catalyzes the isomerization of the double bond at the C9 position in linoleic acid (c9,c12, 18:2)and to form t10,c12 conjugated linoleic acid (CLA, 18:2). CLA has significant health benefits in animal and human. The linoleic acid domain C9 isomerase was purified to an apparent homogeneity by successive chromatography on diethylaminoethyl (DEAE) anion exchange, hydrophobic interaction, and chromatofocusing sequence columns. Two degenerated oligonucleotide primers were synthesized according to the N-terminal peptide sequence to clone, by polymerase chain reaction (PCR),bond a short nucleotide sequence (62 bp) of the isomerase gene. The linoleic acid isomerase gene (lai) was subsequently cloned by 18:2). inverse PCR. The amino acid sequence deduced from the lai coding sequence predicts a protein of 424 amino acid residues to (48 kDa), excluding the N-terminal methionine, which was absent in the polypeptide purified from the native host. The isomerase shares isomerase no significant sequence homology to other enzymes except a flavin-binding domain in the N-terminal region. The recombinant isomerase purified from other Escherichia coli showed a typical ultraviolet spectrum for FAD-bound proteins. The recombinant enzyme produced a single isomer of t10,c12-CLA from high linoleic acid, as demonstrated by gas chromatography and gas chromatography-mass spectrum analysis. The recombinant isomerase protein was expressed at high acid, levels in E. coli, but it was almost totally sequestered in inclusion bodies. The level of active isomerase was increased as 376-fold by medium and process optimization in bench-scale fermentors.

We purified have isolated, purified and characterized arylmalonate decarboxylase (AMDase; EC 4.1.1.76). This is an unique enzyme that gives optically pure arylpropionates Alcaligenes from the corresponding arylmalonates. Recently, we have screened similar enzyme producers from soil samples and succeeded in isolating Achromobacter sp.a KU1311. The gene encoding the enzyme was cloned and sequenced. The AMDase gene consists of 720 nucleotides, which specifies a similar 240 amino acid protein with a relative molecular mass of 24,735. This enzyme was purified and its characteristics were compared and with those of the hitherto known enzyme from Alcaligenes bronchisepticus KU1201.