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101.
Yoo SY Kim KJ Lim SH Kim KW Hwang HK Min BM Choe SJ Kook JK 《FEMS microbiology letters》2005,249(2):323-326
In this study, we isolated four bacterial strains grown on mitis-salivarius sucrose bacitracin agar. The strains had similar biochemical characteristics to biotypes I or II of mutans streptococci. The four isolates were identified as Streptococcus downei by 16S rDNA and dextranase gene (dex) sequencing as well as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) targeting dex. To our knowledge, this is the first report of the isolation and identification of S. downei from dental plaque in humans. The results suggest that S. downei can inhabit the human oral cavity. 相似文献
102.
Cho KJ Shim JH Cho MC Choe YK Hong JT Moon DC Kim JW Yoon DY 《Journal of cellular biochemistry》2005,96(4):869-878
Melanocortins, besides their central roles, have also recently been reported to regulate adipocyte metabolism. In this study, we attempted to characterize the mechanism underlying alpha-melanocyte-stimulating hormone (MSH)-induced lipolysis, and compared it with that of the adrenocorticotrophin hormone (ACTH) in 3T3-L1 adipocytes. Similar to ACTH, MSH treatment resulted in the release of glycerol into the cell supernatant. The activity of hormone-sensitive lipase, a rate-limiting enzyme, which is involved in lipolysis, was significantly increased by MSH treatment. In addition, a variety of kinases, including protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) were also phosphorylated as the result of MSH treatment, and their specific inhibitors caused a reduction in MSH-induced glycerol release and HSL activity, indicating that MSH-induced lipolysis was mediated by these kinases. These results suggest that PKA and ERK constitute the principal signaling pathways implicated in the MSH-induced lipolytic process via the regulation of HSL in 3T3-L1 adipocytes. 相似文献
103.
Characterization of a thermoacidophilic L-arabinose isomerase from Alicyclobacillus acidocaldarius: role of Lys-269 in pH optimum 总被引:2,自引:0,他引:2
Lee SJ Lee DW Choe EA Hong YH Kim SB Kim BC Pyun YR 《Applied and environmental microbiology》2005,71(12):7888-7896
The araA gene encoding L-arabinose isomerase (AI) from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius was cloned, sequenced, and expressed in Escherichia coli. Analysis of the sequence revealed that the open reading frame of the araA gene consists of 1,491 bp that encodes a protein of 497 amino acid residues with a calculated molecular mass of 56,043 Da. Comparison of the deduced amino acid sequence of A. acidocaldarius AI (AAAI) with other AIs demonstrated that AAAI has 97% and 66% identities (99% and 83% similarities) to Geobacillus stearothermophilus AI (GSAI) and Bacillus halodurans AI (BHAI), respectively. The recombinant AAAI was purified to homogeneity by heat treatment, ion-exchange chromatography, and gel filtration. The purified enzyme showed maximal activity at pH 6.0 to 6.5 and 65 degrees C under the assay conditions used, and it required divalent cations such as Mn2+, Co2+, and Mg2+ for its activity. The isoelectric point (pI) of the enzyme was about 5.0 (calculated pI of 5.5). The apparent Km values of the recombinant AAAI for L-arabinose and D-galactose were 48.0 mM (Vmax, 35.5 U/mg) and 129 mM (Vmax, 7.5 U/mg), respectively, at pH 6 and 65 degrees C. Interestingly, although the biochemical properties of AAAI are quite similar to those of GSAI and BHAI, the three AIs from A. acidocaldarius (pH 6), G. stearothermophilus (pH 7), and B. halodurans (pH 8) exhibited different pH activity profiles. Based on alignment of the amino acid sequences of these homologous AIs, we propose that the Lys-269 residue of AAAI may be responsible for the ability of the enzyme to act at low pH. To verify the role of Lys-269, we prepared the mutants AAAI-K269E and BHAI-E268K by site-directed mutagenesis and compared their kinetic parameters with those of wild-type AIs at various pHs. The pH optima of both AAAI-K269E and BHAI-E268K were rendered by 1.0 units (pH 6 to 7 and 8 to 7, respectively) compared to the wild-type enzymes. In addition, the catalytic efficiency (kcat/Km) of each mutant at different pHs was significantly affected by an increase or decrease in Vmax. From these results, we propose that the position corresponding to the Lys-269 residue of AAAI could play an important role in the determination of the pH optima of homologous AIs. 相似文献
104.
Bae WC Lee HK Choe YC Jahng DJ Lee SH Kim SJ Lee JH Jeong BC 《Journal of microbiology (Seoul, Korea)》2005,43(1):21-27
A soluble Cr(VI) reductase was purified from the cytoplasm of Escherichia coli ATCC 33456. The molecular mass was estimated to be 84 and 42 kDa by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, indicating a dimeric structure. The pI was 4.66, and optimal enzyme activity was obtained at pH 6.5 and 37 degrees C. The most stable condition existed at pH 7.0. The purified enzyme used both NADPH and NADH as electron donors for Cr(VI) reduction, while NADPH was the better, conferring 61%; higher activity than NADH. The Km values for NADPH and NADH were determined to be 47.5 and 17.2 micromol, and the Vmax values 322.2 and 130.7 micromol Cr(VI) min(-1)mg(-1) protein, respectively. The activity was strongly inhibited by N-ethylmalemide, Ag2+, Cd2+, Hg2+, and Zn2+. The antibody against the enzyme showed no immunological cross reaction with those of other Cr(VI) reducing strains. 相似文献
105.
Choe H Moore MJ Owens CM Wright PL Vasilieva N Li W Singh AP Shakri R Chitnis CE Farzan M 《Molecular microbiology》2005,55(5):1413-1422
Plasmodium vivax is one of four Plasmodium species that cause human malaria. P. vivax and a related simian malaria parasite, Plasmodium knowlesi, invade erythrocytes by binding the Duffy antigen/receptor for chemokines (DARC) through their respective Duffy binding proteins. Here we show that tyrosines 30 and 41 of DARC are modified by addition of sulphate groups, and that the sulphated tyrosine 41 is essential for association of the Duffy binding proteins of P. vivax (PvDBP) and P. knowlesi (PkDaBP) with DARC-expressing cells. These sulphated tyrosines also participate in the association of DARC with each of its four known chemokine ligands. Alteration of tyrosine 41 to phenylalanine interferes with MCP-1, RANTES and MGSA association with DARC, but not with that of IL8. In contrast, alteration of tyrosine 30 to phenylalanine interferes with the association of IL8 with DARC. A soluble sulphated amino-terminal domain of DARC, but not one modified to phenylalanine at residue 41, can be used to block the association of PvDBP and PkDaBP with red blood cells, with an IC50 of approximately 5 nM. These data are consistent with a role for tyrosine sulphation in the association of many or most chemokines with their receptors, and identify a key molecular determinant of erythrocyte invasion by P. vivax. 相似文献
106.
Choe Y Brinen LS Price MS Engel JC Lange M Grisostomi C Weston SG Pallai PV Cheng H Hardy LW Hartsough DS McMakin M Tilton RF Baldino CM Craik CS 《Bioorganic & medicinal chemistry》2005,13(6):2141-2156
Trypanosoma cruzi, a protozoan parasite, is the causative agent of Chagas disease, a major cause of cardiovascular disease in many Latin American countries. There is an urgent need to develop an improved therapy due to the toxicity of existing drugs and emerging drug resistance. Cruzain, the primary cysteine protease of T. cruzi, is essential for the survival of the parasite in host cells and therefore is an important target for the development of inhibitors as potential therapeutics. A novel series of alpha-ketoamide-, alpha-ketoacid-, alpha-ketoester-, and aldehyde-based inhibitors of cruzain has been developed. The inhibitors were identified by screening protease targeted small molecule libraries and systematically optimizing the P1, P2, P3, and P1' residues using specific structure-guided methods. A total of 20 compounds displayed picomolar potency in in vitro assays and three inhibitors representing different alpha-keto-based inhibitor scaffolds demonstrated anti-trypanosomal activity in cell culture. A 2.3A crystallographic structure of cruzain bound with one of the alpha-ketoester analogs is also reported. The structure and kinetic assay data illustrate the covalent binding, reversible inhibition mechanism of the inhibitor. Information on the compounds reported here will be useful in the development of new lead compounds as potential therapeutic agents for the treatment of Chagas disease and as biological probes to study the role that cruzain plays in the pathology. This study also demonstrates the validity of structure-guided approaches to focused library design and lead compound optimization. 相似文献
107.
108.
109.
Mao L Yang L Arora A Choe ES Zhang G Liu Z Fibuch EE Wang JQ 《The Journal of biological chemistry》2005,280(13):12602-12610
The regulation of protein phosphorylation requires coordinated interaction between protein kinases and protein phosphatases (PPs). Recent evidence has shown that the Galphaq-protein-coupled metabotropic glutamate receptor (mGluR) 5 up-regulates phosphorylation of MAPK/ERK1/2. However, signaling mechanisms linking mGluR5 to ERK are poorly understood. In this study, roles of a major serine/threonine PP, PP2A, in this event were evaluated in cultured neurons. We found that the PP1/2A inhibitors okadaic acid and calyculin A mimicked the effect of the mGluR5 agonists (RS)-3,5-dihydroxyphenylglycine and (RS)-2-chloro-5-hydroxyphenylglycine in facilitating phosphorylation of ERK1/2 and its upstream kinase, MEK1/2, in a PP2A-dependent but not PP1-dependent manner. Co-administration of either inhibitor with an mGluR5 agonist produced additive phosphorylation of ERK1/2. Enzymatic assays showed a basal level of phosphatase activity of PP2A under normal conditions, and activation of mGluR5 selectively inhibited PP2A, but not PP1, activity. In addition, a physical association of the cytoplasmic C terminus of mGluR5 with PP2A was observed, and ligand activation of mGluR5 reduced mGluR5-PP2A binding. Additional mechanistic studies revealed that mGluR5 activation increased tyrosine (Tyr307) phosphorylation of PP2A, which was dependent on activation of a p60c-Src family tyrosine kinase, but not the epidermal growth factor receptor tyrosine kinase and resulted in dissociation of PP2A from mGluR5 and reduced PP2A activity. Together, we have identified a novel, mGluR5-triggered signaling mechanism involving use- and Src-dependent inactivation of PP2A, which contributes to mGluR5 activation of MEK1/2 and ERK1/2. 相似文献
110.
Lee DW Choe EA Kim SB Eom SH Hong YH Lee SJ Lee HS Lee DY Pyun YR 《Archives of biochemistry and biophysics》2005,434(2):333-343
L-Arabinose isomerase (AI) catalyzes the isomerization of L-arabinose to L-ribulose. It can also convert d-galactose to d-tagatose at elevated temperatures in the presence of divalent metal ions. The araA genes, encoding AI, from the mesophilic bacterium Bacillus halodurans and the thermophilic Geobacillus stearothermophilus were cloned and overexpressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The purified enzymes are homotetramers with a molecular mass of 232 kDa and close amino acid sequence identity (67%). However, they exhibit quite different temperature dependence and metal requirements. B. halodurans AI has maximal activity at 50 degrees C under the assay conditions used and is not dependent on divalent metal ions. Its apparent K(m) values are 36 mM for L-arabinose and 167 mM for d-galactose, and the catalytic efficiencies (k(cat)/K(m)) of the enzyme were 51.4 mM(-1)min(-1) (L-arabinose) and 0.4 mM(-1)min(-1) (d-galactose). Unlike B. halodurans AI, G. stearothermophilus AI has maximal activity at 65-70 degrees C, and is strongly activated by Mn(2+). It also has a much higher catalytic efficiency of 4.3 mM(-1)min(-1) for d-galactose and 32.5 mM(-1)min(-1)for L-arabinose, with apparent K(m) values of 117 and 63 mM, respectively. Irreversible thermal denaturation experiments using circular dichroism (CD) spectroscopy showed that the apparent melting temperature of B. halodurans AI (T(m)=65-67 degrees C) was unaffected by the presence of metal ions, whereas EDTA-treated G. stearothermophilus AI had a lower T(m) (72 degrees C) than the holoenzyme (78 degrees C). CD studies of both enzymes demonstrated that metal-mediated significant conformational changes were found in holo G. stearothermophilus AI, and there is an active tertiary structure for G. stearothermophilus AI at elevated temperatures for its catalytic activity. This is in marked contrast to the mesophilic B. halodurans AI where cofactor coordination is not necessary for proper protein folding. The metal dependence of G. stearothermophilus AI seems to be correlated with their catalytic and structural functions. We therefore propose that the metal ion requirement of the thermophilic G. stearothermophilus AI reflects the need to adopt the correct substrate-binding conformation and the structural stability at elevated temperatures. 相似文献