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Find video protocols related to scientific articles indexed in Pubmed.
Novel urushiols with human immunodeficiency virus type 1 reverse transcriptase inhibitory activity from the leaves of Rhus verniciflua.
J Nat Med
PUBLISHED: 06-28-2014
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Two novel urushiols, 1 and 2, and two known urushiols, 3 and 4, were isolated from the leaves of Rhus verniciflua and were examined for their human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitory activity. The novel urushiols were found to be 1,2-dihydroxyphenyl-3-[7'(E),9'(Z),11'(Z)-pentadecatrienyl]-14'-ol (1) and 1,2-dihydroxyphenyl-3-[8'(Z),10'(E),12'(E)-pentadecatrienyl]-14'-ol (2) by spectroscopic analyses. The absolute configuration at C-14' in 1 and 2 was determined to be a racemic mixture of (R) and (S) isomers by ozonolysis. Compound 2 (IC50: 12.6 µM) showed the highest HIV-1 RT inhibitory activity among the four urushiols, being 2.5-fold more potent than the positive control, adriamycin (IC50: 31.9 µM). Although the known urushiols were isolated from the sap and leaves of R. verniciflua, 1 was exclusively present in the leaves, and higher amounts of 2 were found in the leaves than in the sap. Present findings indicate that the leaves of R. verniciflua represent a new biological resource from which novel and known urushiols may be prepared, and the possible use of novel urushiols as bioactive products.
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Multicolor imaging of endoplasmic reticulum-located esterase as a prodrug activation enzyme.
ACS Med Chem Lett
PUBLISHED: 04-10-2014
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The carboxylesterase families of enzymes are key participants in phase I drug metabolism processes. Carboxylesterase families 1 and 2 are of particular clinical relevance. These enzymes produce endoplasmic reticulum localization signals, are primarily localized in the endoplasmic reticulum, and hydrolyze a wide range of ester-containing prodrugs into an activated form. In order to detect enzymes belonging to both families, we developed an optical multicolor imaging technique, which provides a distinct color window for multicolor imaging. This technique required the design and synthesis of three new mechanistic colored probes that fluoresce red, green, or blue and are based on the quinone methide cleavage process. These activity-based probes allow rapid and clear visualization with high specificity against the endoplasmic reticulum in cultured cells based on endoplasmic reticulum localized esterases including both families of carboxylesterase enzymes.
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Enzymatic synthesis of novel oligosaccharides from N-acetylsucrosamine using ?-fructofuranosidase from Aspergillus oryzae.
Carbohydr. Res.
PUBLISHED: 07-22-2013
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Mycelia of Aspergillus oryzae NBRC100959 contain 2 types of ?-fructofuranosidases, transfructosylation-catalyzing enzyme (?FFaseI), and hydrolysis-catalyzing enzyme (?FFaseII). Using ?FFaseI extracted from the mycelia of strain NBRC100959, two novel oligosaccharides consisting of GlcNAc and fructose, ?-d-fructofuranosyl-(2?1)-?-d-fructofuranosyl-(2?1)-2-acetamido-2-deoxy-?-d-glucopyranoside (N-acetyl-1-kestosamine, 1-KesNAc) and ?-d-fructofuranosyl-(2?1)-?-d-fructofuranosyl-(2?1)-?-d-fructofuranosyl-(2?1)-2-acetamido-2-deoxy-?-d-glucopyranoside (N-acetylnystosamine, NysNAc), were synthesized from ?-d-fructofuranosyl-(2?1)-2-acetamido-2-deoxy-?-d-glucopyranoside (N-acetylsucrosamine, SucNAc). We next planned to synthesize 1-KesNAc and NysNAc using A. oryzae mycelia. However, it was thought that the presence of ?FFaseII is disadvantageous for the production of these oligosaccharides by ?FFaseI, because ?FFaseII hydrolyzed 1-KesNAc and NysNAc. We succeeded to produce A. oryzae mycelia containing ?FFaseI as the major ?-fructofuranosidase, by increasing sucrose concentration in the culture medium. Then, using a dried sample of these A. oryzae mycelia, reaction for the oligosaccharide production was performed. As the results, 190mg of 1-KesNAc and 60mg of NysNAc were obtained from 0.6g of SucNAc. This whole-cell catalysis method facilitates the synthesis of 1-KesNAc and NysNAc because extraction and purification of ?FFaseI from mycelia are unnecessary.
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Virtual ligand screening of ?-glucosidase: Identification of a novel potent noncarbohydrate mimetic inhibitor.
Bioorg. Med. Chem. Lett.
PUBLISHED: 08-16-2011
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5-Thiazoleacetamide derivatives of AR122 and AR125 were screened as ?-glucosidase inhibitors by in silico high-throughput screening from commercial drug-like small compound libraries. Inhibition of ?-glucosidase with AR122 and AR125 is time dependent: with no preincubation, AR122 and AR125 are relatively moderate inhibitors, but interestingly, after a 120 min incubation, they were 50-fold more potent (AR122: IC(50)=2.47 ?M and AR125: IC(50)=27.1 ?M). Plots of ln [residual ?-glucosidase activity %] versus preincubation time show a pseudo-first order kinetics for both inhibitors. Through dialysis of enzyme-inhibitor complexes, no activity recovery was shown. These results suggest that AR122 and AR125 constitute a new class of noncarbohydrate mimetic inhibitor with an irreversible mechanism.
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Design and synthesis of an ER-specific fluorescent probe based on carboxylesterase activity with quinone methide cleavage process.
Bioorg. Med. Chem. Lett.
PUBLISHED: 02-12-2011
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CEs are important enzymes that catalyze the hydrolysis of prodrugs. In this Letter, we present a new mechanistic ER-specific fluorescent probe 1 based on CE activity. Permeation of 1 into cells and subsequent hydrolytic activation by CEs causes spontaneously quinone methide cleavage, resulting in bright red fluorescence in ER with high specificity. Probe 1 was developed for CE activity imaging and inhibitor screening at the cellular level.
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Family M42 aminopeptidase from the syntrophic bacterium Symbiobacterium thermophilum: characterization using recombinant protein.
J. Biosci. Bioeng.
PUBLISHED: 07-27-2010
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The chromosomal DNA of the syntrophic thermophile Symbiobacterium thermophilum contains open reading frames of the genes encoding family M42 aminopeptidases, Pep1079, Pep1080, and Pep1081. To characterize these peptidases, the genes were cloned into Escherichia coli and overexpressed. Our experiments using the recombinant proteins confirmed that Pep1079, Pep1080, and Pep1081 are components of arginyl or lysinyl aminopeptidases that require Co²+ for enzymatic activity. Coexistence of Pep1079 and Pep1080 is necessary for expressing high peptidase activity. Pep1081 enhances the activity of Pep1079 and Pep1080.
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Expression, purification, physicochemical characterization and structural analysis of cytochrome c554 from Vibrio parahaemolyticus strain RIMD2210633.
Biosci. Biotechnol. Biochem.
PUBLISHED: 05-07-2010
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The function of cytochrome c(554) of Vibrio parahaemolyticus has not yet been determined. We have determined the physicochemical properties and crystal structure of cytochrome c(554) at 1.8 A in order to help elucidate its function. The physicochemical properties and the tertiary structure of cytochrome c(554) resemble those of dimeric cytochrome c(552) from Pseudomonas nautica, but the Vibrio genus contains no gene for nitrite reductase, cytochrome cd(1), in its genome DNA. These results raise the possibility that both cytochromes denote an electron to an electron carrier and accept an electron from same electron carrier.
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Heterodisaccharide 4-O-(N-acetyl-beta-D-glucosaminyl)-D-glucosamine is a specific inducer of chitinolytic enzyme production in Vibrios harboring chitin oligosaccharide deacetylase genes.
Glycobiology
PUBLISHED: 06-24-2009
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Vibrio parahaemolyticus KN1699 produces 4-O-(N-acetyl-beta-d-glucosaminyl)-d-glucosamine (GlcNAc-GlcN) as a major end product from chitin using two extracellular hydrolases: glycoside hydrolase family 18 chitinase, which produces (GlcNAc)(2) from chitin, and carbohydrate esterase (CE) family 4 chitin oligosaccharide deacetylase (COD), which hydrolyzes the N-acetyl group at the reducing-end GlcNAc residue of (GlcNAc)(2). In this study, we clarified that this heterodisaccharide functions as an inducer of the production of the two above-mentioned chitinolytic enzymes, particularly chitinase. Similar results for chitinase production were obtained with other chitin-decomposing Vibrio strains harboring the CE family 4 COD gene; however, such an increase in chitinase production was not observed in chitinolytic Vibrio strains that did not harbor the COD gene. These results suggest that GlcNAc-GlcN is a unique inducer of chitinase production in Vibrio bacteria that have the COD-producing ability and that the COD involved in the synthesis of this signal compound is one of the key enzymes in the chitin catabolic cascade of these bacteria.
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Design and screening strategies for alpha-glucosidase inhibitors based on enzymological information.
Curr Top Med Chem
PUBLISHED: 02-10-2009
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Alpha-glucosidase inhibitors are marketed as therapeutic drugs for diabetes that act through the inhibition of carbohydrate metabolism. Inhibitors of the alpha-glucosidases that are involved in the biosynthesis of N-linked oligosaccharide chains have been reported to have antitumor, antiviral, and apoptosis-inducing activities, and some have been used clinically. alpha-Glucosidase inhibitors have interesting biological activities, and their design, synthesis, and screening are being actively performed. In quite a few reports, however, alpha-glucosidases with different origins than the target alpha-glucosidases, have been used to evaluate inhibitory activities. There might be confusion regarding the naming of alpha-glucosidases. For example, the term alpha-glucosidase is sometimes used as a generic name for alpha-glucoside hydrolases. Moreover, IUBMB recommends the use of "alpha-glucosidase" (EC 3.2.1.20) for exo-alpha-1,4-glucosidases, which are further classified into four families based on amino acid sequence similarities. Accordingly, substrate specificity and susceptibility to inhibitors varies markedly among enzymes in the IUBMB alpha-glucosidases. The design and screening of inhibitors without consideration of these differences is not efficient. For the development of a practical inhibitor that is operational in cells, HTS using the target alpha-glucosidase and the computer-aided design of inhibitors based on enzymatic information concerning the same alpha-glucosidase are essential.
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Crystallization and structural analysis of cytochrome c(6) from the diatom Phaeodactylum tricornutum at 1.5 A resolution.
Biosci. Biotechnol. Biochem.
PUBLISHED: 01-07-2009
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We determined for the first time the crystal structure of diatom cytochrome c(6) from Phaeodactylum tricornutum at 1.5 A resolution. The overall structure of the protein was classified as a class I c-type cytochrome. The physicochemical properties of the protein were examined by denaturation with guanidine hydrochloride and urea, and compared with those of other algal cytochrome c(6).
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Synthesis of ?-D-fructofuranosyl-(2?1)-2-acetamido-2-deoxy-?-D-glucopyranoside (N-acetylsucrosamine) using ?-fructofuranosidase-containing Aspergillus oryzae mycelia as a whole-cell catalyst.
Carbohydr. Res.
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Using soft granules consisting of Celite 535 and dried Aspergillus oryzae NBRC100959 mycelia containing ?-fructofuranosidase as a whole-cell catalyst, N-acetylsucrosamine [?-D-fructofuranosyl-(2?1)-2-acetamido-2-deoxy-?-D-glucopyranoside] was produced from sucrose and 2-acetamido-2-deoxy-D-glucose by enzymatic transfructosylation. The isolated yield of N-acetylsucrosamine from the reaction mixture was 22.1% (from sucrose). The result of N-terminal amino acid sequence analysis indicated that the enzyme involved in the synthesis of N-acetylsucrosamine is a product from gene (NCBI accession number; NW_001884675, locus tag; AOR_1_1114084) encoding putative ?-fructofuranosidase on chromosome 6 of strain NBRC100959. The N-acetylsucrosamine we produced is highly soluble in water and is more stable in acidic solution than sucrose. The disaccharide was also produced using dried mycelia prepared from another A. oryzae strains.
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