Home About us Contact
|
Home About us Contact | ||
|
|
||
Highest Activity (highest + activity)
Selected AbstractsEssential role of PSM/SH2-B variants in insulin receptor catalytic activation and the resulting cellular responsesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2008Manchao Zhang Abstract The positive regulatory role of PSM/SH2-B downstream of various mitogenic receptor tyrosine kinases or gene disruption experiments in mice support a role of PSM in the regulation of insulin action. Here, four alternative PSM splice variants and individual functional domains were compared for their role in the regulation of specific metabolic insulin responses. We found that individual PSM variants in 3T3-L1 adipocytes potentiated insulin-mediated glucose and amino acid transport, glycogenesis, lipogenesis, and key components in the metabolic insulin response including p70 S6 kinase, glycogen synthase, glycogen synthase kinase 3 (GSK3), Akt, Cbl, and IRS-1. Highest activity was consistently observed for PSM alpha, followed by beta, delta, and gamma with decreasing activity. In contrast, dominant-negative peptide mimetics of the PSM Pro-rich, pleckstrin homology (PH), or src homology 2 (SH2) domains inhibited any tested insulin response. Potentiation of the insulin response originated at the insulin receptor (IR) kinase level by PSM variant-specific regulation of the Km (ATP) whereas the Vmax remained unaffected. IR catalytic activation was inhibited by peptide mimetics of the PSM SH2 or dimerization domain (DD). Either peptide should disrupt the complex of a PSM dimer linked to IR via SH2 domains as proposed for PSM activation of tyrosine kinase JAK2. Either peptide abolished downstream insulin responses indistinguishable from PSM siRNA knockdown. Our results implicate an essential role of the PSM variants in the activation of the IR kinase and the resulting metabolic insulin response. PSM variants act as internal IR ligands that in addition to potentiating the insulin response stimulate IR catalytic activation even in the absence of insulin. J. Cell. Biochem. 103: 162,181, 2008. © 2007 Wiley-Liss, Inc. [source] TMAOase Activity of European Hake (Merluccius merluccius) Organs: Influence of Biological Condition and SeasonJOURNAL OF FOOD SCIENCE, Issue 9 2002M. Rey-Mansilla ABSTRACT: Trimethylamine N-oxide demethylase (TMAOase) activity of several internal organs of hake were studied for 2 consecutive y. The correlation between enzymatic activity and season of year, sex, weight, and length were analyzed. While kidney and spleen showed the highest activities, liver, heart, bile, and gall bladder activities were much lower, and in some cases they were below the detection limit. A correlation between TMAOase activity of kidney and season was found. During winter and spring (February to May), the months matching the spawning peak, high activities were detected, while in summer months the activity level was lower. TMAOase activity in the rest of the organs did not seem to have a seasonal influence. Keywords: TMAOase, season, biological condition, hake, soluble protein [source] Characterization of a developmentally regulated amino acid transporter (AAT1p) of the rust fungus Uromyces fabaeMOLECULAR PLANT PATHOLOGY, Issue 1 2002Christine Struck summary In the rust fungus Uromyces fabae, invasion of the host plant and haustorium formation are accompanied by the activation of many genes (PIGs =in planta induced genes). In addition to the previously described AAT2 (PIG2), AAT1 (PIG27) was found to encode a protein with a high similarity to fungal amino acid permeases. AAT1 transcripts are present in germinated hyphae and throughout the mycelium later in the infection process, but occur at the highest levels in haustoria. Expression of AAT1p in a histidine uptake-defective yeast mutant revealed energy-dependent transport of 14C-histidine, with a KM value of 25.8 µm. In addition, complementation analysis revealed AAT1 -dependent transport for lysine. Using Xenopus oocytes as expression system, AAT1p-dependent symport of protons with a broad spectrum of amino acids was observed, with the highest activities obtained with histidine and lysine. These results confirm that in rust fungi, the expression of amino acid transporters is developmentally regulated and occurs preferentially in the parasitic phase of development. [source] Synthesis and Biological Evaluation of 7-Azaisoindigo DerivativesARCHIV DER PHARMAZIE, Issue 3 2010Zhao-Hui Wang Abstract A series of novel 7-azaisoindigo derivatives 3,14 were designed, synthesized, and structurally characterized by IR, 1H-NMR, 13C-NMR, mass spectra, and elemental analyses. Their antiproliferative activities were evaluated in a hormone-independent prostate cancer cell line DU145. Among them, compounds 8, 9, 14 showed the highest activities. Our study also showed that compounds 7, 11, 12 exhibited higher inhibitory activities on CDK2/cyclin A than that of the positive control meisoindigo. Western blot analysis on DU145 cells treated with compounds 7 and 9 demonstrated that 7-azaisoindigo derivatives could decrease the level of CDK2 activity (phosphorylation) and the expression of cyclin D1, and increase the expression of endogenous cyclin-dependent inhibitor p27. [source] Aminopyridinate-Stabilized Lanthanoid Complexes: Synthesis, Structure and Polymerization of Ethylene and IsopreneEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 18 2010Christian Döring Abstract A series of aminopyridinate-stabilized dialkyl-lanthanoid complexes has been synthesized and characterized. The complexes were prepared by alkane elimination reacting [Ln(CH2SiMe3)3(thf)2] (Ln = Er, Yb, Lu) or [Ln(CH2Ph)3(thf)3] (Ln = Y, Er, Lu) with one equivalent of the bulky aminopyridine (2,6-diisopropylphenyl)-[6-(2,4,6-triisopropylphenyl)pyridin-2-yl]amine. Single-crystal X-ray analyses were carried out for all of the benzyl derivatives. The reaction of these compounds with ammonium borate leads to the elimination of one of the two alkyl functions and affords organolanthanoid cations. The aminopyridinate-stabilized dialkyl-lanthanoid compounds can initiate the polymerization of isoprene after activation with perfluorinated tetraphenyl borates. The obtained polymers have a 3,4-content of 60,95,%. The metal ion size as well as the addition of alkylaluminium compounds influences the microstructure of the obtained polymer. Aminopyridinate-stabilized organolanthanoid cations of Sc, Lu, Er and Y can polymerize ethylene in the presence of alkylaluminium compounds. The Lu, Er and Y complexes act as a CCTP catalyst and the erbium compound exhibits the highest activity. [source] Activation of gelatinolytic/collagenolytic activity in dentin by self-etching adhesivesEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 2 2006Yoshihiro Nishitani Mild acids are known to activate dentin matrix metalloproteinase (MMPs). All self-etching dental adhesives are acidic (pH 1.5,2.7) and may activate dentin MMPs. The purpose of this study was to compare the ability of several all-in-one adhesives to activate gelatinolytic and collagenolytic activities in powdered mineralized dentin. Powdered dentin made from human teeth was mixed with all-in-one adhesives (Clearfil Tri-S Bond, G-Bond, Adper Prompt L-Pop) or a self-etching primer (Clearfil SE Bond primer) for varying times and then the reaction was stopped by extracting the adhesives using acetone. Fresh untreated mineralized dentin powder had a gelatinolytic activity of 3.31 ± 0.39 relative fluorescent units (RFU) per mg dry weight (24 h) that increased, over storage time, to 87.5 RFU mg,1 (24 h) after 6,8 wk. When fresh powder was treated with acidic Tri-S Bond, the gelatinolytic activity increased from 3.24 ± 0.70 RFU mg,1 to >,112.5 RFU mg,1 (24 h) after 20 min and then remained unchanged. Monomers with lower pH values produced less activity. There was a significant, direct correlation between gelatinolytic activity and pH, with Tri-S giving the highest activity. Coating dentin powder with Tri-S resin prevented fluorescent substrates from gaining access to the enzyme, even though it activated the enzyme. In conclusion, self-etch adhesives may activate latent MMP and increase the activity to near-maximum levels and contribute to the degradation of resin,dentin bonds over time. [source] Polyamines and hair: a couple in search of perfectionEXPERIMENTAL DERMATOLOGY, Issue 9 2010Yuval Ramot Please cite this paper as: Polyamines and hair: a couple in search of perfection. Experimental Dermatology 2010; 19: 784,790. Abstract:, Polyamines (spermidine, putrescine and spermine) are multifunctional cationic amines that are indispensable for cellular proliferation; of key significance in the growth of rapidly regenerating tissues and tumors. Given that the hair follicle (HF) is one of the most highly proliferative organs in mammalian biology, it is not surprising that polyamines are crucial to HF growth. Indeed, growing (anagen) HFs show the highest activity of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis, while inhibition of ODC, using eflornithine, results in a decreased rate of excessive facial hair growth in vivo and inhibits human scalp hair growth in organ culture. In sheep, manipulation of dietary intake of polyamines also results in altered wool growth. Polyamine-containing nutraceuticals have therefore been proposed as promoters of human hair growth. Recent progress in polyamine research, coupled with renewed interest in the role of polyamines in skin biology, encourages one to revisit their potential roles in HF biology and highlights the need for a systematic evaluation of their mechanisms of action and clinical applications in the treatment of hair disorders. The present viewpoint essay outlines the key frontiers in polyamine-related hair research and defines the major open questions. Moreover, it argues that a renaissance in polyamine research in hair biology, well beyond the inhibition of ODC activity in hirsutism therapy, is important for the development of novel therapeutic strategies for the manipulation of human hair growth. Such targets could include the manipulation of polyamine biosynthesis and the topical administration of selected polyamines, such as spermidine. [source] Crystal structures of isomaltase from Saccharomyces cerevisiae and in complex with its competitive inhibitor maltoseFEBS JOURNAL, Issue 20 2010Keizo Yamamoto The structures of isomaltase from Saccharomyces cerevisiae and in complex with maltose were determined at resolutions of 1.30 and 1.60 Å, respectively. Isomaltase contains three domains, namely, A, B, and C. Domain A consists of the (,/,)8 -barrel common to glycoside hydrolase family 13. However, the folding of domain C is rarely seen in other glycoside hydrolase family 13 enzymes. An electron density corresponding to a nonreducing end glucose residue was observed in the active site of isomaltase in complex with maltose; however, only incomplete density was observed for the reducing end. The active site pocket contains two water chains. One water chain is a water path from the bottom of the pocket to the surface of the protein, and may act as a water drain during substrate binding. The other water chain, which consists of six water molecules, is located near the catalytic residues Glu277 and Asp352. These water molecules may act as a reservoir that provides water for subsequent hydrolytic events. The best substrate for oligo-1,6-glucosidase is isomaltotriose; other, longer-chain, oligosaccharides are also good substrates. However, isomaltase shows the highest activity towards isomaltose and very little activity towards longer oligosaccharides. This is because the entrance to the active site pocket of isomaltose is severely narrowed by Tyr158, His280, and loop 310,315, and because the isomaltase pocket is shallower than that of other oligo-1,6-glucosidases. These features of the isomaltase active site pocket prevent isomalto-oligosaccharides from binding to the active site effectively. [source] Stage-specific expression of Caenorhabditis elegans ribonuclease H1 enzymes with different substrate specificities and bivalent cation requirementsFEBS JOURNAL, Issue 2 2006Hiromi Kochiwa Ribonuclease H1 (RNase H1) is a widespread enzyme found in a range of organisms from viruses to humans. It is capable of degrading the RNA moiety of DNA,RNA hybrids and requires a bivalent ion for activity. In contrast with most eukaryotes, which have one gene encoding RNase H1, the activity of which depends on Mg2+ ions, Caenorhabditis elegans has four RNase H1-related genes, and one of them has an isoform produced by alternative splicing. However, little is known about the enzymatic features of the proteins encoded by these genes. To determine the differences between these enzymes, we compared the expression patterns of each RNase H1-related gene throughout the development of the nematode and the RNase H activities of their recombinant proteins. We found gene-specific expression patterns and different enzymatic features. In particular, besides the enzyme that displays the highest activity in the presence of Mg2+ ions, C. elegans has another enzyme that shows preference for Mn2+ ion as a cofactor. We characterized this Mn2+ -dependent RNase H1 for the first time in eukaryotes. These results suggest that there are at least two types of RNase H1 in C. elegans depending on the developmental stage of the organism. [source] Determination of the metal ion dependence and substrate specificity of a hydratase involved in the degradation pathway of biphenyl/chlorobiphenylFEBS JOURNAL, Issue 4 2005Pan Wang BphH is a divalent metal ion-dependent hydratase that catalyzes the formation of 2-keto-4-hydroxypentanoate from 2-hydroxypent-2,4-dienoate (HPDA). This reaction lies on the catabolic pathway of numerous aromatics, including the significant environmental pollutant, polychlorinated biphenyls (PCBs). BphH from the PCB degrading bacterium, Burkholderia xenoverans LB400, was overexpressed and purified to homogeneity. Atomic absorption spectroscopy and Scatchard analysis reveal that only one divalent metal ion is bound to each enzyme subunit. The enzyme exhibits the highest activity when Mg2+ was used as cofactor. Other divalent cations activate the enzyme in the following order of effectiveness: Mg2+ > Mn2+ > Co2+ > Zn2+ > Ca2+. This differs from the metal activation profile of the homologous hydratase, MhpD. UV-visible spectroscopy of the Co2+,BphH complex indicates that the divalent metal ion is hexa-coordinated in the enzyme. The nature of the metal ion affected only the kcat and not the Km values in the BphH hydration of HPDA, suggesting that cation has a catalytic rather than just a substrate binding role. BphH is able to transform alternative substrates substituted with methyl- and chlorine groups at the 5-position of HPDA. The specificity constants (kcat/Km) for 5-methyl and 5-chloro substrates are, however, lowered by eight- and 67-fold compared with the unsubstituted substrate. Significantly, kcat for the chloro-substituted substrate is eightfold lower compared with the methyl-substituted substrate, showing that electron withdrawing substituent at the 5-position of the substrate has a negative influence on enzyme catalysis. [source] Possible involvement of an FKBP family member protein from a psychrotrophic bacterium Shewanella sp.FEBS JOURNAL, Issue 7 2004SIB1 in cold-adaptation A psychrotrophic bacterium Shewanella sp. strain SIB1 was grown at 4 and 20 °C, and total soluble proteins extracted from the cells were analyzed by two-dimensional polyacrylamide gel electrophoresis. Comparison of these patterns showed that the cellular content of a protein with a molecular mass of 28 kDa and an isoelectric point of four greatly increased at 4 °C compared to that at 20 °C. Determination of the N-terminal amino acid sequence, followed by the cloning and sequencing of the gene encoding this protein, revealed that this protein is a member of the FKBP family of proteins with an amino acid sequence identity of 56% to Escherichia coli FKBP22. This protein was overproduced in E. coli in a His-tagged form, purified, and analyzed for peptidyl-prolyl cis-trans isomerase activity. When this activity was determined by the protease coupling assay using N -succinyl-Ala-Leu-Pro-Phe- p -nitroanilide as a substrate at various temperatures, the protein exhibited the highest activity at 10 °C with a kcat/Km value of 0.87 µm,1·s,1. When the peptidyl-prolyl cis-trans isomerase activity was determined by the RNase T1 refolding assay at 10 and 20 °C, the protein exhibited higher activity at 10 °C with a kcat/Km value of 0.50 µm,1·s,1. These kcat/Km values are lower but comparable to those of E. coli FKBP22. We propose that a FKBP family protein is involved in cold-adaptation of psychrotrophic bacteria. [source] Chemical composition and antimicrobial activity of essential oils from Tunisian Pituranthos tortuosus (Coss.) MaireFLAVOUR AND FRAGRANCE JOURNAL, Issue 1 2006A. Abdelwahed Abstract The aerial parts of Pituranthos tortuosus, collected during November and April, were analysed by GC and GC,MS. In total, 56 compounds were identified by their retention indices. Antimicrobial assays showed that the November essential oil is more effective than that of April against the Gram-positive bacteria Enterococcus faecalis and Staphylococcus aureus. The April essential oil displayed the highest activity against Staphylococcus aureus. Copyright © 2005 John Wiley & Sons, Ltd. [source] Structure,fungitoxicity relationships of some volatile flavour constituents of the edible mushrooms Agaricus bisporus and Pleurotus floridaFLAVOUR AND FRAGRANCE JOURNAL, Issue 4 2001Eugene Sebastian J. Nidiry Abstract The fungitoxicity of the diethyl ether extracts of two basidiomycete mushrooms, Agaricus bisporus and Pleurotus florida, and 14 flavour constituents present in these mushrooms is being reported. Median effective molar concentrations (EC50) of the compounds for the mycelial growth inhibition of Colletotrichum gloeosporioides on potato,dextrose,agar (PDA) medium were computed and compared. Among the constituents tested for fungitoxicity, 1-octanol exhibited the highest activity. Structure,activity relationship studies of the constituents revealed that high hydrophobicity of the alkyl moiety, the presence of the primary alcoholic group and the absence of branching of the alkyl group are responsible for the high activity of 1-octanol. Copyright © 2001 John Wiley & Sons, Ltd. [source] Targeted delivery of salicylic acid from acne treatment products into and through skin: role of solution and ingredient properties and relationships to irritationINTERNATIONAL JOURNAL OF COSMETIC SCIENCE, Issue 4 2004L. Rhein Salicylic acid (SA) is a beta-hydroxy acid and has multifunctional uses in the treatment of various diseases in skin such as acne, psoriasis, and photoaging. One problem often cited as associated with salicylic acid is that it can be quite irritating at pH 3,4, where it exhibits the highest activity in the treatment of skin diseases. We have identified strategies to control the irritation potential of salicylic acid formulations and have focused on hydroalcoholic solutions used in acne wipes. One strategy is to control the penetration of SA into the skin. Penetration of the drug into various layers of skin, i.e. epidermis, dermis, and receptor fluid, was measured using a modified Franz in vitro diffusion method after various exposure times up to 24 h. A polyurethane polymer (polyolprepolymer-15) was found to be an effective agent in controlling delivery of SA. In a dose-dependent fashion it targeted delivery of more SA to the epidermis as compared to penetration through the skin into the receptor fluid. It also reduced the rapid rate of permeation of a large dose of SA through the skin in the first few hours of exposure. A second strategy that proved successful was incorporation of known mild nonionic surfactants like isoceteth-20. These surfactants cleanse the skin, yet due to their inherent mildness (because of their reduced critical micelle concentration and monomer concentration), keep the barrier intact. Also, they reduce the rate of salicylic acid penetration, presumably through micellar entrapment (either in solution or on the skin surface after the alcohol evaporates). Cumulative irritation studies showed that targeting delivery of SA to the epidermis and reducing the rapid early rate of penetration of large amounts of drug through the skin resulted in a reduced irritation potential. In vivo irritation studies also showed that the surfactant system is the most important factor controlling irritancy. SA delivery is secondary, as formulations with less SA content reduced the rate of delivery to the receptor and yet were some of the most irritating formulations tested, presumably due to the action of the specific anionic surfactant on the barrier. Alcohol content also did not appreciably affect irritation and SA delivery; formulations with considerably low alcohol content but containing anionic versus nonionic surfactant systems exhibited considerably higher irritancy. Thus the surfactant type was again the predominant factor in those studies, although arguably alcohol plays some role (solubilization of SA). Results showed that both polymers and mild surfactants work in concert to provide the optimal formulation benefits of targeted delivery and reduced irritation. Synergistic relationships among hydroalcoholic formulation components will be discussed along with the mechanisms likely involved in controlling delivery of SA to skin. [source] Isolation of a low-temperature adapted lipolytic enzyme from uncultivated micro-organismJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2008C. Roh Abstract Aims:, The aim of the study was to isolate a novel lipolytic enzyme from the activated sludge of uncultured micro-organisms. Methods and Results:, The metagenomic DNA was directly extracted from the activated sludge, and a metagenomic library was constructed by using the pUC vector. The library was screened for lipolytic enzyme activity on 1% tributyrin agar plate. A clone among c. 100 000 recombinant libraries showed the lipolytic activity. The putative lipolytic gene encoding lipo1 from the metagenomic library was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The expressed recombinant enzyme was purified by Ni-nitrilotriacetic acid affinity chromatography and characterized using general substrates of lipolytic property. The gene consisted of 972 bp encoding a polypeptide of 324 amino acids with a molecular mass of 35·6 kDa. Typical residues essential for lipolytic activity such as penta-peptide (GXSXG) and catalytic triad sequences (Ser166, Asp221 and His258) were detected. The deduced amino acid sequence of lipo1 showed low identity with amino acid sequences of esterase/lipase (32%, ZP_01528487) from Pseudomonas mendocina ymp and esterase (31%, AAY45707) from uncultured bacterium. This lipolytic enzyme exhibited the highest activity at pH 7·5 and 10°C. At thermal stability analysis, lipo1 was more unstable at 40°C than 10°C. Conclusions:, An activity based strategy has been an effective method for fishing out a low-temperature adapted lipolytic enzyme from the metagenomic library. This lipo1 enzyme can be considered to belong to the hormone-sensitive lipase family due to the enzyme's oxyanion hole by the sequence HGGG. Significance and Impact of the Study:, Lipo1 is a novel psychrophilic esterase obtained directly from the metagenomic library. Owing its support of significant activity at low temperature, this enzyme is expected to be useful for potential application as a biocatalyst in organic chemistry. [source] Hybrid titanium catalyst supported on core-shell silica/poly(styrene- co -acrylic acid) carrierJOURNAL OF APPLIED POLYMER SCIENCE, Issue 3 2010Lijun Du Abstract Hybrid titanium catalysts supported on silica/poly(styrene- co -acrylic acid) (SiO2/PSA) core-shell carrier were prepared and studied. The resulting catalysts were characterized by Fourier transform infrared (FTIR) spectroscopy, laser scattering particle analyzer and scanning electronic microscope (SEM). The hybrid catalyst (TiCl3/MgCl2/THF/SiO2·TiCl4/MgCl2/PSA) showed core-shell structure and the thickness of the PSA layer in the two different hybrid catalysts was 2.0 ,m and 5.0 ,m, respectively. The activities of the hybrid catalysts were comparable to the conventional titanium-based Ziegler-Natta catalyst (TiCl3/MgCl2/THF/SiO2). The hybrid catalysts showed lower initial polymerization rate and longer polymerization life time compared with TiCl3/MgCl2/THF/SiO2. The activities of the hybrid catalysts were enhanced firstly and then decreased with increasing P/P. Higher molecular weight and broader molecular weight distribution (MWD) of polyethylene produced by the core-shell hybrid catalysts were obtained. Particularly, the hybrid catalyst with a PSA layer of 5.0 ,m obtained the longest polymerization life time with the highest activity (2071 kg PE mol,1 Ti h,1) and the resulting polyethylene had the broadest MWD (polydispersity index = 11.5) under our experimental conditions. The morphology of the polyethylene particles produced by the hybrid catalysts was spherical, but with irregular subparticles due to the influence of PSA layer. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010 [source] L-methioninase production by Aspergillus flavipes under solid-state fermentationJOURNAL OF BASIC MICROBIOLOGY, Issue 4 2009Ashraf S. A. El-Sayed Abstract Solid-state fermentation was carried out for the production of extra-cellular L-methioninase by Aspergillus flavipes (Bain and Sart.) using nine agro-industrial residues, namely wheat bran, rice bran, wheat flour, coconut seeds, cotton seeds, ground nut cake, lentil hulls, soya beans and chicken feathers. Chicken feathers were selected as solid substrate for L-methioninase production by A. flavipes. The maximum L-methioninase productivity (71.0 U/mg protein) and growth (11 mg protein/ml) of A. flavipes was obtained using alkali pretreated chicken feathers of 50% initial moisture content as substrate supplemented with D-glucose (1.0% w/v) and L-methionine (0.2% w/v). External supplementation of the fermentation medium with various vitamin sources has no overinductive effect on L-methioninase biosynthesis. The partially purified A. flavipes L-methioninase preparation showed highest activity (181 U/ml) at pH 8.0 with stability over a pH range (pH 6,8) for 2 h. L-methioninase activity was increased by preincubation of the enzyme for 2 h with Co2+, Mn2+, Cu2+ and Mg2+ and strongly inhibited by the presence of EDTA, NaN3, Li2+, Cd2+, DMSO and 2-mercaptoethanol. The enzyme preparation has a broad substrate spectrum showing a higher affinity to deaminate L-glycine, N -acetylglucosamine and glutamic acid, in addition to their proteolytic activity against bovine serum albumin, casein, gelatin and keratin. The partially purified enzyme was found to be glyco-metalloproteinic in nature as concluded from the analytical and spectroscopic profiles of the enzyme preparation. The demethiolating activity of the enzyme was also visualized chromogenially. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Cytosolic NADP phosphatases I and II from Arthrobacter sp. strain KM: Implication in regulation of NAD+/NADP+ balanceJOURNAL OF BASIC MICROBIOLOGY, Issue 3 2004Shigeyuki Kawai NADP phosphatase (NADPase) is an enzyme that converts NADP+ into NAD+ through dephosphorylation of NADP+, and is considered to be one of the possible candidates for regulation of the NAD+/NADP+ balance in vivo. In order to obtain an intrinsic NADPase, the NADP+ -degrading activity in a membrane-free cell extract of a Gram-positive bacterium, Arthrobacter sp. strain KM, was first assessed and demonstrated to be mainly achieved through the NADPase reaction, indicating NADPase is essential for degradation of NADP+ and therefore for regulation of the NAD+/NADP+ balance in cytosol. Then, the isolation of cytosolic NADPase was attempted using NADP+ as a substrate. Two NADPase isozymes, designated as NADPases I and II, were purified from the cell extract of the bacterium, and were indicated to be the sole cytosolic NADPases regulating the balance of NAD+/NADP+. NADPases I and II are homodimers of 32 and 30 kDa subunits, respectively, and most active at pH 7,8. The N-terminal amino acid sequences of the two enzymes are similar to each other. Among the biological substrates tested, both enzymes showed the highest activity toward NADP+ and NADPH. AMP, ADP, and pyridoxal 5,-phosphate were also dephosphorylated, but to lower extents. Comparison of the features of NADPases I and II with those of other acid phosphatases possessing NADPase activity suggested that NADPases I and II are novel enzymes participating in regulation of the NAD+/NADP+ balance in the cytosol. (© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] CYP3A4 is a Human Microsomal Vitamin D 25-Hydroxylase,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 4 2004Ram P Gupta Abstract The human hepatic microsomal vitamin D 25-hydroxylase protein and gene have not been identified with certainty. Sixteen hepatic recombinant microsomal enzymes were screened for 25-hydroxylase activity; 11 had some 25-hydroxylase activity, but CYP3A4 had the highest activity. In characterized liver microsomes, 25-hydroxylase activity correlated significantly with CYP3A4 testosterone 6,-hydroxylase activity. Activity in pooled liver microsomes was inhibited by known inhibitors of CYP3A4 and by an antibody to CYP3A2. Thus, CYP3A4 is a hepatic microsomal vitamin D 25-hydroxylase. Introduction: Studies were performed to identify human microsomal vitamin D-25 hydroxylase. Materials and Methods: Sixteen major hepatic microsomal recombinant enzymes derived from cytochrome P450 cDNAs expressed in baculovirus-infected insect cells were screened for 25-hydroxylase activity with 1,-hydroxyvitamin D2 [1,(OH)D2], 1,-hydroxyvitamin D3 [1,(OH)D3], vitamin D2, and vitamin D3 as substrates. Activity was correlated with known biological activities of enzymes in a panel of 12 characterized human liver microsomes. The effects of known inhibitors and specific antibodies on activity also were determined. Results: CYP3A4, the most abundant cytochrome P450 enzyme in human liver and intestine, had 7-fold greater activity than that of any of the other enzymes with 1,(OH)D2 as substrate. CYP3A4 25-hydroxylase activity was four times higher with 1,(OH)D2 than with 1,(OH)D3 as substrate, was much less with vitamin D2, and was not detected with vitamin D3. 1,(OH)D2 was the substrate in subsequent experiments. In a panel of characterized human liver microsomes, 25-hydroxylase activity correlated with CYP3A4 testosterone 6,-hydroxylase activity (r = 0.93, p < 0.001) and CYP2C91 diclofenac 4,-hydroxylase activity (r = 0.65, p < 0.05), but not with activity of any of the other enzymes. Activity in recombinant CYP3A4 and pooled liver microsomes was dose-dependently inhibited by ketoconazole, troleandomycin, isoniazid, and ,-naphthoflavone, known inhibitors of CYP3A4. Activity in pooled liver microsomes was inhibited by antibodies to CYP3A2 that are known to inhibit CYP3A4 activity. Conclusion: CYP3A4 is a vitamin D 25-hydroxylase for vitamin D2 in human hepatic microsomes and hydroxylates both 1,(OH)D2 and 1,(OH)D3. [source] Identification and functional analysis of a human homologue of the monkey replication origin ors8JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2006Mario Callejo Abstract We previously isolated from African green monkey (CV-1) cells a replication origin, ors8, that is active at the onset of S-phase. Here, its homologous sequence (hors8, accession number: DQ230978) was amplified from human cells, using the monkey-ors8-specific primers. Sequence alignment between the monkey and the human fragment revealed a 92% identity. Nascent DNA abundance analysis, involving quantification by real-time PCR, indicated that hors8 is an active replication origin, as the abundance of nascent DNA from a genomic region containing it was 97-fold higher relative to a non-origin region in the same locus. Furthermore, the data showed that the hors8 fragment is capable of supporting the episomal replication of its plasmid, when cloned into pBlueScript (pBS), as assayed by the DpnI resistance assay after transfection of HeLa cells. A quantitative chromatin immunoprecipitation (ChIP) assay, using antibodies against Ku, Orc2, and Cdc6, showed that these DNA replication initiator proteins were associated in vivo with the human ors8 (hors8). Finally, nascent DNA abundance experiments from human cells synchronized at different phases of the cell cycle revealed that hors8 is a late-firing origin of DNA replication, having the highest activity 8 h after release from late G1. J. Cell. Biochem. 99: 1606,1615, 2006. © 2006 Wiley-Liss, Inc. [source] AN ESTEROLYTIC ACTIVITY FROM A WILD EDIBLE MUSHROOM, LYCOPERDON PERLATUMJOURNAL OF FOOD BIOCHEMISTRY, Issue 4 2009AHMET COLAK ABSTRACT Lycoperdon perlatum Pers. (Lycoperdaceae, Agaricales, Agaricomycetidae, Agaricomycetes, Basidiomycota, Fungi) was evaluated for its esterolytic potential. Native electrophoresis of the crude extracts showed four bands having Rf values of 0.34, 0.39, 0.52 and 0.59. The esterase showed the highest activity toward a short-chain substrate, p -nitrophenyl acetate. Optimum reaction conditions for L. perlatum crude extract were attained at pH 8.0 and 40C. Esterolytic activity of enzyme extract was stimulated in the presence of Mn2+, Fe2+, Ca2+ and Zn2+ in the reaction mixture. The enzyme activity was stimulated by incubation at pH 6.0 but retained 77% of its original activity at its optimum pH after 24 h. Thermal inactivation was displayed after incubation for 20 min at various temperatures above 30C. At 1 mM final concentration, 2-mercaptoethanol, dithiothreitol, ethylenediamine tetraacetic acid and p -methylphenyl sulfonylfluoride inhibited the esterolytic reaction. These results support that the crude L. perlatum extract possesses an esterolytic activity having properties similar to other esterases. PRACTICAL APPLICATIONS Esterases catalyzing the cleavage and formation of ester bonds are known ,/,-hydrolases (EC 3.1.1.X). Esterases are used for the synthesis of flavor esters for the food industry, modification of triglycerides for fat and oil industry and resolution of racemic mixtures used for the synthesis of fine chemicals for the pharmaceutical industry. Therefore, the search for new enzyme sources is important for the development of new enzymes and applications. [source] COMPARATIVE STUDIES ON PROTEOLYTIC ACTIVITY OF SPLENIC EXTRACT FROM THREE TUNA SPECIES COMMONLY USED IN THAILANDJOURNAL OF FOOD BIOCHEMISTRY, Issue 5 2004SUPPASITH KLOMKLAO ABSTRACT Proteolytic activities of splenic extract from three tuna species including skipjack tuna (Katsuwonus pelamis), yellowfin tuna (Thunnus albacores) and tongol tuna (Thunnus tonggol) were studied. Optimal activity of splenic extract from all tuna species was at pH 9.0 and 55C when casein was used as a substrate. Among all species tested, yellowfin tuna showed the highest activity, followed by skipjack tuna and tongol tuna. The proteolytic activity was strongly inhibited by soybean trypsin inhibitor, TLCK and partially inhibited by ethylenediaminetetraacetic acid. E-64, N-ethylmaleimide, iodoacetic acid, TPCK and pepstatin A showed no inhibition. The effect of NaCl and CaCl2 on proteolytic activity was also investigated. Activities continuously decreased as NaCl concentration increased, and no activity remained in the presence of 30% NaCl. On the other hand, activities increased as CaCl2 concentration increased. The highest activity was obtained in the presence of 1 mM CaCl2. SDS-substrate gel electrophoresis revealed that major proteinases in splenic extract from different tuna species were different in apparent molecular weights and sensitivity to TLCK. Although the major activity bands of all species were strongly inhibited by soybean trypsin inhibitor, varying sensitivity to TLCK probably implied the differences in binding characteristic of enzyme to substrate and/or inhibitors. The results suggest that major proteinases in spleen of all tuna species were trypsin-like serine proteinases. [source] Tuna Pepsin: Characteristics and Its Use for Collagen Extraction from the Skin of Threadfin Bream (Nemipterus spp.)JOURNAL OF FOOD SCIENCE, Issue 5 2008S. Nalinanon ABSTRACT:, Pepsin from the stomach of albacore tuna, skipjack tuna, and tongol tuna was characterized. Pepsin from all tuna species showed maximal activity at pH 2.0 and 50 °C when hemoglobin was used as a substrate. Among the stomach extract of all species tested, that of albacore tuna showed the highest activity (40.55 units/g tissue) (P < 0.05). Substrate-Native-PAGE revealed that pepsin from albacore tuna and tongol tuna consisted of 2 isoforms, whereas pepsin from skipjack tuna had only 1 form. The activity was completely inhibited by pepstatin A, while EDTA (ethylenediaminetetraacetic acid), SBTI (soybean trypsin inhibitor), and E-64 (1-(L -trans-epoxysuccinyl-leucylamino)-4-guanidinobutane) exhibited negligible effect. The activity was strongly inhibited by SDS (sodium dodecyl sulfate) (0.05% to 0.1%, w/v). Cysteine (5 to 50 mM) also showed an inhibitory effect in a concentration dependent manner. ATP, molybdate, NaCl, MgCl2, and CaCl2 had no impact on the activity. When tuna pepsin (10 units/g defatted skin) was used for collagen extraction from the skin of threadfin bream for 12 h, the yield of collagen increased by 1.84- to 2.32-fold and albacore pepsin showed the comparable extraction efficacy to porcine pepsin. The yield generally increased with increasing extraction time (P < 0.05). All collagen obtained with the aid of tuna pepsin showed similar protein patterns compared with those found in acid-solubilized collagen. Nevertheless, pepsin from skipjack tuna caused the degradation of , and , components. All collagens were classified as type I with large portion of ,-chain. However, proteins with molecular weight (MW) greater than 200 kDa were abundant in acid-solubilized collagen. [source] Changes in Radical-scavenging Activity and Components of Mulberry Fruit During MaturationJOURNAL OF FOOD SCIENCE, Issue 1 2006Tomoyuki Oki ABSTRACT Extracts of mulberry fruits (Morus sp.) were prepared from 8 cultivars harvested at 4 stages of maturity, and their radicalscavenging activity, anthocyanin content, and total phenolic content were measured. The radical-scavenging activity was evaluated by a spectrophotometric assay using the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) in a 96-well microplate. Mulberry fruit extracts exhibited the DPPH-scavenging activities, ranging from 2.5 to 20.3 ,mol-Trolox equiv/g-FW. Their activities were variable during maturation, and the highest activity was observed in the fully mature mulberry fruit in all cultivars. Anthocyanin was scarcely present in the immature mulberry fruits; however, its content increased as the fruit matured in all cultivars. On the other hand, all immature mulberry fruits contained non-anthocyanin phenolic compound. An on-line high-performance liquid chromatography (HPLC) method for the detection of DPPH-scavenging compounds revealed the difference in predominant radical scavengers between the immature and fully mature stages in the Miran 5 cultivar. Four major radical scavengers in the Miran 5 cultivar were assigned to 2 caffeoylquinic acids (chlorogenic acid and its isomer) and 2 anthocyanins (cyanidin 3-glucoside and cyanidin 3-rutinoside) in the immature and fully mature stages, respectively, by LC-ESI-MS/MS analysis. The change in the content of 4 compounds in mulberry fruits during maturation demonstrated that the most likely contributors to the DPPH-scavenging activity were caffeoylquinic acids in the immature mulberry and anthocyanins in the mature and fully mature mulberry. [source] Pathogenesis of Potato Gangrene Caused by Phoma exigua var. foveata: II.JOURNAL OF PHYTOPATHOLOGY, Issue 7 2004Activities of some Hydrolases, Dehydrogenases Abstract The location of enzyme activity in gangrene-diseased tubers was determined using the nitrocellulose blotting method. The activity of aminopeptidase and esterase was located in tissues adjacent to dry rot caused by Phoma exigua var. foveata and in other apparently healthy tissues. The activity of glucuronidase, succinic and glucose-6-phosphate dehydrogenases (G-6-PDH), however, was confined to tissues adjacent to the rotted tissue. The pathogen produces very active , - and , -glycosidases, so their highest activity occurred in rotten tissue that was filled with fungal mycelium. Results suggest that all these enzymes are involved in alteration of cell metabolism and the destruction of diseased tuber tissue. [source] Glycosidases in soils as affected by cropping systemsJOURNAL OF PLANT NUTRITION AND SOIL SCIENCE, Issue 6 2005Daniel E. Dodor Abstract Glycosidases are a group of soil enzymes that play a major role in degradation of carbohydrates. This study was conducted to assess the impact of crop rotation and N fertilization on the activities of ,- and ,-glucosidases and ,- and ,-galactosidases in plots of two long-term field experiments at the Clarion-Webster Research Center (CWRC) and Northeast Research Center (NERC) in Iowa. Surface-soil (0,15 cm) samples were taken in 1996 and 1997 in corn (Zeamays L.), soybean (Glycinemax (L.) Merr.), oats (Avenasativa L.), or meadow (alfalfa) (Medicago sativa L.) plots that received 0 or 180,kg N ha,1, applied as urea before corn, and an annual application of 20,kg P ha,1 and 56,kg K ha,1. Activities of the four glycosidases were significantly affected by crop rotations in both years at the two sites but not by nitrogen application. In general, higher activities were observed in plots under meadow or oat and the lowest in continuous corn (CWRC) and soybean (NERC). Four-year rotation showed the highest activity, followed by 2-year rotation and monocropping systems. Linear-regression analyses indicated that, in general, the activities of the glycosidases were significantly correlated with microbial-biomass C (r > 0.302, p , 0.05) and microbial-biomass N (r > 0.321, p , 0.05), organic-C (r > 0.332, p , 0.05) and organic-N (r > 0.399, p , 0.01) contents of the soils. Results of this work suggest that multicropping stimulated the activities of the glycosidases. The specific activities of the glycosidases in soils of the two sites studied, expressed as g p -nitrophenol released per,kg of organic C, differed among the four enzymes. The lowest values were obtained for ,-galactosidase and ,-glucosidase, followed by ,-galactosidase and ,-glucosidase. Glycosidasen in Böden unter dem Einfluss von Bewirtschaftungssystemen Glycosidasen stellen eine Gruppe von Bodenenzymen dar, welche eine entscheidende Rolle im Abbau von Kohlenhydraten spielen. Ziel dieser Untersuchungen war die Erfassung des Einflusses von Fruchtfolge und N-Düngung auf die Aktivitäten von ,- und ,-Glucosidasen und ,- und ,-Galactosidasen in zwei Langzeitfeldversuchen, dem Clarion-Webster-Versuchsfeld (CWRC) und dem Northeast-Versuchsfeld (NERC) in Iowa. In den Jahren 1996 und 1997 wurden Oberbodenproben (0,15 cm) von Parzellen unter Mais (Zeamays L.), Sojabohne (Glycine max (L.) Merr.), Hafer (Avena sativa L.) oder Luzerne (Medicago sativa L.) entnommen, welche vor Mais 0 oder 180,kg N ha,1 in Form von Harnstoff sowie jährliche Düngergaben in Höhe von 20,kg P ha,1 und 56,kg K ha,1 erhielten. Über zwei Jahre wurden die Aktivitäten der vier Glycosidasen in beiden Feldversuchen signifikant von der Bewirtschaftung beeinflusst, jedoch nicht von der N-Düngung. Im allgemeinen wurden höhere Enzymaktivitäten in Parzellen unter Luzerne oder Hafer festgestellt und die geringsten unter Maismonokultur (CWRC) bzw. Sojabohne (NERC). Vierjährige Fruchtfolgen zeigten die höchsten Aktivitäten, gefolgt von zweijährigen Fruchtfolgen und Monokulturen. Analysen mittels linearer Regression weisen auf eine Korrelation zwischen Glycosidaseaktivitäten und C (r > 0.332, p , 0.05) und N der mikrobiellen Biomasse (r > 0.321, p , 0.05) sowie den Gehalten an org. C (r > 0.332, p , 0.05) und N (r > 0.399, p , 0.01) hin. Die Ergebnisse dieser Untersuchungen deuten darauf hin, dass die Aktivitäten von Glycosidasen durch mehrjährige Fruchtfolgen stimuliert wurden. Die spezifischen Glycosidaseaktivitäten in den Böden der zwei Feldversuche, berechnet als freigesetztes p -Nitrophenol (g (kg org. C),1), variierten zwischen den vier Enzymen. Die geringsten Werte wurden für ,-Galactosidase und ,-Glucosidase festgestellt, gefolgt von ,-Galactosidase und ,-Glucosidase. [source] Purification and partial characterization of xyloglucan-hydrolyzing enzymes from watermelon placental tissueJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 4 2009Yasar Karakurt Abstract BACKGROUND: Watermelon (Citrullus lanatus (Thunb.) Matsum & Nakai) fruit matrix glycans are comprised largely of xyloglucans (XGs). As in other fruits, these polymers show significant molecular mass downshifts during ripening. In the present study, we describe the purification and characteristics of a number of xyloglucanases (XGases) from the placental tissue of ripe watermelon. XGases were extracted from watermelon fruit placental tissue and purified by sequential ion-exchange, gel-permeation and concanavalin A chromatography. RESULTS: Five XGases (P1S2, P2S2, P3S1, P3S2, P3S3) were recovered with molecular masses ranging from 30.5 to 77.5 kDa on SDS-PAGE. All XGases showed maximum activity at pH 5,5.5 and 35,40 °C against tamarind seed XG and were also active against XG-rich matrix glycans from watermelon fruit. The enzymes were strongly inhibited by mercury and hydrolyzed XG without generation of oligomers or monomers. P3S3 had the highest activity against XG. The purified enzymes were active toward carboxymethylcellulose, indicating that they were not XG specific. CONCLUSION: The pattern of molecular mass downshifts during XG hydrolysis by the purified XGases and the absence of monomeric and oligomeric products are consistent with endo-type catalysis for the XGases and with a role for these enzymes in the degradation of cell wall XG during ethylene-induced watersoaking of watermelon fruit placental tissues. Copyright © 2009 Society of Chemical Industry [source] Preparation and antioxidant activity of wheat gluten hydrolysates (WGHs) using ultrafiltration membranesJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 5 2008Xiangzhen Kong Abstract BACKGROUD: Many hydrolysates from animal and plant proteins have been found to possess physiological activities. Wheat gluten, an important by-product of the wheat starch industry, is produced worldwide in enormous quantities. In this study, wheat gluten hydrolysates (WGHs) were obtained by enzymatic hydrolysis and fractionated using ultrafiltration membranes. The antioxidant activities of the hydrolysates were investigated by various antioxidant assays, including the ability to inhibit the autoxidation of linoleic acid and the scavenging effect on free radicals. Amino acid composition and molecular weight distribution were also evaluated to determine their relationship with antioxidant activity. RESULTS: The pepsin hydrolysate (PeWGH) had the highest activity and was ultrafiltrated into three major types, PeWGH I (5,10 kDa), PeWGH II (3,5 kDa) and PeWGH III (<3 kDa). PeWGH III showed stronger inhibition of the autoxidation of linoleic acid and higher scavenging activity against 2,2-diphenyl-1-picrylhydrazyl, superoxide and hydroxyl free radicals. Furthermore, PeWGH III had the highest total hydrophobic amino acid content (45.11 g per 100 g protein), and its molecular weight distribution ranged from 1700 to 100 Da. CONCLUSION: The low molecular weight and amino acid composition of PeWGHs were found to be strongly correlated with their antioxidant activity. PeWGH could be used as a natural antioxidant in the pharmaceutical and food industries in the future. Copyright © 2008 Society of Chemical Industry [source] Polyphenol oxidase activity in grass and its effect on plant-mediated lipolysis and proteolysis of Dactylis glomerata (cocksfoot) in a simulated rumen environmentJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 10 2006Michael RF Lee Abstract Little is known about the level or activity of polyphenol oxidase (PPO) in grasses and its potential impact on proteolysis and lipolysis. Six grass species were initially screened for PPO activity (740.6, 291.9, 213.6, 119.0, 16.3 and 6.5 U g,1 fresh weight (FW) for cocksfoot, hybrid ryegrass, Italian ryegrass, perennial ryegrass, timothy and tall fescue respectively). Cocksfoot, which expressed the highest activity, was then used to determine the effect of PPO on plant-mediated proteolysis and lipolysis in a simulated rumen environment. Sourced cocksfoot was macerated and incubated in an antibiotic-containing anaerobic medium with or without ascorbate to deactivate PPO in the dark at 39 °C over five time points. At each time point (0, 1, 2, 6 and 24 h), six replicate samples were destructively harvested; three of the replicates were used for lipid analysis and the other three for protein, free amino acid and bound phenol determination. Characterisation of the herbage showed PPO activities of 649.6 and 0 U g,1 FW, which were reflected in the extent of phenol (derived from quinones) binding to protein after 24 h of incubation, namely 65.1 and 29.6 mg bound phenol g,1 protein (P < 0.001) for cocksfoot and cocksfoot + ascorbate respectively. Proteolysis, measured as free amino acids released into the incubation buffer, was significantly reduced (P < 0.001) with increasing PPO activity, with values after the 24 h incubation of 0.03 and 0.07 mmol L,1 g,1 FW for cocksfoot and cocksfoot + ascorbate respectively. Lipolysis, measured as the proportional decline in the membrane lipid polar fraction, was likewise reduced (P < 0.001) with increasing PPO activity, with values after the 24 h incubation of 0.43 and 0.65 for cocksfoot and cocksfoot + ascorbate respectively. Changes that occurred in protein and the lipid fractions (polar fraction, monoacylglycerol + diacylglycerol, triacylglycerol and free fatty acids) during the incubations are also reported and discussed. These results support the selection of forages high in PPO activity to reduce protein and lipid losses in silo and potentially in the rumen. Copyright © 2006 Society of Chemical Industry [source] Kinetic behaviour and stability of Escherichia coli ATCC27257 alkaline phosphatase immobilised in soil humatesJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 3 2003María C Pilar Abstract Alkaline phosphatase (EC 3.1.3.1) extracted from Escherichia coli ATCC27257 was immobilised by co-flocculation with soil humates in the presence of Ca2+. The effects of time, temperature, pH and concentration of enzyme and support on immobilisation were studied. Between 58 and 92% of the added phosphatase was strongly bound to the humates, depending on the conditions of immobilisation used. Some characteristics of the humate,phosphatase complexes and of the free enzyme were compared. The enzymatic complexes showed values of Km (2.22,mM) and activation energy (33.4,kJ,mol,1) similar to those of the free enzyme (2.00,mM and 27.6,kJ,mol,1). The pH/activity profiles revealed no change in terms of shape or optimum pH (10.5) upon immobilisation of alkaline phosphatase. However, the immobilised enzyme showed maximal activity in the range of 80,100,°C, while the free enzyme had its highest activity at 60,°C. The thermal stability of alkaline phosphatase was enhanced by complexation to the soil humates. © 2003 Society of Chemical Industry [source] | |||||||||||||||||||||||||||||||