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Good Substrates (good + substrate)
Selected AbstractsPurification and molecular characterization of subtilisin-like alkaline protease BPP-A from Bacillus pumilus strain MS-1LETTERS IN APPLIED MICROBIOLOGY, Issue 3 2006T. Miyaji Abstract Aims:, The present study was conducted by screening zein-degrading bacteria in an attempt to obtain zein-degrading protease. Methods and Results:, Soil bacteria were screened by formation of a clear zone on zein plates. Characterization of a zein-degrading bacterium indicated a taxonomic affiliation to Bacillus pumilus, and was named MS-1 strain. The strain produced two different types of extracellular proteases, BPP-A and BPP-B. In this study, we purified and characterized BPP-A because it exhibited a higher ability to hydrolyze zein than BPP-B. When casein was used as the substrate, the optimal pH for BPP-A was 11·0. In BPP-A, zein was better substrate than casein at pH 13·0, whereas casein was better one than zein at pH 11·0. The bppA gene encoded a 383-amino acid pre-pro form of BPP-A, and mature BPP-A contained 275 amino acid residues. It was concluded that BPP-A belonged to the subtilisin family. Conclusion:, A zein-degrading bacterium assigned to B. pumilus produced two different types of extracellular proteases, BPP-A and BPP-B. BPP-A exhibited an ability to hydrolyze zein in an extreme alkaline condition. Significance and Impact of the Study:, This is a first report on screening for zein-degrading micro-organisms. The subtilisin-like protease BPP-A is possible to utilize as an industrial enzyme for the production of zein hydrolysates. [source] The structure of human deoxycytidine kinase in complex with clofarabine reveals key interactions for prodrug activationACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2006Yan Zhang Clofarabine [2-chloro-9-(2-deoxy-2-fluoro-,- d -arabinofuranosyl)-9H -purin-6-amine] is a hybrid of the widely used anticancer drugs cladribine and fludarabine. It is the precursor of an effective chemotherapeutic agent for leukemias and other hematological malignancies and received accelerated approval by the FDA for the treatment of pediatric patients with relapsed or refractory acute lymphoblastic leukemia. Clofarabine is phosphorylated intracellularly by human deoxycytidine kinase (dCK) to the 5,-monophosphate, which is the rate-limiting step in activation of the prodrug. dCK has a broad substrate specificity, with a much higher activity to deoxycytidine than to deoxyadenosine and deoxyguanosine. As a purine-nucleoside analog, clofarabine is a better substrate of dCK than deoxycytidine. The crystal structure of dCK has been solved previously in complex with pyrimidine nucleosides and ADP [Sabini et al. (2003), Nature Struct. Biol.10, 513,519]. In the current study, the crystal structure of clofarabine- and ADP-bound dCK was solved to 2.55,Å by molecular replacement. It appears that the enzyme takes the same conformation as in the structures of the pyrimidine nucleoside-bound complexes. The interactions between 2-Cl and its surrounding hydrophobic residues contribute to the high catalytic efficiency of dCK for clofarabine. [source] Adhesion pattern and growth of primary human osteoblastic cells on five commercially available titanium surfacesCLINICAL ORAL IMPLANTS RESEARCH, Issue 7 2010Giovanni Passeri Abstract Objective: The aim of this study is to analyze the morphology and proliferation of human osteoblastic cells in vitro on five commercially available titanium surfaces. Materials and methods: Human primary cells of the osteoblastic lineage were obtained from bone explants. The cells were plated on polished (T1), machined (T2), sand-blasted/acid-etched (T3), sand-blasted/acid-etched, modified with hydrogen peroxide rinse (T4), and plasma-sprayed titanium (T5) disks. Cell morphology was studied after 6, 24, 72 h, 7 and 14 days of culture by scanning electron microscopy. The formation and distribution of focal adhesions was investigated by immunocytochemical staining at 3, 6 and 24 h. Cell growth was measured by an MTT assay after 3, 7 and 9 days of culture. Moreover, the production of osteocalcin and osteoprotegerin (OPG) was evaluated in the supernatants by ELISA. Results: Morphological analysis revealed that substrate topography profoundly affected cells' shape and their anchoring structures. Large lamellipodia were formed on polished and machined surfaces, while thin filopodia were more frequently observed on T3 and T4 samples. Moreover, cells formed stronger focal adhesions on T3 and T4 surfaces, and cell proliferation was higher on rough surfaces. Osteocalcin production was higher on the T4 surface, whereas OPG steadily increased on every surface. Conclusions: Taken together, these data show that all the surfaces allowed cell attachment, adhesion and proliferation, but T4 and T5 surfaces appeared to be a better substrate for the adhesion, proliferation and differentiation of cells of the osteoblastic lineage. To cite this article: Passeri G, Cacchioli A, Ravanetti F, Galli C, Elezi E, Macaluso GM. Adhesion pattern and growth of primary human osteoblastic cells on five commercially available titanium surfaces. Clin. Oral Impl. Res. 21, 2010; 756,765. doi: 10.1111/j.1600-0501.2009.01906.x [source] Deciphering interactions of the aminoglycoside phosphotransferase(3,)-IIIa with its ligandsBIOPOLYMERS, Issue 9 2009Lingzhi Wu Abstract Aminoglycoside phosphotransferase(3,)-IIIa (APH) is the enzyme with broadest substrate range among the phosphotransferases that cause resistance to aminoglycoside antibiotics. In this study, the thermodynamic characterization of interactions of APH with its ligands are done by determining dissociation constants of enzyme,substrate complexes using electron paramagnetic resonance and fluorescence spectroscopy. Metal binding studies showed that three divalent cations bind to the apo-enzyme with low affinity. In the presence of AMPPCP, binding of the divalent cations occurs with 7-to-37-fold higher affinity to three additional sites dependent on the presence and absence of different aminoglycosides. Surprisingly, when both ligands, AMPPCP and aminoglycoside, are present, the number of high affinity metal binding sites is reduced to two with a 2-fold increase in binding affinity. The presence of divalent cations, with or without aminoglycoside present, shows only a small effect (<3-fold) on binding affinity of the nucleotide to the enzyme. The presence of metal,nucleotide, but not nucleotide alone, increases the binding affinity of aminoglycosides to APH. Replacement of magnesium (II) with manganese (II) lowered the catalytic rates significantly while affecting the substrate selectivity of the enzyme such that the aminoglycosides with 2,-NH2 become better substrates (higher Vmax) than those with 2,-OH. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 801,809, 2009. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Soyprotein fibers with high strength and water stability for potential medical applicationsBIOTECHNOLOGY PROGRESS, Issue 6 2009Narendra Reddy Abstract Fibers with mechanical properties and water stability suitable for tissue engineering have been developed from soyproteins. Proteins are biocompatible and biodegradable and are preferred over synthetic polymers for medical applications. Although plant proteins are abundant and inexpensive and can be made into various types of scaffolds, very few attempts have been made to understand the suitability of using plant proteins for medical applications, especially as fibrous substrates for tissue engineering. So far, it has not been able to obtain good quality soyprotein fibers without using toxic crosslinking agents or blending soyprotein with synthetic polymers. In this research, we have developed 100% soyprotein fibers with good strength and water stability without using any external crosslinking agents. The soyprotein fibers have better wet strength than collagen fibers and are conducive to the attachment, growth, and proliferation of mouse fibroblasts. Fibers are better substrates than films for growth and orientation of cells and are therefore preferable for tissue engineering applications. Soyprotein fibers show good potential to be novel biomaterials with properties suitable for tissue engineering and other medical applications. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Amplification of low quantity bacterial RNA for microarray studies: time-course analysis of Leptospirillum ferrooxidans under nitrogen-fixing conditions,ENVIRONMENTAL MICROBIOLOGY, Issue 6 2006Mercedes Moreno-Paz Summary We have developed a method for the amplification of low quantity total bacterial RNA for DNA microarrays analysis. Current methods are based on the linear amplification by the in vitro transcription from the T7 promoter, similar to that used for eukaryotic mRNA amplification. For the incorporation of T7 promoter, the prokaryotic RNA must be enzymatically modified for the incorporation of a polyA tail at the 3, end to emulate the eukaryotic mRNA. The method we describe and validate herein avoids this step by the direct and random incorporation of the T7 promoter. From 500 ng of total bacterial RNA, we obtained 130,150 µg of antisense RNA, such products being good substrate for fluorescent labelling and DNA microarray analysis. The method was validated with bacterial samples from which it is very difficult to obtain sufficient amounts and quality of total RNA for global gene expression analysis. This is critical for low cell density growing microorganisms, environmental samples, or many extremophiles where the composition of the cultural media severely affects the RNA yield, like in the case of the acidophile and iron oxidizer Gram-negative bacterium Leptospirillum ferrooxidans. We further validated our amplification method in parallel experiments with non-amplified RNA by following the expression of the L. ferrooxidans nif regulon along the time-course of growth. [source] Study of in vitro glucuronidation of hydroxyquinolines with bovine liver microsomesFUNDAMENTAL & CLINICAL PHARMACOLOGY, Issue 6 2002Masanobu Kanou Abstract Glucuronidation of drugs by UDP-glucuronosyltransferase (UGT) is a major phase II conjugation reaction. Defects in UGT are associated with Crigler,Najjar syndrome and Gilbert's syndrome with severe hyperbilirubinaemias and jaundice. We analysed the reactivities of some hydroxyquinoline derivatives, which are naturally produced from quinoline by cytochrome P450. The analyses were carried out using a microassay system for UGT activity in bovine liver microsomes in the range 0.5,100 pmol/assay with the highly sensitive radio-image analyser Fuji BAS2500 (Fujifilm, Tokyo, Japan). 3-Hydroxylquinoline is a good substrate for glucuronidation, and the relative Kcat values were 3.1-fold higher than the values for p-nitrophenol. 5,6-Dihydroquinoline-5,6- trans -diol gave a similar Km value to that of 3-hydroxyquinoline, but the Vmax value was approximately 1/15 of that of p-nitrophenol and showed weak reactivity. Quinoline N-oxide gave a low Vmax value and showed marginal activity. The Kcat values of 6-hydroxyquinoline and 5-hydroxyquinoline were 2.1- and 1.2-fold higher than that of p-nitrophenol, respectively. Fluoroquinoline (FQ) derivatives, such as 3FQ, 7,8diFQ and 6,7,8triFQ, did not show any substrate activities. These results suggest that there are therapeutic problems in administration of some quinoline drugs to patients with jaundice. [source] Structural insight of human DEAD-box protein rck/p54 into its substrate recognition with conformational changesGENES TO CELLS, Issue 4 2006Tsutomu Matsui Human rck/p54, a product of the gene cloned at the breakpoint of t(11; 14) (q23;q32) chromosomal translocation on 11q23 in B-cell lymphoma, is a member of the DEAD-box RNA helicase family. Here, the crystal structure of Nc-rck/p54, the N-terminal core domain of rck/p54, revealed that the P-loop in motif I formed a closed conformation, which was induced by Asn131, a residue unique to the RCK subfamily. It appears that ATP does not bind to the P-loop. The results of dynamic light scattering revealed to ATP-induced conformational change of rck/p54. It was demonstrated that free rck/p54 is a distended molecule in solution, and that the approach between N-terminal core and C-terminal domains for ATP binding would be essential when unwinding RNA. The results from helicase assay using electron micrograph, ATP hydrolytic and luciferase assay showed that c-myc IRES RNA, whose secondary structure regulates IRES-dependant translation, was unwound by rck/p54 and indicated that it is a good substrate for rck/p54. Over-expression of rck/p54 in HeLa cells caused growth inhibition and cell cycle arrest at G2/M with down-regulation of c-myc expression. These findings altogether suggest that rck/p54 may affect the IRES-dependent translation of c-myc even in the cells. [source] Steric and Electronic Effects on an Antibody-Catalyzed Diels,Alder ReactionHELVETICA CHIMICA ACTA, Issue 12 2002Yael Gozin A series of substituted thiophene dioxides was tested as diene substrates for the antibody 1E9, which was elicited with a hexachloronorbornene derivative and normally catalyzes the inverse electron-demand Diels,Alder reaction between 2,3,4,5-tetrachlorothiophene dioxide (TCTD) and N -ethylmaleimide (NEM). Previous structural and computational studies had suggested that the catalytic efficiency of this system derives in part from a very snug fit between the apolar active site and the transition state of this reaction. Nevertheless, replacing all the Cl-atoms in the hapten with Br-atoms leads to no loss in affinity (Kd=0.1,nM), indicating substantial conformational flexibility in the residues that line the binding pocket. Consistent with this observation, the 2,3,4,5-tetrabromothiophene dioxide is a good substrate for the antibody (kcat=1.8,min,1, KNEM=14,,M), despite being considerably larger than TCTD. In contrast, normal electron-demand Diels,Alder reactions between NEM and unsubstituted thiophene dioxide or 2,3,4,5-tetramethylthiophene dioxide, which are much smaller or nearly isosteric with TCTD, respectively, are not detectably accelerated. These results show that the electronic properties of the 1E9 active site are optimized to a remarkable degree for the inverse electron-demand Diels,Alder reaction for which it was designed. Indeed, they appear to play a more important role in catalysis than simple proximity effects. [source] Ruthenium-catalyzed carbonylative cycloaddition reactions involving carbonyl and imino groups as assembling unitsTHE CHEMICAL RECORD, Issue 4 2008Naoto Chatani Abstract This paper describes carbonylative cycloaddition reactions catalyzed by Ru3(CO)12. Ru3(CO)12 was found to catalyze an intramolecular Pauson,Khand-type reaction. Carbonylative cycloaddition reactions involving a carbonyl group in aldehydes, ketones, and esters as a two-atom assembling unit were also achieved in the presence of Ru3(CO)12 as the catalyst. The reaction of 5-hexyn-1-al and 6-heptyn-1-al derivatives with CO in the presence of Ru3(CO)12 resulted in cyclocarbonylation from which bicyclic ,, ,-unsaturated lactones were obtained. Intermolecular [2,+,2,+,1] carbonylative cycloaddition of alkenes, ketones, and CO was also catalyzed by Ru3(CO)12 as the catalyst to give saturated ,-lactone derivatives. Simple ketones were not applicable, but ketones having a CO or CN group at the ,-position served as a good substrate. These reactions could be extended to carbonylative cycloaddition of the corresponding imines leading to ,-butyrolactam derivatives. The [4,+,1] carbonylative addition of ,,,-unsaturated imines leading to unsaturated ,-lactams was achieved with Ru3(CO)12. © 2008 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 8: 201,212; 2008: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.20149 [source] Regulation of the epididymal receptor tyrosine kinase ros by the protein tyrosine phosphatase SHP-1AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 3 2002Rico Pusch The SH2 domain PTP SHP-1 was recently identified as a potent negative regulator of the orphan receptor tyrosine kinase Ros, an important regulator of epidimys differentiation (Keilhack et al. J Cell Biol 2001; 152:325,334). Phosphorylated Ros strongly and directly associates with SHP-1 in yeast-two-hybrid, GST pull-down, and coimmunoprecipitation experiments. Catalytically inactive SHP-1C455S exhibits greatly elevated binding to phosphorylated Ros. Direct Ros,SHP-1 interaction is mediated by the SHP-1 N-terminal SH2 domain and Ros phosphotyrosine 2267. Overexpression of SHP-1 results in Ros dephosphorylation and effectively down-regulates Ros-dependent proliferation and transformation. Elevated phosphorylation of Ros in ,viable motheaten (me-v)' mice which, have strongly reduced SHP-1 activity, suggests that Ros signaling is under control of SHP-1 in vivo. Thus sterility of male me-v mice seems to be related to dysregulation of Ros. A synthetic phosphopeptide derived from the Ros sequence around Y2267 potently activates recombinant SHP-1 in vitro but is not a good substrate for SHP-1. In contrast, phosphorylation sites in the activation loop of Ros are effectively dephosphorylated. Based on these observations we propose a mechanistic model of Ros,SHP-1 interaction. Using fusion proteins of SHP-1 variants and of Ros with GFP-proteins of different spectral characteristics the interaction of Ros and SHP-1 can be visualized in intact cells by different microscopic techniques. [source] Interaction of the Catalytic Domain of Inositol 1,4,5-Trisphosphate 3-Kinase A with Inositol Phosphate AnaloguesCHEMBIOCHEM, Issue 8 2005Alexandra Poinas Dr. Abstract The levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in the cytoplasm are tightly regulated by two enzymes, Ins(1,4,5)P3 3-kinase and type I Ins(1,4,5)P3 5-phosphatase. The catalytic domain of Ins(1,4,5)P3 3-kinase (isoenzymes A, B and C) is restricted to approximately 275 amino acids at the C-terminal end. We were interested in understanding the catalytic mechanism of this key family of enzymes in order to exploit this in inhibitor design. We expressed the catalytic domain of rat Ins(1,4,5)P3 3-kinase A in Escherichia coli as a His- and S-tagged fusion protein. The purified enzyme was used in an Ins(1,4,5)P3 kinase assay to phosphorylate a series of inositol phosphate analogues with three or four phosphate groups. A synthetic route to D -2-deoxy-Ins(1,4,5)P3 was devised. D -2-Deoxy-Ins(1,4,5)P3 and D -3-deoxy-Ins(1,4,6)P3 were potent inhibitors of the enzyme, with IC50 values in the micromolar range. Amongst all analogues tested, only D -2-deoxy-Ins(1,4,5)P3 appears to be a good substrate of the Ins(1,4,5)P3 3-kinase. Therefore, the axial 2-hydroxy group of Ins(1,4,5)P3 is not involved in recognition of the substrate nor does it participate in the phosphorylation mechanism of Ins(1,4,5)P3. In contrast, the equatorial 3-hydroxy function must be present in that configuration for phosphorylation to occur. Our data indicate the importance of the 3-hydroxy function in the mechanism of inositol trisphosphate phosphorylation rather than in substrate binding. [source] Hepatic microsomal cytochrome P450 enzyme activity in relation to in vitro metabolism/inhibition of polychlorinated biphenyls and testosterone in Baltic grey seal (Halichoerus grypus)ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2003Hongxia Li Abstract Among other factors, cytochrome P450 (CYP) enzyme activity determines polychlorinated biphenyl (PCB) bioaccu-mulation, biotransformation, and toxicity in exposed species. We measured the oxidative metabolism in vitro of 12 PCB congeners, representing structural groups based on the number and position of the chlorine atoms, by the hepatic microsomes of one Baltic grey seal (Halichoerus grypus). Microsomal metabolism was observed for several PCBs with vicinal H atoms exclusively in the ortho and meta positions and without any ortho -Cl substituents (CB-15 [4,4,-Cl2] and CB-77 [3,3,,4,4,-Cl4]), vicinal meta and para -H atoms (CB-52 [2,2,,5,5,-Cl4], and ,101 [2,2,,4,5,5,-Cl5]) or with both characteristics in combination with either only one ortho -Cl (CB-26 [2,3,,5-Cl3], CB-31 [2,4,,5-Cl3]) or two ortho -Cl substituents (CB-44 [2,2,,3,5,-Cl4]). To allocate PCB biotransformation to specific CYPs, the inhibitive effect of compounds with known CYP-specific inhibition properties was assessed on in vitro PCB metabolism and on regio- and stereospecific testosterone hydroxylase activities. Metabolic inhibition was considered relevant at concentrations ,1.0 ,M because these inhibitors became decreasingly selective at higher concentrations. At <1.0 ,M, ellipticine (CYP1A1/2 inhibitor) selectively inhibited CB-15, ,26, ,31, and ,77 metabolism, with no significant inhibition of CB-44, ,52, and ,101 metabolism. Inhibition of CB-52 and ,101 metabolism by chloramphenicol (CYP2B inhibitor) started at 1.0 ,M and maximized at about 100% at 10 ,M. Ketoconazole (CYP3A inhibitor) appeared to selectively inhibit CB-26, ,31, and ,44 metabolism relative to CB-15, ,77, and ,52 at concentrations ,1.0 ,M. Major testosterone metabolites formed in vitro were 2,-(CYP3A), 6,- (CYP3A, CYP1A), and 16,- (CYP2B) hydroxytestosterone and androstenedione (CYP2B, CYP2C11). The CYP forms indicated are associated with the specific metabolism of testosterone in laboratory animals. Inhibition of 2,- and 6,-hydroxytestosterone formation at ellipticine and ketoconazole concentrations ,1.0,M suggested that both inhibitors were good substrates of CYP3A-like enzymes in grey seal. Chloramphenicol (model for CYP2B) is apparently not a good inhibitor of CYP1A and CYP3A activities in grey seal because the chemical did not inhibit any metabolic route of testosterone at concentrations from 0.1 to 10 ,M. Our findings demonstrated that at least CYP1A- and CYP3A-like enzymes in the liver of grey seals are capable of metabolizing PCBs with ortho - meta and/or meta - para vicinal hydrogens. A CYP2B form might also be involved, but this could not be proven by the results of our experiments. Defining the profiles of CYP enzymes that are responsible for PCB biotransformation is necessary to fully understand the bioaccumulation, toxicokinetics, and risk of PCB exposure in seals and other free-ranging marine mammals. [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] Novel diadenosine polyphosphate analogs with oxymethylene bridges replacing oxygen in the polyphosphate chainFEBS JOURNAL, Issue 6 2009Potential substrates and/or inhibitors of Ap4A hydrolases Dinucleoside polyphosphates (NpnN,s; where N and N, are nucleosides and n = 3,6 phosphate residues) are naturally occurring compounds that may act as signaling molecules. One of the most successful approaches to understand their biological functions has been through the use of NpnN, analogs. Here, we present the results of studies using novel diadenosine polyphosphate analogs, with an oxymethylene group replacing one or two bridging oxygen(s) in the polyphosphate chain. These have been tested as potential substrates and/or inhibitors of the symmetrically acting Ap4A hydrolase [bis(5,-nucleosyl)-tetraphosphatase (symmetrical); EC 3.6.1.41] from E. coli and of two asymmetrically acting Ap4A hydrolases [bis(5,-nucleosyl)-tetraphosphatase (asymmetrical); EC 3.6.1.17] from humans and narrow-leaved lupin. The six chemically synthesized analogs were: ApCH2OpOCH2pA (1), ApOCH2pCH2OpA (2), ApOpCH2OpOpA (3), ApCH2OpOpOCH2pA (4), ApOCH2pOpCH2OpA (5) and ApOpOCH2pCH2OpOpA (6). The eukaryotic asymmetrical Ap4A hydrolases degrade two compounds, 3 and 5, as anticipated in their design. Analog 3 was cleaved to AMP (pA) and ,,,-methyleneoxy-ATP (pOCH2pOpA), whereas hydrolysis of analog 5 gave two molecules of ,,,-oxymethylene ADP (pCH2OpA). The relative rates of hydrolysis of these analogs were estimated. Some of the novel nucleotides were moderately good inhibitors of the asymmetrical hydrolases, having Ki values within the range of the Km for Ap4A. By contrast, none of the six analogs were good substrates or inhibitors of the bacterial symmetrical Ap4A hydrolase. [source] Development of recombinant inhibitors specific to human kallikrein 2 using phage-display selected substratesFEBS JOURNAL, Issue 3 2004Sylvain M. Cloutier The reactive site loop of serpins undoubtedly defines in part their ability to inhibit a particular enzyme. Exchanges in the reactive loop of serpins might reassign the targets and modify the serpin,protease interaction kinetics. Based on this concept, we have developed a procedure to change the specificity of known serpins. First, reactive loops are very good substrates for the target enzymes. Therefore, we have used the phage-display technology to select from a pentapeptide phage library the best substrates for the human prostate kallikrein hK2 [Cloutier, S.M., Chagas, J.R., Mach, J.P., Gygi, C.M., Leisinger, H.J. & Deperthes, D. (2002) Eur. J. Biochem. 269, 2747,2754]. Selected substrates were then transplanted into the reactive site loop of ,1-antichymotrypsin to generate new variants of this serpin, able to inhibit the serine protease. Thus, we have developed some highly specific ,1-antichymotrypsin variants toward human kallikrein 2 which also show high reactivity. These inhibitors might be useful to help elucidate the importance of hK2 in prostate cancer progression. [source] Escherichia coli tRNase Z can shut down growth probably by removing amino acids from aminoacyl-tRNAsGENES TO CELLS, Issue 11 2008Hiroaki Takaku In most organisms, tRNase Z is considered to be essential for 3, processing of tRNA molecules. The Escherichia coli tRNase Z gene, however, appears to be dispensable under normal growth conditions, and its existence remained an enigma. Here we intensively examined various (pre-)tRNAs for good substrates of E. coli tRNase Z in vitro, and found that the enzyme can remove the 3, terminal CCA residues from mature tRNAs regardless of their nucleotide modifications. Furthermore, we discovered that E. coli tRNase Z, when sufficiently expressed in the cell, can shut down growth probably by removing amino acids from aminoacyl-tRNAs. We confirmed in vitro that E. coli tRNase Z exceptionally possesses the activity that cleaves off the 3, terminal residues charging an amino acid from an aminoacyl-tRNA molecule. The current data suggest that tRNase Z might help modulate a cell growth rate by repressing translation under some stressful conditions. [source] Fluorescent, internally quenched, peptides for exploring the pH-dependent substrate specificity of cathepsin BJOURNAL OF PEPTIDE SCIENCE, Issue 7 2006Paolo Ruzza Abstract Cathepsin B is a cysteine protease that in tumor tissues is localized in both acidic lysosomes and extracellular spaces. It can catalyze the cleavage of peptide bonds by two mechanisms: endoproteolytic attack with a pH optimum around 7.4, and attack from the C -terminus with a pH optimum at 4.5,5.5. In this work, seven fluorescent, internally quenched, decapeptides have been synthesized using the prototypical cathepsin B selective substrate Z-Phe-Arg-AMC as a lead, and used to identify the structural factors determining the susceptibility of peptides to hydrolysis at acidic and neutral pH values. Each peptide differs from the others in one amino acid (residue 6) and contains a highly fluorescent Nma group linked to the ,-amino function of the N -terminal Orn residue and a Dnp group linked to the side chain of the Lys8 residue acting as a quencher. Proteolytic cleavage was monitored by measuring the increase of fluorescence at 440 nm upon excitation at 340 nm, and the cleavage sites were determined by HPLC followed by ESI-MS analysis. Peptides containing Ala or Phe at position 6 are good substrates for the enzyme at both pH 5.0 and 7.4. By contrast, those containing Glu, Asp, Lys or Val are not cleaved at all by cathepsin B at pH 7.4, and are poorly hydrolyzed at pH 5.0. These findings provide new information for the rational design of cathepsin B-activated peptide-containing anticancer drugs. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd. [source] Identification of gas-producing components in different varieties of Phaseolus vulgaris by in vitro fermentationJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 6 2001M Granito Abstract Phaseolus vulgaris consumption has been limited as part of the occidental diet owing to flatulence production. Raffinose, stachyose and verbascose have been shown to be the main components responsible for flatulence; however, it is thought that soluble fibre could also be involved in this phenomenon. The aim of the present study was to identify the main components of beans influencing flatus. Ten varieties of P vulgaris originating from South America were first analysed for their main nutrient and carbohydrate fractions. Three of the varieties were then fractionated to extract soluble and insoluble fibres. Various combinations of ,-galactosides and soluble and insoluble fibre fractions, in similar proportions to those contained in cooked grains, were used as substrates for in vitro fermentation studies using human faecal inoculum to determine the fermentative capacity of each of the three fractions. Considering the white varieties, total gas production and acidification of the medium were correlated with fermented organic matter from soluble fibre (R2,=,1)) and with ,-galactosides (R2,=,0.75). On the other hand, tannins present in pigmented varieties did not seem to interfere significantly in fermentation of soluble fibre. The total production of gas per gram of mixed fractions of soluble fibre and ,-galactosides in proportions found in cooked grains was lower than that expected from each substrate separately. It can be concluded that soluble fibre and ,-galactosides are good substrates for endogenous colonic flora subjected to in vitro fermentation studies and are thus responsible for flatulence induced by legume consumption. © 2001 Society of Chemical Industry [source] Antitumor activity of sugar-modified cytosine nucleosidesCANCER SCIENCE, Issue 2 2004Akira Matsuda Nucleoside analogues which show antimetabolic activity in cells have been successfully used in the treatment of various tumors. Nucleosides such as 1-,-D-arabinofuranosylcytosine (araC), 6-mercaptopurine, fludarabine and cladribine play an important role in the treatment of leukemias, while gemcitabine, 5-fluorouracil and its prodrugs are used extensively in the treatment of many types of solid tumors. All of these compounds are metabolized similarly to endogenous nucleosides and nucleotides. Active metabolites interfere with the de novo synthesis of nucleosides and nucleotides or inhibit the DNA chain elongation after being incorporated into the DNA strand as terminators. Furthermore, nucleoside antimetabolites incorporated into the DNA strand induce strand-breaks and finally cause apoptosis. Nucleoside antimetabolites target one or more specific enzyme(s). The mode of inhibitory action on the target enzyme is not always similar even among nucleoside antimetabolites which have the same nucleoside base, such as araC and gemcitabine. Although both nucleosides are phosphorylated by deoxycytidine kinase and are also good substrates of cytidine deaminase, only gemcitabine shows antitumor activity against solid tumors. This suggests that differences in the pharmacological activity of these nucleoside antimetabolites may reflect different modes of action on target molecules. The design, in vitro cytotoxicity, in vivo antitumor activity, metabolism and mechanism of action of sugar-modified cytosine nucleosides, such as (2,S)-2,-deoxy-2,-C-methylcytidine (SMDC), 1-(2-deoxy-2-methylene-,-D-erythro-pentofuranosyl)cytosine (DMDC), 1-(2-C-cyano-2-deoxy-1-,-D-arabino-pentofuranosyl)cytosine (CNDAC) and 1-(3-C-ethynyl-,-D-ribo-pentofuranosyl)cytosine (ECyd), developed by our groups, are discussed here. [source] The Interaction of Heteroaryl-Acrylates and Alanines with Phenylalanine Ammonia-Lyase from ParsleyCHEMISTRY - A EUROPEAN JOURNAL, Issue 10 2006Csaba Paizs Dr. Abstract Acrylic acids and alanines substituted with heteroaryl groups at the ,-position were synthesized and spectroscopically characterized (UV, HRMS, 1H NMR, and 13C NMR spectroscopy). The heteroaryl groups were furanyl, thiophenyl, benzofuranyl, and benzothiophenyl and contained the alanyl side chains either at the 2- or 3-positions. While the former are good substrates for phenylalanine ammonia-lyase (PAL), the latter compounds are inhibitors. Exceptions are thiophen-3-yl-alanine, a moderate substrate and furan-3-yl-alanine, which is inert. Possible reasons for these exceptions are discussed. Starting from racemic heteroaryl-2-alanines their D -enantiomers were prepared by using a stereodestructive procedure. From the heteroaryl-2-acrylates, the L -enantiomers of the heteroaryl-2-alanines were prepared at high ammonia concentration. These results can be best explained by a Friedel,Crafts-type electrophilic attack at the aromatic part of the substrates as the initial step of the PAL reaction. [source] | ||||||||||||||||||||||||||||