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Sample records for carboxypeptidases

  1. Catalysis of carboxypeptidase A

    DEFF Research Database (Denmark)

    Wu, Shanshan; Zhang, Chunchun; Xu, Dingguo;

    2010-01-01

    The catalytic mechanism of carboxypeptidase A (CPA) for the hydrolysis of ester substrates is investigated using hybrid quantum mechanical/molecular mechanical (QM/MM) methods and high-level density functional theory. The prevailing mechanism was found to utilize an active-site water molecule ass...... here and in our earlier publication, a unified model is proposed to account for nearly all experimental observations concerning the catalysis of CPA....

  2. Detection and quantitation of glutamate carboxypeptidase II in human blood

    Czech Academy of Sciences Publication Activity Database

    Knedlík, Tomáš; Navrátil, Václav; Vik, V.; Pacík, D.; Šácha, Pavel; Konvalinka, Jan

    2014-01-01

    Roč. 74, č. 7 (2014), s. 768-780. ISSN 0270-4137 R&D Projects: GA ČR GAP304/12/0847 Grant ostatní: OPPC(CZ) CZ.2.16/3.1.00/24016 Institutional support: RVO:61388963 Keywords : serum marker * glutamate carboxypeptidase II * plasma glutamate carboxypeptidase * prostate cancer * prostate -specific membrane antigen Subject RIV: CE - Biochemistry Impact factor: 3.565, year: 2014

  3. Genome-Wide Identification and Characterization of Carboxypeptidase Genes in Silkworm (Bombyx mori)

    Science.gov (United States)

    Ye, Junhong; Li, Yi; Liu, Hua-Wei; Li, Jifu; Dong, Zhaoming; Xia, Qingyou; Zhao, Ping

    2016-01-01

    The silkworm (Bombyx mori) is an economically-important insect that can secrete silk. Carboxypeptidases have been found in various metazoan species and play important roles in physiological and biochemical reactions. Here, we analyzed the silkworm genome database and characterized 48 carboxypeptidases, including 34 metal carboxypeptidases (BmMCP1–BmMCP34) and 14 serine carboxypeptidases (BmSCP1–BmSCP14), to better understand their diverse functions. Compared to other insects, our results indicated that carboxypeptidases from silkworm have more family members. These silkworm carboxypeptidases could be divided into four families: Peptidase_M2 carboxypeptidases, Peptidase_M14 carboxypeptidases, Peptidase_S10 carboxypeptidases and Peptidase_S28 carboxypeptidases. Microarray analysis showed that the carboxypeptidases had distinct expression patterns, whereas quantitative real-time PCR demonstrated that the expression level of 13 carboxypeptidases significantly decreased after starvation and restored after re-feeding. Overall, our study provides new insights into the functional and evolutionary features of silkworm carboxypeptidases. PMID:27483237

  4. Isolation and characterization of carboxypeptidase Ⅲ from germinating triticale grains

    Institute of Scientific and Technical Information of China (English)

    Adam Drzymala; Wieslaw Bielawski

    2009-01-01

    Carboxypeptidase Ⅲ from germinating triticale grains was purified 434.2-fold with a six-step procedure including:homogenization,ammonium sulfate precipitation,cation-exchange chromatography on CM-cellulose,gel filtration chromatography on Sephadex G-150,cation-exchange chromatography on SP8HR column(HPLC),and affinity chromatography on CABSSepharose 4B.Triticale carboxypeptidase Ⅲ is a monomer with a molecular weight of 45 kDa,which optimally hydrolyzes peptides at temperature 30-50℃and pH 4.6.N-CBZ-Ala-Phe,N-CBZ-Ala-Leu,and N-CBZ-Ala-Met are hydrolyzed at the highest rates.Amino acids with aromatic or large aliphatic side chains are preferred in position P1',whereas the presence of these types of groups in position P1 of the substrate results in a lower rate of hydrolysis.Peptides containing glutamic acid in positions P1 are poor substrates for the enzyme.This phenomenon suggests the hydrophobic substrate-binding sites S1 and S1'.The active site contains serine since diisopropylfluorophosphate and phenylmethanesulfonyl fluoride reduce the activity by 89.9%and 81.5%,respectively.Moreover,the activity of triticale carboxypeptidase Ⅲ is reduced by mercury ions and organomercurial compounds,which suggests the presence of a sulfhydryl group adjacent to the active site of the enzyme.Identification of purified enzyme by mass spectrometry method demonstrated that the enzyme is a homolog of barley carboxypeptidase Ⅲ.

  5. Production, purification, and properties of serine carboxypeptidase from Paecilomyces carneus.

    Science.gov (United States)

    Umetsu, H; Hishinuma, K; Wake, H; Ichishima, E

    1996-07-01

    Seventeen strains of the genus Paecilomyces were examined for their ability to produce serine carboxypeptidase. Paecilomyces carneus IFO 7012 exhibited the highest potency for serine carboxypeptidase production. A maximum yield of serine carboxypeptidase was obtained by koji culture of the strain at 22 degrees C for 7 days. The serine carboxypeptidase was purified to homogeneity from an extract of the koji culture. The molecular weight of the enzyme was estimated to be 47,000 by HPLC. The isoelectric point of the enzyme was determined to be 4.0, and the optimum pH was 4.0 toward benzyloxycarbonyl-L-glutamyl-L-tyrosine (Z-Glu-Tyr) and benzyloxycarbonyl-L-phenylalanyl-L-alanine (Z-Phe-Ala), respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and p-chloromercurybenzoate. Relative hydrolysis rates of N-acylpeptides and kinetic studies indicated that the enzyme preferred substrates having bulky amino acids in the penultimate position from their carboxy-termini. PMID:8661688

  6. Isolation and characterization of human membrane carboxypeptidase (HMCP)

    International Nuclear Information System (INIS)

    The authors detected a membrane-bound carboxypeptidase in human placenta and other tissues which cleaves C-terminal Lys or Arg of peptides such as Lys6-Met5-enkephalin. The enzyme was solubilized from placental microvilli with 0.8% CHAPS and purified 427-fold by ion-exchange chromatography, Sepharose-arginine affinity chromatography, chromatofocusing and gel filtration on HPLC. HMCP had a mol. wt. of 67,000 in SDS-PAGE and 65,300 in gel filtration and a pH optimum of 7.0. HMCP cleaved Bz-Gly-argininic acid the fastest (90 μmol/min/mg) followed by Bz-Ala-Lys (41), Bz-Phe-Lys (26), Bz-Gly-Arg (1.7) and Bz-Gly-Lys (1.6). Activity was stimulated by CoCl2 and inhibited by cadmium acet., o-phenanthroline and 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid but not by phenylmethylsulfonyl fluoride, aprotinin or p-chloromercuriphenylsulfonate. The enzyme was stable for 1 hr at room temp. at pH 4.25, but lost 31% activity at pH 4.0. HMCP did not react with antiserum to human plasma carboxypeptidase N in Western blotting. This study shows that human placental microvilli contain a membrane carboxypeptidase, that differs from other carboxypeptidases, and cleaves C-terminal basic amino acids from peptides. This enzyme could be involved in regulating the level of peptide hormones in the placenta and other tissues

  7. Cloning of cDNAs that encode human mast cell carboxypeptidase A, and comparison of the protein with mouse mast cell carboxypeptidase A and rat pancreatic carboxypeptidases

    International Nuclear Information System (INIS)

    Human skin and lung mast cells and rodent peritoneal cells contain a carboxypeptidase in their secretory granules. The authors have screened human lung cDNA libraries with a mouse mast cell carboxypeptidase A (MC-CPA) cDNA probe to isolate a near-full-length cDNA that encodes human MC-CPA. The 5' end of the human MC-CPA transcript was defined by direct mRNA sequencing and by isolation and partial sequencing of the human MC-CPA gene. Human MC-CPA is predicted to be translated as a 417 amino acid preproenzyme which includes a 15 amino acid signal peptide and a 94-amino acid activation peptide. The mature human MC-CPA enzyme has a predicted size of 36.1 kDa, a net positive charge of 16 at neutral pH, and 86% amino acid sequence identity with mouse MC-CPA. DNA blot analyses showed that human MC-CPA mRNA is transcribed from a single locus in the human genome. Comparison of the human MC-CPA with mouse MC-CPA and with three rat pancreatic carboxypeptidases shows that these enzymes are encoded by distinct but homologous genes

  8. Structure and function of carboxypeptidase A alpha in supercooled water.

    OpenAIRE

    Thompson, J. S.; Gehring, H; Vallee, B L

    1980-01-01

    The spectral and enzymatic characteristics of chromophoric derivatives of carboxypeptidase A alpha (EC 3.4.17.1) have been examined at subzero temperatures in supercooled water-in-oil emulsions. Substrate and temperature dependencies of enzyme kinetics indicated the existence of a solution-like enzyme phase that greatly extends the temperature range (greater than 60 degrees C) over which the activity of this enzyme can be measured. The emulsion spectra were virtually identical to those of sol...

  9. Cloning and expression of human colon mast cell carboxypeptidase

    Institute of Scientific and Technical Information of China (English)

    Zhang-Quan Chen; Shao-Heng He

    2004-01-01

    AIM: To clone and express the human colon mast cell METHODS: Total RNA was extracted from colon tissue, and the cDNA encoding human colon mast cell carboxypeptidase was amplified by reverse-transcription PCR (RT-PCR). The product cDNA was subcloned into the prokaryotic expression vector pMAL-c2x and eukaryotic expression vector pPIC9K to conrtruct prokaryotic expression vector pMAL/human MC-CP (hMC-CP) and eukaryotic pPIC9K/hMC-CP. The recombinant fusion protein expressed in E.coli was induced with IPTG and purified by amylose affinity chromatography. After digestion with factor Xa, recombinant hMC-CP was purified by heparin agarose chromatography. The recombinant hMC-CP expressed in Pichia pastoris (P.pastoris) was induced with methanol and analyzed by SDS-PAGE, Western blot, N-terminal amino acid RESULTS: The cDNA encoding the human colon mast cell carboxypeptidase was cloned, which had five nucleotide variations compared with skin MC-CP cDNA. The recombinant hMC-CP protein expressed in E.coli was purified with amylose affinity chromatography and heparin agarose chromatogphy.SDS-PAGE and Western blot analysis showed that the recombinant protein expressed by E. coli had a molecular weight of 36 kDa and reacted to the anti-native hMC-CP monoclonal antibody (CA5). The N-terminal amino acid sequence confirmed further the product was hMC-CP. E. coli generated hMC-CP showed a very low level of enzymatic activity, but P. pastoris produced hMC-CP had a relatively high enzymatic activity towards a synthetic substrate hippuryl-L-phenylalanine.carboxypeptidase can be successfully cloned and expressed in E.coli and P. pastoris, which will contribute greatly to the fonctional study on hMC-CP.

  10. Structural basis of the resistance of an insect carboxypeptidase to plant protease inhibitors

    OpenAIRE

    Bayés, A.; Comellas-Bigler, M.; Rodriquez de la Vega, M.; Maskos, K.; Bode, W; Aviles, F. X.; Beekwilder, M.J.; Vendrell, J

    2005-01-01

    Corn earworm (Helicoverpa zea), also called tomato fruitworm, is a common pest of many Solanaceous plants. This insect is known to adapt to the ingestion of plant serine protease inhibitors by using digestive proteases that are insensitive to inhibition. We have now identified a B-type carboxypeptidase of H. zea (CPBHz) insensitive to potato carboxypeptidase inhibitor (PCI) in corn earworm. To elucidate the structural features leading to the adaptation of the insect enzyme, the crystal struct...

  11. Preparation, crystallization, and preliminary X-ray diffraction study of mutant carboxypeptidase T containing the primary specificity pocket of carboxypeptidase B

    International Nuclear Information System (INIS)

    Recombinant G215S, A251G, T257A, D260G, T262D mutant carboxypeptidase T from Thermoactinomyces vulgaris containing mutations in the primary specificity pocket was prepared and crystallized. Single crystals with a size of up to 0.3 mm were grown and investigated by X-ray diffraction. Recombinant mutant carboxypeptidase T containing the primary specificity subsite compositionally identical to that of pancreatic carboxypeptidase B crystallizes in the same space group as the natural enzyme. The crystals belong to sp. gr. P6322; the unit-cell parameters are a = b = 157.867 A, c = 104.304 A, α = β = 90 deg., γ = 120 deg. X-ray diffraction data suitable for determining the three-dimensional structure at atomic resolution were collected from one crystal.

  12. Preparation, crystallization, and preliminary X-ray diffraction study of mutant carboxypeptidase T containing the primary specificity pocket of carboxypeptidase B

    Science.gov (United States)

    Akparov, V. Kh.; Grishin, A. M.; Timofeev, V. I.; Kuranova, I. P.

    2010-09-01

    Recombinant G215S, A251G, T257A, D260G, T262D mutant carboxypeptidase T from Thermoactinomyces vulgaris containing mutations in the primary specificity pocket was prepared and crystallized. Single crystals with a size of up to 0.3 mm were grown and investigated by X-ray diffraction. Recombinant mutant carboxypeptidase T containing the primary specificity subsite compositionally identical to that of pancreatic carboxypeptidase B crystallizes in the same space group as the natural enzyme. The crystals belong to sp. gr. P6322; the unit-cell parameters are a = b = 157.867 Å, c = 104.304 Å, α = β = 90°, γ = 120°. X-ray diffraction data suitable for determining the three-dimensional structure at atomic resolution were collected from one crystal.

  13. Prolactin/Stat5 and androgen R1881 coactivate carboxypeptidase-D gene in breast cancer cells.

    Science.gov (United States)

    Koirala, Samir; Thomas, Lynn N; Too, Catherine K L

    2014-03-01

    Plasma membrane-bound carboxypeptidase-D (CPD) cleaves C-terminal arginine from extracellular substrates. In the cell, arginine is converted to nitric oxide (NO). We have reported that up-regulation of CPD mRNA/protein levels by 17β-estradiol and prolactin (PRL) in breast cancer cells, and by testosterone in prostate cancer cells, increased NO production and cell survival. The CPD promoter contains a consensus γ-interferon-activated sequence (GAS) and 3 putative androgen response elements (ARE.1, ARE.2, ARE.3) that could potentially bind PRL-activated transcription factor Stat5 (signal transducer and activator of transcription 5) and the liganded androgen receptor (AR), respectively. This study showed that synthetic androgen R1881 and PRL elevated CPD mRNA/protein levels in human MCF-7 and T47D breast cancer cells in a time-/dose-dependent manner. PRL/R1881-elevated CPD expression was blocked by actinomycin-D, and a CPD promoter construct containing these GAS and AREs was stimulated by PRL or R1881, indicating transcriptional regulation by both hormones. Luciferase reporter assays showed that GAS and the adjacent ARE.1 only were active. Mutation of GAS in the ΔGAS-CPD construct (ARE.1 intact) abolished CPD promoter activity in response to PRL and, surprisingly, to R1881 as well. ΔGAS-CPD promoter activity was restored by PRL+R1881 in combination, and enhanced by ectopic Stat5, but abolished by Stat5 gene knockdown. Chromatin immunoprecipitation analysis confirmed binding of activated Stat5 and liganded AR to GAS and ARE.1, respectively. Activated Stat5 also induced binding of unliganded AR to ARE.1, and liganded AR induced binding of unactivated Stat5 to GAS. In summary, PRL and R1881, acting through Stat5 and AR, act cooperatively to stimulate CPD gene transcription in breast cancer cells. PMID:24433040

  14. Regulation of dopamine transporter activity by carboxypeptidase E

    Directory of Open Access Journals (Sweden)

    Zhang Heping

    2009-05-01

    Full Text Available Abstract Background The dopamine transporter (DAT plays a critical role in terminating the action of dopamine by rapid reuptake into the presynaptic neuron. Previous studies have revealed that the DAT carboxyl terminus (DAT-CT can directly interact with other cellular proteins and regulate DAT function and trafficking. Results Here, we have identified that carboxypeptidase E (CPE, a prohormone processing exopeptidase and sorting receptor for the regulated secretory pathway, interacts with the DAT-CT and affects DAT function. Mammalian cell lines coexpressing CPE and DAT exhibited increased DAT-mediated dopamine uptake activity compared to cells expressing DAT alone. Moreover, coexpression of an interfering DAT-CT minigene inhibited the effects of CPE on DAT. Functional changes caused by CPE could be attributed to enhanced DAT expression and subsequent increase in DAT cell surface localization, due to decreased DAT degradation. In addition, CPE association could reduce the phosphorylation state of DAT on serine residues, potentially leading to reduced internalization, thus stabilizing plasmalemmal DAT localization. Conclusion Taken together, our results reveal a novel role for CPE in the regulation of DAT trafficking and DAT-mediated DA uptake, which may provide a novel target in the treatment of dopamine-governed diseases such as drug addiction and obesity.

  15. Refolding of a carboxypeptidase Y folding intermediate in vitro by low-affinity binding of the proregion

    DEFF Research Database (Denmark)

    Winther, Jakob R.; Sørensen, P; Kielland-Brandt, Morten

    1994-01-01

    Efficient folding of carboxypeptidase Y is dependent on the presence of the proregion. Thus, denatured procarboxypeptidase Y, in contrast to the mature enzyme, refolds efficiently in vitro in low ionic strength buffers. Under these conditions denatured mature carboxypeptidase Y forms an inactive,...

  16. Identification of a Dithiazoline Inhibitor of Escherichia coli l,d-Carboxypeptidase A

    OpenAIRE

    Baum, Ellen Z.; Crespo-Carbone, Steven M.; Foleno, Barbara; Peng, Sean; Hilliard, Jamese J.; Abbanat, Darren; Goldschmidt, Raul; Bush, Karen

    2005-01-01

    The enzyme l,d-carboxypeptidase A is involved in the recycling of bacterial peptidoglycan and is essential in Escherichia coli during stationary phase. By high-throughput screening, we have identified a dithiazoline inhibitor of the enzyme with a 50% inhibitory concentration of 3 μM. The inhibitor appeared to cause lysis of E. coli during stationary phase, behavior that is similar to a previously described deletion mutant of l,d-carboxypeptidase A (M. F. Templin, A. Ursinus, and J.-V. Holtje,...

  17. Requirement of the propeptide for in vivo formation of active yeast carboxypeptidase Y

    DEFF Research Database (Denmark)

    Ramos, C; Winther, Jakob R.; Kielland-Brandt, Morten

    1994-01-01

    Deletions have been constructed in the region encoding the 91-amino acid propeptide of the vacuolar enzyme carboxypeptidase Y of Saccharomyces cerevisiae, and in vivo effects of these mutations on the intracellular transport of the mutant proenzymes have been examined. Deletions did not include t...

  18. A high-resolution structure of ligand-free human glutamate carboxypeptidase II

    Czech Academy of Sciences Publication Activity Database

    Bařinka, C.; Starková, Jana; Konvalinka, Jan; Lubkowski, J.

    F63, č. 3 (2007), s. 150-153. ISSN 1744-3091 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : human glutamate carboxypeptidase II * X-ray structure * prostate - cancer Subject RIV: CE - Biochemistry Impact factor: 0.645, year: 2007

  19. Glutamate Carboxypeptidase II in Diagnosis and Treatment of Neurologic Disorders and Prostate Cancer

    Czech Academy of Sciences Publication Activity Database

    Bařinka, Cyril; Rojas, C.; Slusher, B.; Pomper, M.

    2012-01-01

    Roč. 19, č. 6 (2012), s. 856-870. ISSN 0929-8673 Institutional research plan: CEZ:AV0Z50520701 Keywords : Glutamate carboxypeptidase II * X-ray crystallography * prostate -specific membrane antigen Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.070, year: 2012

  20. Sub-zero temperature inactivation of carboxypeptidase Y under high hydrostatic pressure.

    Science.gov (United States)

    Kinsho, Toshihiko; Ueno, Hiroshi; Hayashi, Rikimaru; Hashizume, Chieko; Kimura, Kunio

    2002-09-01

    High hydrostatic pressure induced cold inactivation of carboxypeptidase Y. Carboxypeptidase Y was fully active when exposed to subzero temperature at 0.1 MPa; however, the enzyme became inactive when high hydrostatic pressure and subzero temperature were both applied. When the enzyme was treated at pressures higher than 300 MPa and temperatures lower than -5 degrees C, it underwent an irreversible inactivation in which nearly 50% of the alpha-helical structure was lost as judged by circular dichroism spectral analysis. When the applied pressure was limited to below 200 MPa, the cold inactivation process appeared to be reversible. In the presence of reducing agent, this reversible phenomenon, observed at below 200 MPa, diminished to give an inactive enzyme; the agent reduces some of disulfide bridge(s) in an area of the structure that is newly exposed area because of the cold inactivation. Such an area is unavailable if carboxypeptidase Y is in its native conformation. Because all the disulfide bridges in carboxypeptidase Y locate near the active site cleft, it is suggested that the structural destruction, if any, occurs preferentially in this disulfide rich area. A possible mechanism of pressure-dependent cold inactivation of CPY is to destroy the alpha-helix rich region, which creates an hydrophobic environment. This destruction is probably a result of the reallocation of water molecules. Experiments carried out in the presence of denaturing agents (SDS, urea, GdnHCl), salts, glycerol, and sucrose led to a conclusion consistent with the idea of water reallocation. PMID:12230580

  1. Exchange of regions of the carboxypeptidase Y propeptide. Sequence specificity and function in folding in vivo

    DEFF Research Database (Denmark)

    Ramos, C; Winther, Jakob R.

    1996-01-01

    Candida albicans. This propeptide was partially functional when combined with carboxypeptidase Y. Analysis of the biosynthesis of the mutant forms of the zymogen showed that a fraction of the molecules proceeded from the endoplasmic reticulum with fairly rapid kinetics, while the rest was degraded....

  2. Discovery of Orally Available Prodrugs of the Glutamate Carboxypeptidase II (GCPII) Inhibitor 2-Phosphonomethylpentanedioic Acid (2-PMPA)

    Czech Academy of Sciences Publication Activity Database

    Majer, Pavel; Jančařík, Andrej; Krečmerová, Marcela; Tichý, Tomáš; Tenora, Lukáš; Wozniak, K.; Wu, Y.; Pommier, E.; Ferraris, D.; Rais, R.; Slusher, B. S.

    2016-01-01

    Roč. 59, č. 6 (2016), s. 2810-2819. ISSN 0022-2623 Institutional support: RVO:61388963 Keywords : glutamate carboxypeptidase II * glutamate * 2-PMPA * prodrug Subject RIV: CC - Organic Chemistry Impact factor: 5.447, year: 2014

  3. Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoes.

    Science.gov (United States)

    Lufrano, Daniela; Cotabarren, Juliana; Garcia-Pardo, Javier; Fernandez-Alvarez, Roberto; Tort, Olivia; Tanco, Sebastián; Avilés, Francesc Xavier; Lorenzo, Julia; Obregón, Walter D

    2015-12-01

    Natural protease inhibitors of metallocarboxypeptidases are rarely reported. In this work, the cloning, expression and characterization of a proteinaceous inhibitor of the A/B-type metallocarboxypeptidases, naturally occurring in tubers of Solanum tuberosum, subsp. andigenum cv. Imilla morada, are described. The obtained cDNA encoded a polypeptide of 80 residues, which displayed the features of metallocarboxypeptidase inhibitor precursors from the Potato Carboxypeptidase Inhibitor (PCI) family. The mature polypeptide (39 residues) was named imaPCI and in comparison with the prototype molecule of the family (PCI from S. tuberosum subsp. tuberosum), its sequence showed one difference at its N-terminus and another three located at the secondary binding site, a region described to contribute to the stabilization of the complex inhibitor-target enzyme. In order to gain insights into the relevance of the secondary binding site in nature, a recombinant form of imaPCI (rimaPCI) having only differences at the secondary binding site with respect to recombinant PCI (rPCI) was cloned and expressed in Escherichia coli. The rimaPCI exhibited a molecular mass of 4234.8Da by MALDI-TOF/MS. It displayed potent inhibitory activity towards A/B-type carboxypeptidases (with a Ki in the nanomolar range), albeit 2-4-fold lower inhibitory capacity compared to its counterpart rPCI. This result is in agreement with our bioinformatic analysis, which showed that the main interaction established between the secondary binding site of rPCI and the bovine carboxypeptidase A is likely lost in the case of rimaPCI. These observations reinforce the importance of the secondary binding site of PCI-family members on inhibitory effects towards A/B-type metallocarboxypeptidases. Furthermore, as a simple proof of concept of its applicability in biotechnology and biomedicine, the ability of rimaPCI to protect human epidermal growth factor from C-terminal cleavage and inactivation by carboxypeptidases A and B

  4. Novel Substrate-Based Inhibitors of Human Glutamate Carboxypeptidase II with Enhanced Lipophilicity

    Czech Academy of Sciences Publication Activity Database

    Plechanovová, Anna; Byun, Y.; Alquicer, Glenda; Škultétyová, Ĺubica; Mlčochová, Petra; Němcová, Adriana; Kim, H.-J.; Navrátil, Michal; Mease, R.; Lubkowski, J.; Pomper, M.; Konvalinka, Jan; Rulíšek, Lubomír; Bařinka, Cyril

    2011-01-01

    Roč. 54, č. 21 (2011), s. 7535-7546. ISSN 0022-2623 R&D Projects: GA MŠk(CZ) ME10031; GA MŠk LC512 Grant ostatní: EMBO(DE) 1978 Institutional research plan: CEZ:AV0Z50520701; CEZ:AV0Z40550506 Keywords : Glutamate carboxypeptidase II. * QM/MM calculations * X-ray crystallography * lipophilicity * inhibitors Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.248, year: 2011

  5. A high-affinity inhibitor of yeast carboxypeptidase Y is encoded by TFS1 and shows homology to a family of lipid binding proteins

    DEFF Research Database (Denmark)

    Bruun, A W; Svendsen, I; Sørensen, S O;

    1998-01-01

    degree of specificity, showing a 200-fold higher Ki toward a carboxypeptidase from Candida albicans which is highly homologous to carboxypeptidase Y. The TFS1 gene product shows extensive similarity to a class of proteins termed "21-23-kDa lipid binding proteins", members of which are found in several...

  6. Carborane-containing urea-based inhibitors of glutamate carboxypeptidase II: Synthesis and structural characterization

    Czech Academy of Sciences Publication Activity Database

    Youn, S.; Kim, K.I.; Ptáček, Jakub; Ok, K.; Nováková, Zora; Kim, Y.; Koo, J.; Bařinka, Cyril; Byun, Y.

    2015-01-01

    Roč. 25, č. 22 (2015), s. 5232-5236. ISSN 0960-894X R&D Projects: GA ČR GAP301/12/1513; GA MŠk(CZ) ED1.1.00/02.0109 Grant ostatní: GA MŠk(CZ) CZ.1.07/2.3.00/30.0045 Institutional support: RVO:86652036 Keywords : Carborane * Glutamate carboxypeptidase II * X-ray crystal structure Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 2.420, year: 2014

  7. Identification of the catalytic histidine residue participating in the charge-relay system of carboxypeptidase Y.

    OpenAIRE

    Jung, G.; Ueno, H; Hayashi, R.; Liao, T. H.

    1995-01-01

    The essential histidine residue of carboxypeptidase Y (CPY) was modified by a site-specific reagent, a chloromethylketone derivative of benzyloxycarbonyl-L-phenylalanine. The single modified histidine residue was converted to N tau-carboxy-methyl histidine (cmHis) upon performic acid oxidation. A peptide containing cmHis was isolated from the tryptic-thermolytic digest. Based on the amino acid composition and sequence analysis, the peptide is shown to be Val-Phe-Asp-Gly-Gly-cmHis-MetO2-Val-Pr...

  8. Crystallization and preliminary X-ray diffraction study of porcine carboxypeptidase B

    Energy Technology Data Exchange (ETDEWEB)

    Akparov, V. Kh., E-mail: valery@akparov.ru [Scientific Center of Russian Federation Research Institute for Genetics and Selection of Industrial Microorganisms (Russian Federation); Timofeev, V. I., E-mail: inna@ns.crys.ras.ru; Kuranova, I. P., E-mail: tostars@mail.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    2015-05-15

    Crystals of porcine pancreatic carboxypeptidase B have been grown in microgravity by the capillary counter-diffusion method through a gel layer. The X-ray diffraction study showed that the crystals belong to sp. gr. P4{sub 1}2{sub 1}2 and have the following unit-cell parameters: a = b = 79.58 Å, c = 100.51 Å; α = β = γ = 90.00°. The X-ray diffraction data set suitable for the determination of the three-dimensional structure at atomic resolution was collected from one of the grown crystals at the SPring 8 synchrotron facility to 0.98 Å resolution.

  9. Cloning and nucleotide sequence of the Salmonella typhimurium dcp gene encoding dipeptidyl carboxypeptidase.

    OpenAIRE

    Hamilton, S.; Miller, C G

    1992-01-01

    Plasmids carrying the Salmonella typhimurium dcp gene were isolated from a pBR328 library of Salmonella chromosomal DNA by screening for complementation of a peptide utilization defect conferred by a dcp mutation. Strains carrying these plasmids overproduced dipeptidyl carboxypeptidase approximately 50-fold. The nucleotide sequence of a 2.8-kb region of one of these plasmids contained an open reading frame coding for a protein of 77,269 Da, in agreement with the 80-kDa size for dipeptidyl car...

  10. Propeptide of carboxypeptidase Y provides a chaperone-like function as well as inhibition of the enzymatic activity

    DEFF Research Database (Denmark)

    Winther, Jakob R.; Sørensen, P

    1991-01-01

    The zymogen of the vacuolar carboxypeptidase Y from Saccharomyces cerevisiae was purified and characterized with respect to activation as well as refolding in vitro. The purified procarboxypeptidase Y has no detectable activity but can be efficiently activated by proteinase K from Tritirachium al...

  11. High-resolution structure of the M14-type cytosolic carboxypeptidase from Burkholderia cenocepacia refined exploiting PDB-REDO strategies

    International Nuclear Information System (INIS)

    The structure of a bacterial M14-family carboxypeptidase determined exploiting microfocus synchrotron radiation and highly automated refinement protocols reveals its potential to act as a polyglutamylase. A potential cytosolic metallocarboxypeptidase from Burkholderia cenocepacia has been crystallized and a synchrotron-radiation microfocus beamline allowed the acquisition of diffraction data to 1.9 Å resolution. The asymmetric unit comprises a tetramer containing over 1500 amino acids, and the high-throughput automated protocols embedded in PDB-REDO were coupled with model–map inspections in refinement. This approach has highlighted the value of such protocols for efficient analyses. The subunit is constructed from two domains. The N-terminal domain has previously only been observed in cytosolic carboxypeptidase (CCP) proteins. The C-terminal domain, which carries the Zn2+-containing active site, serves to classify this protein as a member of the M14D subfamily of carboxypeptidases. Although eukaryotic CCPs possess deglutamylase activity and are implicated in processing modified tubulin, the function and substrates of the bacterial family members remain unknown. The B. cenocepacia protein did not display deglutamylase activity towards a furylacryloyl glutamate derivative, a potential substrate. Residues previously shown to coordinate the divalent cation and that contribute to peptide-bond cleavage in related enzymes such as bovine carboxypeptidase are conserved. The location of a conserved basic patch in the active site adjacent to the catalytic Zn2+, where an acetate ion is identified, suggests recognition of the carboxy-terminus in a similar fashion to other carboxypeptidases. However, there are significant differences that indicate the recognition of substrates with different properties. Of note is the presence of a lysine in the S1′ recognition subsite that suggests specificity towards an acidic substrate

  12. High-resolution structure of the M14-type cytosolic carboxypeptidase from Burkholderia cenocepacia refined exploiting PDB-REDO strategies

    Energy Technology Data Exchange (ETDEWEB)

    Rimsa, Vadim; Eadsforth, Thomas C. [University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom); Joosten, Robbie P. [Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam (Netherlands); Hunter, William N., E-mail: w.n.hunter@dundee.ac.uk [University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom)

    2014-02-01

    The structure of a bacterial M14-family carboxypeptidase determined exploiting microfocus synchrotron radiation and highly automated refinement protocols reveals its potential to act as a polyglutamylase. A potential cytosolic metallocarboxypeptidase from Burkholderia cenocepacia has been crystallized and a synchrotron-radiation microfocus beamline allowed the acquisition of diffraction data to 1.9 Å resolution. The asymmetric unit comprises a tetramer containing over 1500 amino acids, and the high-throughput automated protocols embedded in PDB-REDO were coupled with model–map inspections in refinement. This approach has highlighted the value of such protocols for efficient analyses. The subunit is constructed from two domains. The N-terminal domain has previously only been observed in cytosolic carboxypeptidase (CCP) proteins. The C-terminal domain, which carries the Zn{sup 2+}-containing active site, serves to classify this protein as a member of the M14D subfamily of carboxypeptidases. Although eukaryotic CCPs possess deglutamylase activity and are implicated in processing modified tubulin, the function and substrates of the bacterial family members remain unknown. The B. cenocepacia protein did not display deglutamylase activity towards a furylacryloyl glutamate derivative, a potential substrate. Residues previously shown to coordinate the divalent cation and that contribute to peptide-bond cleavage in related enzymes such as bovine carboxypeptidase are conserved. The location of a conserved basic patch in the active site adjacent to the catalytic Zn{sup 2+}, where an acetate ion is identified, suggests recognition of the carboxy-terminus in a similar fashion to other carboxypeptidases. However, there are significant differences that indicate the recognition of substrates with different properties. Of note is the presence of a lysine in the S1′ recognition subsite that suggests specificity towards an acidic substrate.

  13. Truncating Homozygous Mutation of Carboxypeptidase E (CPE) in a Morbidly Obese Female with Type 2 Diabetes Mellitus, Intellectual Disability and Hypogonadotrophic Hypogonadism

    NARCIS (Netherlands)

    Alsters, Suzanne I M; Goldstone, Anthony P; Buxton, Jessica L; Zekavati, Anna; Sosinsky, Alona; Yiorkas, Andrianos M; Holder, Susan; Klaber, Robert E; Bridges, Nicola; van Haelst, Mieke M; le Roux, Carel W; Walley, Andrew J; Walters, Robin G; Mueller, Michael; Blakemore, Alexandra I F

    2015-01-01

    Carboxypeptidase E is a peptide processing enzyme, involved in cleaving numerous peptide precursors, including neuropeptides and hormones involved in appetite control and glucose metabolism. Exome sequencing of a morbidly obese female from a consanguineous family revealed homozygosity for a truncati

  14. Over-expression of carboxypeptidase of extreme thermophile pyrococcus furiosus in escherichia coli

    International Nuclear Information System (INIS)

    Thermophiles and extreme thermophiles are potential source of thermostable proteases for economical application. This study deals with cloning and over-expression of a carboxypeptidase (CBP) from the extreme thermophile archaeon Pyrococcus furiosus in E. coli. Using the forward and the reverse primers designed according to the putative CBP gene sequence analysed from the published genome sequence of P. furiosus, 1.5 kb fragment of CBP gene was PCR amplified. After TA-cloning in pTZ57R/T vector, the gene was ligated into pET-22b(+) and the recombinant plasmid thus obtained was used to transform E. coli BL21 (DE3)RIPL. On induction with IPTG for 6-8 hours CBP was expressed up to 30% of the total cell proteins. The enzyme, however, was expressed in an insoluble form which was refolded to an active state by treatment with urea. (author)

  15. Three-dimensional structure of recombinant carboxypeptidase T from Thermoactinomyces vulgaris without calcium ions

    International Nuclear Information System (INIS)

    Crystals of recombinant carboxypeptidase T (CPT) from Thermoactinomyces vulgaris were grown in a capillary by the counterdiffusion method in the absence of calcium ions. The three-dimensional structure of CPT was solved at 1.69-Å resolution using the X-ray diffraction data collected from the crystals of the enzyme on the SPring-8 synchrotron radiation facility and was then refined to Rfact = 16.903% and Rfree = 18.165%. The coordinates of the refined model were deposited in the Protein Data Bank (PDB ID: 3QNV). A comparison of this structure with the structure of wild-type CPT containing bound calcium ions, which was determined earlier, revealed a number of conformational changes both in the calcium-binding sites and the enzyme active site. Based on the results of this comparison, the possible factors responsible for the difference in the catalytic activity of the two forms of the enzyme are considered.

  16. Effects of pH on the Structure and Function of Carboxypeptidase A: Crystallographic Studies

    OpenAIRE

    Shoham, G; Rees, D. C.; Lipscomb, W N

    1984-01-01

    High-resolution crystal structures are described for carboxypeptidase A (EC 3.4.17.1) in crystals grown at pH 8.5, 9.0, and 9.5 and compared with the structure at pH 7.5. The comparison shows that in the pH range of 7.5-9.5 the enzyme structure is practically unchanged, and, most importantly, that the flexible side chain of Tyr-248 remains exclusively in the "up" position, away from the Zn atom, throughout the pH range. There is no evidence for binding of Tyr-248 to Zn at any of these pH valu...

  17. Decreased synthesis of serum carboxypeptidase N (SCPN) in familial SCPN deficiency

    Energy Technology Data Exchange (ETDEWEB)

    Mathews, K.P.; Curd, J.G.; Hugli, T.E.

    1986-01-01

    Serum carboxypeptidase N (SCPN) is the primary inactivator of the C3a, C4a, and C5a anaphylatoxins as well as an inactivator of bradykinin. Thus, SCPN deficiency potentially could result in significant pathophysiologic consequences. Previous studies identified a deficient subject afflicted with frequent episodes of angioedema, and other family members also had SCPN deficiency. To delineate this abnormality further, the fractional catabolic rate (FRC) and enzyme synthesis were determined in three members of the afflicted kindred as well as in five normal persons following the infusion of homogeneous /sup 125/I-SCPN. The mean FCR and synthesis rates for SCPN in the normal subjects were 1.3%/hr and 20,793 U/kg/hr, respectively. Reduced synthesis was concluded to be primarily responsible for the low SCPN levels in the afflicted kindred. The high FRC of SCPN discourages attempted maintenance therapy with infusions of enriched SCPN preparations.

  18. Decreased synthesis of serum carboxypeptidase N (SCPN) in familial SCPN deficiency

    International Nuclear Information System (INIS)

    Serum carboxypeptidase N (SCPN) is the primary inactivator of the C3a, C4a, and C5a anaphylatoxins as well as an inactivator of bradykinin. Thus, SCPN deficiency potentially could result in significant pathophysiologic consequences. Previous studies identified a deficient subject afflicted with frequent episodes of angioedema, and other family members also had SCPN deficiency. To delineate this abnormality further, the fractional catabolic rate (FRC) and enzyme synthesis were determined in three members of the afflicted kindred as well as in five normal persons following the infusion of homogeneous 125I-SCPN. The mean FCR and synthesis rates for SCPN in the normal subjects were 1.3%/hr and 20,793 U/kg/hr, respectively. Reduced synthesis was concluded to be primarily responsible for the low SCPN levels in the afflicted kindred. The high FRC of SCPN discourages attempted maintenance therapy with infusions of enriched SCPN preparations

  19. Carborane-containing urea-based inhibitors of glutamate carboxypeptidase II: Synthesis and structural characterization.

    Science.gov (United States)

    Youn, Sihyun; Kim, Kyung Im; Ptacek, Jakub; Ok, Kiwon; Novakova, Zora; Kim, YunHye; Koo, JaeHyung; Barinka, Cyril; Byun, Youngjoo

    2015-11-15

    Glutamate carboxypeptidase II (GCPII) is a zinc metalloprotease on the surface of astrocytes which cleaves N-acetylaspartylglutamate to release N-acetylaspartate and glutamate. GCPII inhibitors can decrease glutamate concentration and play a protective role against apoptosis or degradation of brain neurons. Herein, we report the synthesis and structural analysis of novel carborane-based GCPII inhibitors. We determined the X-ray crystal structure of GCPII in complex with a carborane-containing inhibitor at 1.79Å resolution. The X-ray analysis revealed that the bulky closo-carborane cluster is located in the spacious entrance funnel region of GCPII, indicating that the carborane cluster can be further structurally modified to identify promising lead structures of novel GCPII inhibitors. PMID:26459214

  20. Molecular Identification of β-Citrylglutamate Hydrolase as Glutamate Carboxypeptidase 3*

    Science.gov (United States)

    Collard, François; Vertommen, Didier; Constantinescu, Stefan; Buts, Lieven; Van Schaftingen, Emile

    2011-01-01

    β-Citrylglutamate (BCG), a compound present in adult testis and in the CNS during the pre- and perinatal periods is synthesized by an intracellular enzyme encoded by the RIMKLB gene and hydrolyzed by an as yet unidentified ectoenzyme. To identify β-citrylglutamate hydrolase, this enzyme was partially purified from mouse testis and characterized. Interestingly, in the presence of Ca2+, the purified enzyme specifically hydrolyzed β-citrylglutamate and did not act on N-acetyl-aspartylglutamate (NAAG). However, both compounds were hydrolyzed in the presence of Mn2+. This behavior and the fact that the enzyme was glycosylated and membrane-bound suggested that β-citrylglutamate hydrolase belonged to the same family of protein as glutamate carboxypeptidase 2 (GCP2), the enzyme that catalyzes the hydrolysis of N-acetyl-aspartylglutamate. The mouse tissue distribution of β-citrylglutamate hydrolase was strikingly similar to that of the glutamate carboxypeptidase 3 (GCP3) mRNA, but not that of the GCP2 mRNA. Furthermore, similarly to β-citrylglutamate hydrolase purified from testis, recombinant GCP3 specifically hydrolyzed β-citrylglutamate in the presence of Ca2+, and acted on both N-acetyl-aspartylglutamate and β-citrylglutamate in the presence of Mn2+, whereas recombinant GCP2 only hydrolyzed N-acetyl-aspartylglutamate and this, in a metal-independent manner. A comparison of the structures of the catalytic sites of GCP2 and GCP3, as well as mutagenesis experiments revealed that a single amino acid substitution (Asn-519 in GCP2, Ser-509 in GCP3) is largely responsible for GCP3 being able to hydrolyze β-citrylglutamate. Based on the crystal structure of GCP3 and kinetic analysis, we propose that GCP3 forms a labile catalytic Zn-Ca cluster that is critical for its β-citrylglutamate hydrolase activity. PMID:21908619

  1. Molecular identification of β-citrylglutamate hydrolase as glutamate carboxypeptidase 3.

    Science.gov (United States)

    Collard, François; Vertommen, Didier; Constantinescu, Stefan; Buts, Lieven; Van Schaftingen, Emile

    2011-11-01

    β-Citrylglutamate (BCG), a compound present in adult testis and in the CNS during the pre- and perinatal periods is synthesized by an intracellular enzyme encoded by the RIMKLB gene and hydrolyzed by an as yet unidentified ectoenzyme. To identify β-citrylglutamate hydrolase, this enzyme was partially purified from mouse testis and characterized. Interestingly, in the presence of Ca(2+), the purified enzyme specifically hydrolyzed β-citrylglutamate and did not act on N-acetyl-aspartylglutamate (NAAG). However, both compounds were hydrolyzed in the presence of Mn(2+). This behavior and the fact that the enzyme was glycosylated and membrane-bound suggested that β-citrylglutamate hydrolase belonged to the same family of protein as glutamate carboxypeptidase 2 (GCP2), the enzyme that catalyzes the hydrolysis of N-acetyl-aspartylglutamate. The mouse tissue distribution of β-citrylglutamate hydrolase was strikingly similar to that of the glutamate carboxypeptidase 3 (GCP3) mRNA, but not that of the GCP2 mRNA. Furthermore, similarly to β-citrylglutamate hydrolase purified from testis, recombinant GCP3 specifically hydrolyzed β-citrylglutamate in the presence of Ca(2+), and acted on both N-acetyl-aspartylglutamate and β-citrylglutamate in the presence of Mn(2+), whereas recombinant GCP2 only hydrolyzed N-acetyl-aspartylglutamate and this, in a metal-independent manner. A comparison of the structures of the catalytic sites of GCP2 and GCP3, as well as mutagenesis experiments revealed that a single amino acid substitution (Asn-519 in GCP2, Ser-509 in GCP3) is largely responsible for GCP3 being able to hydrolyze β-citrylglutamate. Based on the crystal structure of GCP3 and kinetic analysis, we propose that GCP3 forms a labile catalytic Zn-Ca cluster that is critical for its β-citrylglutamate hydrolase activity. PMID:21908619

  2. Development of a High-Throughput Fluorescence Polarization Assay to Identify Novel Ligands of Glutamate Carboxypeptidase II

    Czech Academy of Sciences Publication Activity Database

    Alquicer, Glenda; Sedlák, David; Byun, Y.; Pavlíček, Jiří; Stathis, M.; Rojas, C.; Slusher, B.; Pomper, M.G.; Bartůněk, Petr; Bařinka, Cyril

    2012-01-01

    Roč. 17, č. 8 (2012), s. 1030-1040. ISSN 1087-0571 R&D Projects: GA MŠk(CZ) ME10031; GA MŠk(CZ) LC06077 Institutional research plan: CEZ:AV0Z50520701 Institutional support: RVO:68378050 Keywords : fluorescence polarization * glutamate carboxypeptidase II * high-throughput screening Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.207, year: 2012

  3. Modelling the structure of latexin-carboxypeptidase A complex based on chemical cross-linking and molecular docking

    OpenAIRE

    Mouradov, Dmitri; Craven, Ari; Forwood, Jade Kenneth; Flanagan, Jack U.; García-Castellanos, Raquel; Gomis-Rüth, F. Xavier; Hume, David A.; Martin, Jennifer Lynn; Kobe, Bostjan; Huber, Thomas

    2006-01-01

    We have determined the three-dimensional structure of the protein complex between latexin and carboxypeptidase A using a combination of chemical cross-linking, mass spectrometry and molecular docking. The locations of three intermolecular cross-links were identified using mass spectrometry and these constraints were used in combination with a speed-optimised docking algorithm allowing us to evaluate more than 3 × 1011 possible conformations. While cross-links represent only limited structural...

  4. Phosphonate analogues of carboxypeptidase A substrates are potent transition-state analogue inhibitors.

    Science.gov (United States)

    Hanson, J E; Kaplan, A P; Bartlett, P A

    1989-07-25

    Analogues of tri- and tetrapeptide substrates of carboxypeptidase A in which the scissile peptide linkage is replaced with a phosphonate moiety (-PO2--O-) were synthesized and evaluated as inhibitors of the enzyme. The inhibitors terminated with either L-lactate or L-phenyllactate [designated (O) Ala and (O) Phe, respectively] in the P1' position. Transition-state analogy was shown for a series of 14 tri- and tetrapeptide derivatives containing the structure RCO-AlaP-(O)Ala [RCO-AP(O)A, AP indicates the phosphonic acid analogue of alanine] by the correlation of the Ki values for the inhibitors and the Km/kcat values for the corresponding amide substrates. This correlation supports a transition state for the enzymatic reaction that resembles the tetrahedral intermediate formed upon addition of water to the scissile carbonyl group. The inhibitors containing (O) Phe at the P1' position proved to be the most potent reversible inhibitors of carboxypeptidase A reported to date: the dissociation constants of ZAFP(O)F, ZAAP(O)F, and ZFAP(O)F are 4, 3, and 1 pM, respectively. Because of the high affinity of these inhibitors, their dissociation constants could not be determined by steady-state methods. Instead, the course of the association and dissociation processes was monitored for each inhibitor as its equilibrium with the enzyme was established in both the forward and reverse directions. A phosphonamidate analogue, ZAAPF, in which the peptide linkage is replaced with a -PO2-NH- moiety, was prepared and shown to hydrolyze rapidly at neutral pH (t1/2 = 20 min at pH 7.5). This inhibitor is bound an order of magnitude less tightly than the corresponding phosphonate, ZAAP(O)F, a result that contrasts with the 840-fold higher affinity of phosphonamidates for thermolysin [Bartlett, P. A., & Marlowe, C. K. (1987) Science 235, 569-571], a zinc peptidase with a similar arrangement of active-site catalytic residues. PMID:2790000

  5. Active Compounds Against Anopheles minimus Carboxypeptidase B for Malaria Transmission-Blocking Strategy.

    Science.gov (United States)

    Mongkol, Watcharakorn; Arunyawat, Uraiwan; Surat, Wunrada; Kubera, Anchanee

    2015-11-01

    Malaria transmission-blocking compounds have been studied to block the transmission of malaria parasites, especially the drug-resistant Plasmodium. Carboxypeptidase B (CPB) in the midgut of Anopheline mosquitoes has been demonstrated to be essential for the sexual development of Plasmodium in the mosquito. Thus, the CPB is a potential target for blocking compounds. The aim of this research was to screen compounds from the National Cancer Institute (NCI) diversity dataset and U.S. Food and Drug Administration (FDA)-approved drugs that could reduce the Anopheles CPB activity. The cDNA fragment of cpb gene from An. minimus (cpbAmi) was amplified and sequenced. The three-dimensional structure of CPB was predicted from the deduced amino acid sequence. The virtual screening of the compounds from NCI diversity set IV and FDA-approved drugs was performed against CPBAmi. The inhibition activity against CPBAmi of the top-scoring molecules was characterized in vitro. Three compounds-NSC-1014, NSC-332670, and aminopterin with IC50 at 0.99 mM, 1.55 mM, and 0.062 mM, respectively-were found to significantly reduce the CPBAmi activity. PMID:26352934

  6. Novel Substrate-Based Inhibitors of Human Glutamate Carboxypeptidase II with Enhanced Lipophilicity

    Energy Technology Data Exchange (ETDEWEB)

    Plechanovová, Anna; Byun, Youngjoo; Alquicer, Glenda; Škultétyová, L; ubica; Ml; #269; ochová, Petra; N; #283; mcová, Adriana; Kim, Hyung-Joon; Navrátil, Michal; Mease, Ronnie; Lubkowski, Jacek; Pomper, Martin; Konvalinka, Jan; Rulíšek, Lubomír; Ba; #345; inka, Cyril (NCI); (Czech Academy); (JHMI)

    2012-10-09

    Virtually all low molecular weight inhibitors of human glutamate carboxypeptidase II (GCPII) are highly polar compounds that have limited use in settings where more lipophilic molecules are desired. Here we report the identification and characterization of GCPII inhibitors with enhanced liphophilicity that are derived from a series of newly identified dipeptidic GCPII substrates featuring nonpolar aliphatic side chains at the C-terminus. To analyze the interactions governing the substrate recognition by GCPII, we determined crystal structures of the inactive GCPII(E424A) mutant in complex with selected dipeptides and complemented the structural data with quantum mechanics/molecular mechanics calculations. Results reveal the importance of nonpolar interactions governing GCPII affinity toward novel substrates as well as formerly unnoticed plasticity of the S1' specificity pocket. On the basis of those data, we designed, synthesized, and evaluated a series of novel GCPII inhibitors with enhanced lipophilicity, with the best candidates having low nanomolar inhibition constants and clogD > -0.3. Our findings offer new insights into the design of more lipophilic inhibitors targeting GCPII.

  7. In silico approaches to identify the potential inhibitors of glutamate carboxypeptidase II (GCPII) for neuroprotection.

    Science.gov (United States)

    Naushad, Shaik Mohammad; Janaki Ramaiah, M; Stanley, Balraj Alex; Prasanna Lakshmi, S; Vishnu Priya, J; Hussain, Tajamul; Alrokayan, Salman A; Kutala, Vijay Kumar

    2016-10-01

    To develop a potential inhibitor for glutamate carboxypeptidase II (GCPII) effective against all the eight common genetic variants reported, PyMOL molecular visualization system was used to generate models of variants using the crystal structure of GCPII i.e. 2OOT as a template. High-throughput virtual screening of 29 compounds revealed differential efficacy across the eight genetic variants (pIC50: 4.70 to 10.22). Pharmacophore analysis and quantitative structure-activity relationship (QSAR) studies revealed a urea-based N-acetyl aspartyl glutamate (NAAG) analogue as more potent inhibitor, which was effective across all the genetic variants of GCPII as evidenced by glide scores (-4.32 to -7.08) and protein-ligand interaction plots (13 interactions in wild GCPII). This molecule satisfied Lipinski rule of five and rule of three for drug-likeliness. Being a NAAG-analogue, this molecule might confer neuroprotection by inhibiting glutamatergic neurotransmission mediated by N-acetylated alpha-linked acidic dipeptidase (NAALADase), a splice variant of GCPII. PMID:27430729

  8. Soluble expression, purification and functional characterisation of carboxypeptidase G2 and its individual domains.

    Science.gov (United States)

    Jeyaharan, Dhadchayini; Aston, Philip; Garcia-Perez, Angela; Schouten, James; Davis, Paul; Dixon, Ann M

    2016-11-01

    Due to its applications in the treatment of cancer and autoimmune diseases, the 42 kDa zinc-dependent metalloenzyme carboxypeptidase G2 (CPG2) is of great therapeutic interest. An X-ray crystal structure of unliganded CPG2 reported in 1997 revealed the domain architecture and informed early rational drug design efforts, however further efforts at co-crystallization of CPG2 with ligands, substrates or inhibitors have not been reported. Thus key features of CPG2 such as the location of the active site, the presence of additional ligand-binding sites, stability, oligomeric state, and the molecular basis of activity remain largely unknown, with the current working understanding of CPG2 activity based primarily on computational modelling. To facilitate renewed efforts in CPG2 structural biology, we report the first high-yield (250 mg L(-1)) recombinant expression (and purification) of soluble and active CPG2 using the Escherichia coli expression system. We used this protocol to produce full-length enzyme, as well as protein fragments corresponding to the individual catalytic and dimerization domains, and the activity and stability of each construct was characterised. We adapted our protocol to allow for uniform incorporation of NMR labels ((13)C, (15)N and (2)H) and present preliminary solution-state NMR spectra of high quality. Taken together, our results offer a route for production and solution-state characterization that supports renewed effort in CPG2 structural biology as well as design of significantly truncated CPG2 proteins, which retain activity while yielding (potentially) improved immunogenicity. PMID:27374188

  9. High expression of human carboxypeptidase M in Pichia pastoris: Purification and partial characterization

    Directory of Open Access Journals (Sweden)

    R.B. Craveiro

    2006-02-01

    Full Text Available Carboxypeptidase M (CPM is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein-1 min-1. This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa, suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes

  10. High expression of human carboxypeptidase M in Pichia pastoris: Purification and partial characterization

    Directory of Open Access Journals (Sweden)

    Craveiro R.B.

    2006-01-01

    Full Text Available Carboxypeptidase M (CPM is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein-1 min-1. This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa, suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes

  11. Synthesis and evaluation of an inhibitor of carboxypeptidase A with a Ki value in the femtomolar range.

    Science.gov (United States)

    Kaplan, A P; Bartlett, P A

    1991-08-20

    Comparative studies among a series of tripeptide phosphonate inhibitors of the zinc peptidase carboxypeptidase A indicate that incorporation of the phosphonic acid analogue of valine at the P1 position results in significantly higher affinity than the glycine, alanine, or phenylalanine analogues. When applied to the tripeptide analogue Cbz-Phe-ValP-(O)Phe [ZFVP(O)F], determination of the inhibition constant Ki was complicated by the very slow rate of dissociation. The rate of exchange of [3H]ZFVP(O)F with enzyme-bound [14C]ZFVP(O)F was followed for periods of 3-4 months to measure dissociation rate constants in the range of (1.7-4.4) x 10(-9) s-1, corresponding to half-lives of 5-13 years. Although the on- and off-rate constants differ for different carboxypeptidase isozymes, their ratios, corresponding to the inhibition constants Ki, are consistently in the range of 10-27 fM. Both the inhibition constants and the dissociation rate constants appear to be the lowest values yet determined for an enzyme-small inhibitor interaction. PMID:1868091

  12. Nitrogen source and growth stage of Candida albicans influence expression level of vacuolar aspartic protease Apr1p and carboxypeptidase Cpy1p

    Czech Academy of Sciences Publication Activity Database

    Bauerová, Václava; Pichová, Iva; Hrušková-Heidingsfeldová, Olga

    2012-01-01

    Roč. 58, č. 5 (2012), s. 678-681. ISSN 0008-4166 R&D Projects: GA MŠk(CZ) LC531; GA ČR GA310/09/1945 Institutional research plan: CEZ:AV0Z40550506 Keywords : Candida albicans * carboxypeptidase Y * nitrogen starvation * vacuole Subject RIV: CE - Biochemistry Impact factor: 1.199, year: 2012

  13. Characterization of monoclonal antibodies against glutamate carboxypeptidase II, the diagnostic and potential therapeutic prostate cancer marker, via the method of surface plasmon resonance

    Czech Academy of Sciences Publication Activity Database

    Tykvart, Jan; Šácha, Pavel; Konvalinka, Jan

    2012-01-01

    Roč. 279, SI suppl. 1 (2012), s. 90-90. ISSN 1742-464X. [IUBMB Congress /22./ and FEBS Congress /37./. 04.09.2012-09.09.2012, Seville] Institutional support: RVO:61388963 Keywords : glutamate carboxypeptidase II Subject RIV: CE - Biochemistry

  14. The pro region required for folding of carboxypeptidase Y is a partially folded domain with little regular structural core

    DEFF Research Database (Denmark)

    Sørensen, P; Winther, Jakob R.; Kaarsholm, N C; Poulsen, F M

    1993-01-01

    pro region is a partially folded protein domain under the conditions where it is functional. It is characterized by a relatively high content of secondary structural elements but a very low content of defined tertiary structure. Although these characteristics are reminiscent of the compact denatured...... states that have been identified as intermediates in protein folding ("molten globules"), the pro region exhibits only very weak binding of the hydrophobic probe 1-anilino-8-naphthalenesulfonate, and it is resistant toward complete thermal unfolding. Altogether the data indicate an extremely flexible......The pro region of carboxypeptidase Y (CPY) from yeast is necessary for the correct folding of the enzyme [Winther, J. R., & Sørensen P. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 9330-9334]. Using fluorescence, circular dichroism, and heteronuclear NMR analyses, it is demonstrated that the isolated...

  15. The Cell Shape-determining Csd6 Protein from Helicobacter pylori Constitutes a New Family of L,D-Carboxypeptidase.

    Science.gov (United States)

    Kim, Hyoun Sook; Im, Ha Na; An, Doo Ri; Yoon, Ji Young; Jang, Jun Young; Mobashery, Shahriar; Hesek, Dusan; Lee, Mijoon; Yoo, Jakyung; Cui, Minghua; Choi, Sun; Kim, Cheolhee; Lee, Nam Ki; Kim, Soon-Jong; Kim, Jin Young; Bang, Geul; Han, Byung Woo; Lee, Byung Il; Yoon, Hye Jin; Suh, Se Won

    2015-10-01

    Helicobacter pylori causes gastrointestinal diseases, including gastric cancer. Its high motility in the viscous gastric mucosa facilitates colonization of the human stomach and depends on the helical cell shape and the flagella. In H. pylori, Csd6 is one of the cell shape-determining proteins that play key roles in alteration of cross-linking or by trimming of peptidoglycan muropeptides. Csd6 is also involved in deglycosylation of the flagellar protein FlaA. To better understand its function, biochemical, biophysical, and structural characterizations were carried out. We show that Csd6 has a three-domain architecture and exists as a dimer in solution. The N-terminal domain plays a key role in dimerization. The middle catalytic domain resembles those of l,d-transpeptidases, but its pocket-shaped active site is uniquely defined by the four loops I to IV, among which loops I and III show the most distinct variations from the known l,d-transpeptidases. Mass analyses confirm that Csd6 functions only as an l,d-carboxypeptidase and not as an l,d-transpeptidase. The d-Ala-complexed structure suggests possible binding modes of both the substrate and product to the catalytic domain. The C-terminal nuclear transport factor 2-like domain possesses a deep pocket for possible binding of pseudaminic acid, and in silico docking supports its role in deglycosylation of flagellin. On the basis of these findings, it is proposed that H. pylori Csd6 and its homologs constitute a new family of l,d-carboxypeptidase. This work provides insights into the function of Csd6 in regulating the helical cell shape and motility of H. pylori. PMID:26306031

  16. Peptide synthesis by enzymatic catalysis: new application to the total radiosynthesis of the tritiated leucine-enkephalin hormone, using Y carboxypeptidase

    International Nuclear Information System (INIS)

    A new method of enzymatic labelling of peptide hormones is described. The enzyme used, a protease, Y carboxypeptidase is able, in some conditions, to catalyze the formation of peptide bounds. This property has been used for the synthesis of a pentapeptide, the tritiated leucine-enkephalin, with the incorporation of every radioactive amino acid. The specific radioactivity of the labelled molecule is 139 Ci/mmole and its biological properties (receptor binding and immunoreactivity) are identical with native leucine-enkephalin properties

  17. Carboxypeptidase E, an essential element of the regulated secretory pathway, is expressed and partially co-localized with chromogranin A in chicken thymus

    OpenAIRE

    Zhang, Xiaodong; Zhu, James; Loh, Y. Peng; Luc R. Berghman

    2009-01-01

    Although the functions of hormones and neuropeptides in the thymus have been extensively studied, we still do not know whether these intrathymic humoral elements are released in a stimulated manner via the regulated secretory pathway or in a constitutive manner. Carboxypeptidase E (CpE) and chromogranin A (CgA) are functional and structural hallmarks of the regulated secretory pathway in (neuro) endocrine cells. Whereas we have previously shown a CgA-positive neuroendocrine population in the ...

  18. Carboxypeptidase E Protects Hippocampal Neurons During Stress in Male Mice by Up-regulating Pro-survival BCL2 Protein Expression

    OpenAIRE

    Murthy, S. R. K.; Thouennon, E.; Li, W.-S.; Cheng, Y; Bhupatkar, J.; Cawley, N.X.; Lane, M.; Merchenthaler, I; Loh, Y P

    2013-01-01

    Prolonged chronic stress causing elevated plasma glucocorticoids leads to neurodegeneration. Adaptation to stress (allostasis) through neuroprotective mechanisms can delay this process. Studies on hippocampal neurons have identified carboxypeptidase E (CPE) as a novel neuroprotective protein that acts extracellularly, independent of its enzymatic activity, although the mechanism of action is unclear. Here, we aim to determine if CPE plays a neuroprotective role in allostasis in mouse hippocam...

  19. A germination-related gene encoding a serine carboxypeptidase is expressed during the differentiation of the vascular tissue in wheat grains and seedlings.

    Science.gov (United States)

    Domínguez, Fernando; González, María Cruz; Cejudo, Francisco J

    2002-09-01

    Carboxypeptidases expressed in the aleurone layer participate in the mobilization of endosperm storage proteins during cereal grain germination. The genes encoding these proteins are also expressed in the scutellum of germinating grains, but their function in this organ is not yet clear. We have analyzed the expression of a carboxypeptidase III (CPIII) gene in germinating wheat (Triticum aestivum L.) grains. CPIII transcripts accumulated transiently in the scutellum showing a maximum at 2-3 days after imbibition and were exclusively localized to the scutellar vascular tissue. The analysis of CPIII expression in developing shoots and roots from growing seedlings confirmed the localization of CPIII transcripts to differentiating vascular tissue. The TUNEL assay detected in situ nuclear DNA fragmentation in cells showing CPIII expression, indicating that they undergo programmed cell death. Relative RT-PCR analysis showed that the CPIII gene expressed at high level in aleurone cells is the one expressed in vegetative tissues, and allowed the use of this gene as a molecular marker of tracheary element differentiation in wheat seedlings. These results are indicative of the involvement of serine carboxypeptidases in programmed cell death during the development of the vascular tissue in wheat, a new role for these enzymes, besides the mobilization of starchy-endosperm proteins during germination. PMID:12244437

  20. Association of human carboxypeptidase E exon5 gene polymorphisms with angiographical characteristics of coronary atherosclerosis in a Chinese population

    Institute of Scientific and Technical Information of China (English)

    Jie WANG; En-zhi JIA; Yun ZHANG; Zhi-jian YANG; Tie-bing ZHU; Lian-sheng WANG; Bo CHEN; Ke-jiang CAO; Jun HUANG; Wen-zhu MA

    2008-01-01

    Aim: To explore the association between 801C>T and 847C>T polymorphisms of the human carboxypeptidase E (CPE) gene exon5, which could cause hyperproinsulinemia, and the angiographical characteristics of coronary atherosclerosis. Methods: In total, 1044 consecutive patients who underwent coronary angiography for suspected or known coronary atherosclerosis were examined with respect to their genotypes, insulin, proinsulin level, and other risk factors of coronary atherosclerosis. The angiographical characteristics of coro-nary atherosclerosis (ie the severity of coronary heart disease) were defined by Gensini's score (GS) system. Results: The results showed that the genotype frequencies ofCC, CT, and TT at 801C>T locus were significantly different among the patients of the 4 groups who were classified by quartile values of GS (P=0.033). However, the frequency of the 847T allele was 0 for all the patients. The ordinal logistic regression analysis revealed that the increased risk of angiographical characteristics of coronary atherosclerosis were associated with CPE 801CT/TT variant genotypes [adjusted odds ratio (OR)=1.23, 95% confidence interval (CI) =0.93-1.63 for 801CT and adjusted OR=3.13, 95% CI=1.18-8.28 for 801TT] com-pared with the 801CC wild-type homozygotes. A stratification analysis showed that the effects of the CPE 801TT genotype were more evident among subgroups with relatively older (>=60 years) patients, males, and smokers. Furthermore, an analysis of covariance controlling age, sex, and body mass index indicated that differences of blood glucose, insulin, insulin resistance, and the proinsulin level between 801C>T genotype groups were not statistically significant. Conclusion: These findings indicate that the 801C>T polymorphism in the CPE exon5 gene may contribute to the angiographical characteristics of coronary atherosclerosis in the Chinese population.

  1. Truncating Homozygous Mutation of Carboxypeptidase E (CPE in a Morbidly Obese Female with Type 2 Diabetes Mellitus, Intellectual Disability and Hypogonadotrophic Hypogonadism.

    Directory of Open Access Journals (Sweden)

    Suzanne I M Alsters

    Full Text Available Carboxypeptidase E is a peptide processing enzyme, involved in cleaving numerous peptide precursors, including neuropeptides and hormones involved in appetite control and glucose metabolism. Exome sequencing of a morbidly obese female from a consanguineous family revealed homozygosity for a truncating mutation of the CPE gene (c.76_98del; p.E26RfsX68. Analysis detected no CPE expression in whole blood-derived RNA from the proband, consistent with nonsense-mediated decay. The morbid obesity, intellectual disability, abnormal glucose homeostasis and hypogonadotrophic hypogonadism seen in this individual recapitulates phenotypes in the previously described fat/fat and Cpe knockout mouse models, evidencing the importance of this peptide/hormone-processing enzyme in regulating body weight, metabolism, and brain and reproductive function in humans.

  2. Crystal growth of phosphopantetheine adenylyltransferase, carboxypeptidase t, and thymidine phosphorylase on the international space station by the capillary counter-diffusion method

    International Nuclear Information System (INIS)

    Crystals of phosphopantetheine adenylyltransferase from Mycobacterium tuberculosis, thymidine phosphorylase from Escherichia coli, carboxypeptidase T from Thermoactinomyces vulgaris and its mutant forms, and crystals of complexes of these proteins with functional ligands and inhibitors were grown by the capillary counter-diffusion method in the Japanese Experimental Module Kibo on the International Space Station. The high-resolution X-ray diffraction data sets suitable for the determination of high-resolution three-dimensional structures of these proteins were collected from the grown crystals on the SPring-8 synchrotron radiation facility. The conditions of crystal growth for the proteins and the data-collection statistics are reported. The crystals grown in microgravity diffracted to a higher resolution than crystals of the same proteins grown on Earth.

  3. Characterization of SCO4439, a D-alanyl-D-alanine carboxypeptidase involved in spore cell wall maturation, resistance, and germination in Streptomyces coelicolor.

    Science.gov (United States)

    Rioseras, Beatriz; Yagüe, Paula; López-García, María Teresa; Gonzalez-Quiñonez, Nathaly; Binda, Elisa; Marinelli, Flavia; Manteca, Angel

    2016-01-01

    This work contributes to the understanding of cell wall modifications during sporulation and germination in Streptomyces by assessing the biological function and biochemical properties of SCO4439, a D-alanyl-D-alanine carboxypeptidase (DD-CPase) constitutively expressed during development. SCO4439 harbors a DD-CPase domain and a putative transcriptional regulator domain, separated by a putative transmembrane region. The recombinant protein shows that DD-CPase activity is inhibited by penicillin G. The spores of the SCO4439::Tn5062 mutant are affected in their resistance to heat and acid and showed a dramatic increase in swelling during germination. The mycelium of the SCO4439::Tn5062 mutant is more sensitive to glycopeptide antibiotics (vancomycin and teicoplanin). The DD-CPase domain and the hydrophobic transmembrane region are highly conserved in Streptomyces, and both are essential for complementing the wild type phenotypes in the mutant. A model for the biological mechanism behind the observed phenotypes is proposed, in which SCO4439 DD-CPase releases D-Ala from peptidoglycan (PG) precursors, thereby reducing the substrate pool for PG crosslinking (transpeptidation). PG crosslinking regulates spore physical resistance and germination, and modulates mycelium resistance to glycopeptides. This study is the first demonstration of the role of a DD-CPase in the maturation of the spore cell wall. PMID:26867711

  4. Sequential on-line C-terminal sequencing of peptides based on carboxypeptidase Y digestion and optically gated capillary electrophoresis with laser-induced fluorescence detection.

    Science.gov (United States)

    Tian, Miaomiao; Zhang, Ning; Liu, Xiaoxia; Guo, Liping; Yang, Li

    2016-08-12

    We report a novel method for sequential on-line C-terminal sequencing of peptides, which combines carboxypeptidase Y (CPY) digestion with on-line derivatization and optically gated capillary electrophoresis with laser-induced fluorescence detection (OGCE-LIF). Various factors that may affect the C-terminal sequencing were investigated and optimized. High repeatability of on-line derivatization and the sequential OGCE-LIF assay of amino acids (AAs) was achieved with relative standard deviation (RSD) (n=20) less than 1.5% and 3.2% for migration time and peak height, respectively. A total of 13 AAs was efficiently separated in the present study, indicating that the method can be used for sequencing of peptides consisting of the 13 AAs studied. Using two synthesized N-terminally blocked peptides as test examples, we show that the present method can on-line monitor the released AAs with a temporal resolution of 50s during the entire CPY digestion process. The rates of AA release as a function of digestion time were easily measured; thus, the AA sequence of the peptide was determined with just one OGCE assay. Our study indicates the present approach is an effective, reliable, and convenient method for rapid analysis of the C-terminal sequence of peptides, with potential application in peptide analysis and proteome research. PMID:27425760

  5. Synthesis and Evaluation of Optical 2-Benzyl-5-bromo-4- oxopentanoic Acids as Transition-state Analog Inhibitors against Carboxypeptidase A

    Institute of Scientific and Technical Information of China (English)

    JIN,Jing-Yi; WANG,Shou-Feng; XUAN,Wei; SHENG,Ji-Wen; WANG,Si-Hong; TIAN,Guan-Rong

    2008-01-01

    Both enantiomers of 2-benzyl-5-bromo-4-oxopentanoic acid were prepared utilizing the diazo ketones as the key intermediates. The compounds were assayed for inhibitory activity against carboxypeptidase A (CPA, EC 3.4.17.1). The (R)-form is 260-fold more potent than the corresponding (S)-form. The finding that (R)-form, which belongs to the L-series, is mostly responsible for the inhibitory activity accords with the substrate specificity of CPA.For comparison, both the optical forms of 2-benzyl-4-oxopentanoic acid were also synthesized and evaluated as the inhibitors against CPA. These results reveal that the introduction of a bromo group at the α-position of ketones can significantly enhance the electrophilicity of the carbonyl group. Further molecular docking study suggested that the gem-diol form of the α-bromo ketone, which mimics the transition state in the CPA catalytic process, could chelate the zinc ion in the active site of CPA and thus result in the strong inhibition.

  6. Knockdown of Carboxypeptidase A6 in Zebrafish Larvae Reduces Response to Seizure-Inducing Drugs and Causes Changes in the Level of mRNAs Encoding Signaling Molecules

    Science.gov (United States)

    Lopes, Mark William; Sapio, Matthew R.; Leal, Rodrigo B.; Fricker, Lloyd D.

    2016-01-01

    Carboxypeptidase A6 (CPA6) is an extracellular matrix metallocarboxypeptidase that modulates peptide and protein function by removal of hydrophobic C-terminal amino acids. Mutations in the human CPA6 gene that reduce enzymatic activity in the extracellular matrix are associated with febrile seizures, temporal lobe epilepsy, and juvenile myoclonic epilepsy. The characterization of these human mutations suggests a dominant mode of inheritance by haploinsufficiency through loss of function mutations, however the total number of humans with pathologic mutations in CPA6 identified to date remains small. To better understand the relationship between CPA6 and seizures we investigated the effects of morpholino knockdown of cpa6 mRNA in zebrafish (Danio rerio) larvae. Knockdown of cpa6 mRNA resulted in resistance to the effect of seizure-inducing drugs pentylenetetrazole and pilocarpine on swimming behaviors. Knockdown of cpa6 mRNA also reduced the levels of mRNAs encoding neuropeptide precursors (bdnf, npy, chga, pcsk1nl, tac1, nts, edn1), a neuropeptide processing enzyme (cpe), transcription factor (c-fos), and molecules implicated in glutamatergic signaling (grin1a and slc1a2b). Treatment of zebrafish embryos with 60 mM pilocarpine for 1 hour led to reductions in levels of many of the same mRNAs when measured 1 day after pilocarpine exposure, except for c-fos which was elevated 1 day after pilocarpine treatment. Pilocarpine treatment, like cpa6 knockdown, led to a reduced sensitivity to pentylenetetrazole when tested 1 day after pilocarpine treatment. Taken together, these results add to mounting evidence that peptidergic systems participate in the biological effects of seizure-inducing drugs, and are the first in vivo demonstration of the molecular and behavioral consequences of cpa6 insufficiency. PMID:27050163

  7. Knockdown of Carboxypeptidase A6 in Zebrafish Larvae Reduces Response to Seizure-Inducing Drugs and Causes Changes in the Level of mRNAs Encoding Signaling Molecules.

    Science.gov (United States)

    Lopes, Mark William; Sapio, Matthew R; Leal, Rodrigo B; Fricker, Lloyd D

    2016-01-01

    Carboxypeptidase A6 (CPA6) is an extracellular matrix metallocarboxypeptidase that modulates peptide and protein function by removal of hydrophobic C-terminal amino acids. Mutations in the human CPA6 gene that reduce enzymatic activity in the extracellular matrix are associated with febrile seizures, temporal lobe epilepsy, and juvenile myoclonic epilepsy. The characterization of these human mutations suggests a dominant mode of inheritance by haploinsufficiency through loss of function mutations, however the total number of humans with pathologic mutations in CPA6 identified to date remains small. To better understand the relationship between CPA6 and seizures we investigated the effects of morpholino knockdown of cpa6 mRNA in zebrafish (Danio rerio) larvae. Knockdown of cpa6 mRNA resulted in resistance to the effect of seizure-inducing drugs pentylenetetrazole and pilocarpine on swimming behaviors. Knockdown of cpa6 mRNA also reduced the levels of mRNAs encoding neuropeptide precursors (bdnf, npy, chga, pcsk1nl, tac1, nts, edn1), a neuropeptide processing enzyme (cpe), transcription factor (c-fos), and molecules implicated in glutamatergic signaling (grin1a and slc1a2b). Treatment of zebrafish embryos with 60 mM pilocarpine for 1 hour led to reductions in levels of many of the same mRNAs when measured 1 day after pilocarpine exposure, except for c-fos which was elevated 1 day after pilocarpine treatment. Pilocarpine treatment, like cpa6 knockdown, led to a reduced sensitivity to pentylenetetrazole when tested 1 day after pilocarpine treatment. Taken together, these results add to mounting evidence that peptidergic systems participate in the biological effects of seizure-inducing drugs, and are the first in vivo demonstration of the molecular and behavioral consequences of cpa6 insufficiency. PMID:27050163

  8. Genome-wide comparative study of rice and Arabidopsis serine carboxypeptidase-like protein families%水稻与拟南芥全基因组丝氨酸羧肽酶类蛋白家族比较分析

    Institute of Scientific and Technical Information of China (English)

    冯英; 俞朝

    2009-01-01

    基于水稻与拟南芥全基因组序列,在基因组与蛋白质组水平上对这2种模式植物的丝氨酸羧肽酶 (SCPs)基因家族进行比较分析.利用隐马可夫模型(hidden Markov models,HMM),发现水稻与拟南芥中分别存在71个与54个丝氨酸羧肽酶类(serine carboxypeptidases like,SCPL)蛋白编码基因,它们广泛分布于基因组中各条染色体上,并且存在多个基因簇聚区.基因结构分析显示,拟南芥的SCPL基因存在广泛的交替剪接方式,而这种现象在水稻SCPL基因中却不常见.蛋白结构分析表明,所有SCPL家族成员均具有α/β水解酶折叠亚族与S10家族典型的保守结构域与二级结构特征.系统进化分析表明,这125个SCPL蛋白可以分成3大类,与水稻不同,大多数拟南芥SCPL (88.9%)可归属于双链羧肽酶Ⅰ或Ⅱ.%The genomes from the model plants rice and Arabidopsis thaliana were systematically mined for serine carboxypeptidases (SCPs) genes and a comparative analysis of them were done at both gene and protein levels. Based on hidden Markov models (HMM), 71 and 54 SCPL (serine carboxypeptidases like) proteins coding genes were identified from rice and Arabidopsis thaliana, respectively. Those genes were widely distributed across the genomes, with the exception of several gene clusters probably resulted from recently gene duplication. Gene structure analysis indicated that remarkable number of Arabidopsis thaliana SCPL genes harbored alternative splice sites contrasting to seldom alternative splicing happened in those of rice genes. All SCPL family members shared conserved sequence motifs and secondary structure characteristics with S10 family enzymes and other members of the larger α/β hydrolase fold superfamily of enzymes. Phylogenetics analysis of the 125 putative genes and other SCPs distinguished three distinct clades, and although most Arabidopsis thaliana genes (88.9%) belong to two chains carboxypeptidase or Ⅱ, the 71 rice genes show

  9. Characterization of the porcine carboxypeptidase E cDNA

    DEFF Research Database (Denmark)

    Hreidarsdôttir, G.E.; Cirera, Susanna; Fredholm, Merete

    2007-01-01

    very low nonsynonymousl/synonymous substitution ratios between the proteins was found indicating that purifying selection os acting on the CPE gene. A nonsynonymous SNP identified at position 1272 in the transcript resulting in a codon change from TCA (Serine) to TTA (Leucine) was genotyped in the...

  10. Glutamate carboxypeptidase II does not process amyloid-beta peptide

    Czech Academy of Sciences Publication Activity Database

    Sedlák, František; Šácha, Pavel; Blechová, Miroslava; Březinová, Anna; Šafařík, Martin; Šebestík, Jaroslav; Konvalinka, Jan

    2013-01-01

    Roč. 27, č. 7 (2013), s. 2626-2632. ISSN 0892-6638 R&D Projects: GA ČR GAP304/12/0847 Institutional support: RVO:61388963 Keywords : PSMA * Alzheimer's disease * disaggregation * exopeptidase * substrate specificity * depsipeptide Subject RIV: CE - Biochemistry Impact factor: 5.480, year: 2013

  11. Functional Annotation of Two New Carboxypeptidases from the Amidohydrolase Superfamily of Enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, D.; Xu, C; Kumaran, D; Brown, A; Sauder, M; Burley, S; Swaminathan, S; Raushel, F

    2009-01-01

    Two proteins from the amidohydrolase superfamily of enzymes were cloned, expressed, and purified to homogeneity. The first protein, Cc0300, was from Caulobacter crescentus CB-15 (Cc0300), while the second one (Sgx9355e) was derived from an environmental DNA sequence originally isolated from the Sargasso Sea (gi|44371129). The catalytic functions and the substrate profiles for the two enzymes were determined with the aid of combinatorial dipeptide libraries. Both enzymes were shown to catalyze the hydrolysis of l-Xaa-l-Xaa dipeptides in which the amino acid at the N-terminus was relatively unimportant. These enzymes were specific for hydrophobic amino acids at the C-terminus. With Cc0300, substrates terminating in isoleucine, leucine, phenylalanine, tyrosine, valine, methionine, and tryptophan were hydrolyzed. The same specificity was observed with Sgx9355e, but this protein was also able to hydrolyze peptides terminating in threonine. Both enzymes were able to hydrolyze N-acetyl and N-formyl derivatives of the hydrophobic amino acids and tripeptides. The best substrates identified for Cc0300 were l-Ala-l-Leu with kcat and kcat/Km values of 37 s-1 and 1.1 x 105 M-1 s-1, respectively, and N-formyl-l-Tyr with kcat and kcat/Km values of 33 s-1 and 3.9 x 105 M-1 s-1, respectively. The best substrate identified for Sgx9355e was l-Ala-l-Phe with kcat and kcat/Km values of 0.41 s-1 and 5.8 x 103 M-1 s-1. The three-dimensional structure of Sgx9355e was determined to a resolution of 2.33 Angstroms with l-methionine bound in the active site. The a-carboxylate of the methionine is ion-paired to His-237 and also hydrogen bonded to the backbone amide groups of Val-201 and Leu-202. The a-amino group of the bound methionine interacts with Asp-328. The structural determinants for substrate recognition were identified and compared with other enzymes in this superfamily that hydrolyze dipeptides with different specificities.

  12. delta-Thiolactones as Prodrugs of Thiol-Based Glutamate Carboxypeptidase II (GCPII) Inhibitors

    Czech Academy of Sciences Publication Activity Database

    Ferraris, D. V.; Majer, Pavel; Ni, C.; Slusher, C. E.; Rais, R.; Wu, Y.; Wozniak, K. M.; Alt, J.; Rojas, C.; Slusher, B. S.; Tsukamoto, T.

    2014-01-01

    Roč. 57, č. 1 (2014), s. 243-247. ISSN 0022-2623 Institutional support: RVO:61388963 Keywords : pharmacokinetics * discovery * disorders * diagnosis * pain Subject RIV: CC - Organic Chemistry Impact factor: 5.447, year: 2014

  13. Structural characterization of P1 '-diversified urea-based inhibitors of glutamate carboxypeptidase II

    Czech Academy of Sciences Publication Activity Database

    Pavlíček, Jiří; Ptáček, Jakub; Černý, Jiří; Byun, Y.; Škultétyová, Ĺubica; Pomper, M.G.; Lubkowski, J.; Bařinka, Cyril

    2014-01-01

    Roč. 24, č. 10 (2014), s. 2340-2345. ISSN 0960-894X R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109 Grant ostatní: EMBO(DE) 1978 Institutional support: RVO:86652036 Keywords : GCPII * Prostate-specific membrane antigen * PSMA Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 2.420, year: 2014

  14. Structural basis of the resistance of an insect carboxypeptidase to plant protease inhibitors

    NARCIS (Netherlands)

    Bayés, A.; Comellas-Bigler, M.; Rodriquez de la Vega, M.; Maskos, K.; Bode, W.; Aviles, F.X.; Beekwilder, M.J.; Vendrell, J.

    2005-01-01

    Corn earworm (Helicoverpa zea), also called tomato fruitworm, is a common pest of many Solanaceous plants. This insect is known to adapt to the ingestion of plant serine protease inhibitors by using digestive proteases that are insensitive to inhibition. We have now identified a B-type carboxypeptid

  15. Generation and basic characterization of glutamate carboxypeptidase II knock-out mice

    Czech Academy of Sciences Publication Activity Database

    Vorlová, Barbora; Kašpárek, Petr; Šácha, Pavel; Sedláček, Radislav; Konvalinka, Jan

    2016-01-01

    Roč. 25, č. 2 (2016), s. 267. ISSN 0962-8819. [Transgenic Technology Meeting (TT2016) /13./. 20.03.2016-23.03.2016, Praha] Institutional support: RVO:61388963 ; RVO:68378050 Keywords : GCPII * PSMA * FolhI * knock-out mice Subject RIV: CE - Biochemistry

  16. Design of composite inhibitors targeting glutamate carboxypeptidase II: the importance of effector functionalities

    Czech Academy of Sciences Publication Activity Database

    Nováková, Zora; Černý, Jiří; Choy, C.J.; Nedrow, J.R.; Choi, J.K.; Lubkowski, J.; Berkman, C.E.; Bařinka, Cyril

    2016-01-01

    Roč. 283, č. 1 (2016), s. 130-143. ISSN 1742-464X R&D Projects: GA ČR GAP301/12/1513; GA MŠk(CZ) ED1.1.00/02.0109 Grant ostatní: GA MŠk(CZ) CZ.1.07/2.3.00/30.0045 Institutional support: RVO:86652036 Keywords : molecular modeling * NAALADase * prostate-specific membrane antigen * X-ray crystallography Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.001, year: 2014

  17. Design of Highly Potent Urea-Based, Exosite-Binding Inhibitors Selective for Glutamate Carboxypeptidase II

    Czech Academy of Sciences Publication Activity Database

    Tykvart, Jan; Schimer, Jiří; Jančařík, Andrej; Bařinková, Jitka; Navrátil, Václav; Starková, Jana; Šrámková, Karolína; Konvalinka, Jan; Majer, Pavel; Šácha, Pavel

    2015-01-01

    Roč. 58, č. 10 (2015), s. 4357-4363. ISSN 0022-2623 R&D Projects: GA ČR GBP208/12/G016; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : GCPII selective inhibitors * GCPIII * GCPII Subject RIV: CE - Biochemistry Impact factor: 5.447, year: 2014

  18. Structure of glutamate carboxypeptidase II, a drug target in neuronal damage and prostate cancer

    Czech Academy of Sciences Publication Activity Database

    Mesters, J. R.; Bařinka, Cyril; Li, W.; Tsukamoto, T.; Majer, P.; Slusher, B.; Konvalinka, Jan; Hilgenfeld, R.

    2006-01-01

    Roč. 25, č. 6 (2006), s. 1375-1384. ISSN 0261-4189 R&D Projects: GA ČR(CZ) GA301/03/0784; GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : NAALADase * neurodegenerative disease * peptidase * prostate cancer Subject RIV: CE - Biochemistry Impact factor: 10.086, year: 2006

  19. Structural basis of interactions between human glutamate carboxypeptidase II and its substrate analogs

    Czech Academy of Sciences Publication Activity Database

    Bařinka, C.; Hlouchová, Klára; Rovenská, Miroslava; Majer, P.; Dauter, M.; Hin, N.; Ko, Y. S.; Tsukamoto, T.; Slusher, B. S.; Konvalinka, Jan; Lubkowski, J.

    2008-01-01

    Roč. 376, č. 5 (2008), s. 1438-1450. ISSN 0022-2836 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : prostate-specific membrane antigen * metallopeptidase * folate hydrolase * NAALADase * phosphapeptide Subject RIV: CE - Biochemistry Impact factor: 4.146, year: 2008

  20. A t(3;8) chromosomal translocation associated with hepatitis B virus intergration involves the carboxypeptidase N locus.

    OpenAIRE

    Pineau, P.; Marchio, A; Terris, B; Mattei, M G; Tu, Z X; Tiollais, P; Dejean, A

    1996-01-01

    Integrated hepatitis B virus (HBV) DNA is found in the great majority of human hepatocellular carcinomas, suggesting that these viral integrations may be implicated in liver oncogenesis. Besides the insertional mutagenesis characterized in a few selected cases and the contribution of viral transactivators to cell transformation to malignancy, HBV has been shown to generate gross chromosomal rearrangements potentially involved in carcinogenesis. Here, we report a t(3;8) chromosomal translocati...

  1. Onset of obesity in carboxypeptidase E-deficient mice and effect on airway responsiveness and pulmonary responses to ozone

    OpenAIRE

    Johnston, Richard A.; Zhu, Ming; Hernandez, Christopher B.; Williams, Erin S.; Shore, Stephanie A.

    2010-01-01

    When compared with lean, wild-type mice, obese Cpefat mice, 14 wk of age and older, manifest innate airway hyperresponsiveness (AHR) to intravenous methacholine and enhanced pulmonary inflammation following acute exposure to ozone (O3). The purpose of this study was to examine the onset of these augmented pulmonary responses during the onset of obesity. Thus airway responsiveness and O3-induced pulmonary inflammation and injury were examined in 7- and 10-wk-old Cpefat and age-matched, wild-ty...

  2. Insulin regulates carboxypeptidase E by modulating translation initiation scaffolding protein eIF4G1 in pancreatic β cells

    OpenAIRE

    Liew, Chong Wee; Assmann, Anke; Templin, Andrew T.; Raum, Jeffrey C.; Kathryn L Lipson; Rajan, Sindhu; Qiang, Guifen; Hu, Jiang; Kawamori, Dan; Lindberg, Iris; Philipson, Louis H.; Sonenberg, Nahum; Goldfine, Allison B.; Stoffers, Doris A.; Mirmira, Raghavendra G.

    2014-01-01

    Elevated circulating proinsulin and a poor biological response to insulin are observed early in individuals with type 2 diabetes. Genome-wide association studies have recently identified genes associated with proinsulin processing, and clinical observations suggest that elevated proinsulin and its intermediates are markers of dysfunctional insulin-secreting β cells. Here, we propose a previously unidentified mechanism in the regulation of an enzyme that is involved in proinsulin processing ca...

  3. Rational design of urea-based glutamate carboxypeptidase II (GCPII) inhibitors as versatile tools for specific drug targeting and delivery

    Czech Academy of Sciences Publication Activity Database

    Tykvart, Jan; Schimer, Jiří; Bařinková, Jitka; Pachl, Petr; Poštová Slavětínská, Lenka; Majer, Pavel; Konvalinka, Jan; Šácha, Pavel

    2014-01-01

    Roč. 22, č. 15 (2014), s. 4099-4108. ISSN 0968-0896 R&D Projects: GA ČR GBP208/12/G016 Grant ostatní: OPPK(CZ) CZ.2.16/3.1.00/24016 Institutional support: RVO:61388963 Keywords : GCPII * PSMA * structure-aided drug design * specific drug targeting Subject RIV: CE - Biochemistry Impact factor: 2.793, year: 2014

  4. Structural and biochemical characterization of the folyl-poly-gamma-L-glutamate hydrolyzing activity of human glutamate carboxypeptidase II

    Czech Academy of Sciences Publication Activity Database

    Navrátil, Michal; Ptáček, Jakub; Šácha, Pavel; Starková, Jana; Lubkowski, J.; Bařinka, Cyril; Konvalinka, Jan

    2014-01-01

    Roč. 281, č. 14 (2014), s. 3228-3242. ISSN 1742-464X R&D Projects: GA ČR GAP304/12/0847; GA MŠk(CZ) ED1.1.00/02.0109 Grant ostatní: OPPC(CZ) CZ.2.16/3.1.00/24016 Institutional support: RVO:61388963 ; RVO:86652036 Keywords : arene-binding site * crystal structure * folate hydrolase 1 * H475Y(1561C -> T) * polymorphism * zinc metalloprotease Subject RIV: CE - Biochemistry Impact factor: 4.001, year: 2014

  5. Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II

    Czech Academy of Sciences Publication Activity Database

    Tykvart, Jan; Šácha, Pavel; Bařinka, Cyril; Knedlík, Tomáš; Starková, Jana; Lubkowski, J.; Konvalinka, Jan

    2012-01-01

    Roč. 82, č. 1 (2012), s. 106-115. ISSN 1046-5928 R&D Projects: GA MŠk 1M0508; GA MŠk LC512 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520701 Keywords : affinity purification * biotin acceptor peptide * recombinant protein expression * biotin-protein ligase (BirA) * co-localization * PSMA Subject RIV: CE - Biochemistry Impact factor: 1.429, year: 2012

  6. Structure and Dynamics of the Metal Site of Cadmium-Substituted Carboxypeptidase A in Solution and Crystalline States and under Steady-State peptide Catalysis

    DEFF Research Database (Denmark)

    Bauer, R.; Danielsen, E.; Hemmingsen, L.;

    1997-01-01

    geometry with an OH- ligand at the solvent site and a carbonyl oxygen at an additional ligand site. In marked contrast, conformational rigidity is not induced by the inhibitor/poor substrate Gly-L-Tyr nor by the products of high turnover substrates, Bz-Gly, Bz-Gly-Gly, and L-Phe. These results...

  7. Amino acids at the N- and C-termini of human glutamate carboxypeptidase II are required for enzymatic activity and proper folding

    Czech Academy of Sciences Publication Activity Database

    Bařinka, Cyril; Mlčochová, Petra; Šácha, Pavel; Hilgert, Ivan; Majer, P.; Slusher, B. S.; Hořejší, Václav; Konvalinka, Jan

    2004-01-01

    Roč. 271, č. 13 (2004), s. 2782-2790. ISSN 0014-2956 R&D Projects: GA AV ČR IAA5055108 Institutional research plan: CEZ:AV0Z4055905 Keywords : metallopeptidase * prostate cancer * mutagenesis Subject RIV: CE - Biochemistry Impact factor: 3.260, year: 2004

  8. Glutamate carboxypeptidase II and folate deficiencies result in reciprocal protection against cognitive and social deficits in mice: implications for neurodevelopmental disorders

    OpenAIRE

    Schaevitz, LS; Picker, JD; J. Rana; Kolodny, NH; Shane, B; Berger-Sweeney, JE; Coyle, JT

    2012-01-01

    Interactions between genetic and environmental risk factors underlie a number of neuropsychiatric disorders, including schizophrenia (SZ) and autism (AD). Due to the complexity and multitude of the genetic and environmental factors attributed to these disorders, recent research strategies focus on elucidating the common molecular pathways through which these multiple risk factors may function. In this study, we examine the combined effects of a haplo-insufficiency of glutamate carboxypeptidas...

  9. Secretory Granule Proteases in Rat Mast Cells. Cloning of 10 Different Serine Proteases and a Carboxypeptidase A from Various Rat Mast Cell Populations

    OpenAIRE

    Lützelschwab, Claudia; Pejler, Gunnar; Aveskogh, Maria; Hellman, Lars

    1997-01-01

    Two of the major rat mast cell proteases, rat mast cell protease 1 (RMCP-1) and RMCP-2, have for many years served as important phenotypic markers for studies of various aspects of mast cell (MC) biology. However, except for these proteases only fragmentary information has been available on the structure and complexity of proteases expressed by different subpopulations of rat MCs. To address these questions, cDNA libraries were constructed from freshly isolated rat peritoneal MCs and from the...

  10. Disease: H01136 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available H01136 Carboxypeptidase N deficiency Carboxypeptidase N (CPN) is a plasma zinc metalloprotease t ... dema or chronic urticaria, as well as hay fever or asthma . It has been reported that mutations in CPN1, whic ...

  11. Protein (Cyanobacteria): 29411 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available carboxypeptidase 3 Prochlorococcus marinus str. MIT 9313 MRILPACSKIAKRNHSQLLIRILGAGGILIGSNVSAAPTVLSPPPPVAIQG...WPSLQSGRLCPSLQRSFKSLLGQGSSAWSVSVLDRHGQLLADINGTIAKVPASNQKLITTAFALDKLGPDFKLRTQLLRRPDGVLEISGEGDPDLGITEIQRFAMAALGQGGSRTFRSHDDLQLLIR...LSLVNHDQVKTSSAQSVLLHEEDSAPMHALVSLANAESHNFTAEVLLRHAAKSWDVRLASREAMRWMQRQNLPLTGLRIADGSGLSRNNRMSSQTLATLLMRMGHHPLAPYYQASMAIA...GQRGTLRKLFRGTSLEGKFWGKTGTLNGVRSISGILETEDGPRYVSAIANGASSPNRTIGLLLKAIQRFSPCSSSLSTSK ... ...EEPQQRWWPSDWHPADRIYAYGAPITRLALTSNALDVAVTNPIGRLQRLLEREIHRQGGSAH

  12. Sequence Classification: 390004 [

    Lifescience Database Archive (English)

    Full Text Available IN-BINDING PROTEIN DACB1 (D-ALANYL-D-ALANINE CARBOXYPEPTIDASE) (DD-PEPTIDASE) (DD-CARBOXYPEPTIDASE) (PBP) (D...D-TRANSPEPTIDASE) (SERINE-TYPE D-ALA-D-ALA CARBOXYPEPTIDASE) || http://www.ncbi.nlm.nih.gov/protein/31794515 ...

  13. Sequence Classification: 399770 [

    Lifescience Database Archive (English)

    Full Text Available IN-BINDING PROTEIN DACB1 (D-ALANYL-D-ALANINE CARBOXYPEPTIDASE) (DD-PEPTIDASE) (DD-CARBOXYPEPTIDASE) (PBP) (D...D-TRANSPEPTIDASE) (SERINE-TYPE D-ALA-D-ALA CARBOXYPEPTIDASE) (D-AMINO ACID HYDROLASE) || http://www.ncbi.nlm.nih.gov/protein/15610466 ...

  14. Investigation of peptidyl synthase activity of the Y Carboxypeptidase: influence of the primary structure of the substrate C-terminal fragment and application to radio-marking (3H or 14C) of three neuro hypophysis hormones

    International Nuclear Information System (INIS)

    As radio-marking of peptides of biological interest induced significant advances in the metabolism study, radio-immunology and molecular endocrinology, this research thesis reports investigation performed on hormonal peptides released by the post-hypophysis. The ultimate objective is to substitute in these hormones the C-terminal Gly-NH2 by its tritiated radioactive homologue to provide these peptides with a R.A.S equal to that of the tritiated Gly-NH2 in order to study reaction mechanisms and to generalize this method to a larger number of peptides

  15. Sequence Classification: 389576 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB TMH Non-TMB Non-TMB TMB TMB >gi|31794087|ref|NP_856580.1| PROBABLE D-ALANYL-D-ALANINE CARB...OXYPEPTIDASE DACB2 (PENICILLIN-BINDING PROTEIN) (DD-PEPTIDASE) (DD-CARBOXYPEPTIDASE) (PBP) (D...D-TRANSPEPTIDASE) (SERINE-TYPE D-ALA-D-ALA CARBOXYPEPTIDASE) || http://www.ncbi.nlm.nih.gov/protein/31794087 ...

  16. Sequence Classification: 399338 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB TMH Non-TMB Non-TMB TMB TMB >gi|57117035|ref|YP_177914.1| PROBABLE D-ALANYL-D-ALANINE CARB...OXYPEPTIDASE DACB2 (PENICILLIN-BINDING PROTEIN) (DD-PEPTIDASE) (DD-CARBOXYPEPTIDASE) (PBP) (D...D-TRANSPEPTIDASE) (SERINE-TYPE D-ALA-D-ALA CARBOXYPEPTIDASE) (D-AMINO ACID HYDROLASE) || http://www.ncbi.nlm.nih.gov/protein/57117035 ...

  17. Protein (Cyanobacteria): 187174 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available ZP_10228843.1 1117:3736 1118:4133 1125:120 1160279:2131 Carboxypeptidase (fragment)...EPSISLYILVHGMANGIFTGRRLGQYVSVNRTDFVNARKVINDMDRAHHIANLAQQWLTKLNNFPVPEALPEAVNLGAGMPAFIEENVQLSEDELFLFEQIMSS ...

  18. Protein (Cyanobacteria): 29412 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available Ala carboxypeptidase 3 (S13) family protein Prochlorococcus marinus str. MIT 9303 MKIIPASFQITKRTHSQLLIRILGAGGILI...VLEISGEGDPDLGITEIQRFAMAALGQGGSRTFRSHDDLQLLIREEPQQRWWPSDWHPADRIYAYGAPITRLALTSNALDV...AVTNPIGRLQRLLEREIHRQGGKAHLSLVNHDQVKTSSAQSVLLHEEDSAPMHALVSLANAESHNFTAEVLLRHAAESWDVRLASREAMRWMQRQNLPLTGLRIADGS...GLSRNNRMSSQTLATLLMRMGHHPLAPYYQASMAIAGQRGTLRKLFRGTSLEGKFWGKTGTLNGVRSISGILETVDGPRYVSAIANGASSPNRTIGLLLKATQRFSPCSSSLSTSN ... ...GSNVNAAPTVLSPPPPVAVQGWPSLQSGRLCPSLQRSFKSLLGQGSSAWSVSVVDRHGQLLADINGTVAKVPASNQKLITTAFALDKLGPDFKLRTQLLRRPDG

  19. Protein (Viridiplantae): 357469291 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 989 3880:1989 Serine carboxypeptidase family protein Medicago truncatula MGSSWNHNQERFSCTWKPTSLQNNIKTYTRENERRMEIICHEAPVQERER...ATVKGSGHTAPAVTPEQCLAMFTRWTSNLPFGRGSNPDLKLECGIASYSLGEDSQYVTSLLVAELATNMLAHALRIPYIWSMVSIRSQREAMYAYLRRDRMERLAVEGVVPMCGGRSRGRAPHADER...TDIWQDTYLCCRLGYFSTSRLWVPRMHWVDLRTITIHVTHLDQLDLGNVYMSPYAKHRQICPFEQTSLYSGWLRYGTRRVRDLLAQVGHIMSPAEQEALDEVVAEQEGERGYLELTIRLGHIRYHVLSVMANSVVERGIGEWNSLESILTEVHGGGFIVVGVV ... ...TTRIDTSYNIIDHMLLSAFVERWHKETSRACSGGITQQKRGQSLRIYLLYLVGITLFTNKSATYMDVTYLKYFRDLDLVSDYAWGVAALYRELNNASHYK...LPLGTGFSYAKNVTYHRSDWKLVHNTYQFLRKWLIDHPEFLSNEFYIGADSYSGIPVPAVLQEISNGNEKGLQPLINLQGYLLGNPYTTHKEDNYQIQYAHGMGLISDELYASLQRNCKGEYIDVDYRNE

  20. NCBI nr-aa BLAST: CBRC-ETEL-01-1421 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ETEL-01-1421 ref|XP_001392987.1| carboxypeptidase Y cpy from patent WO9609397-...A1-Aspergillus niger emb|CAK96655.1| carboxypeptidase Y cpy from patent WO9609397-A1-Aspergillus niger XP_001392987.1 0.10 34% ...

  1. Main: 3SC2 [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available n D.-I.Liao, K.Breddam, R.M.Sweet, T.Bullock, S.J.Remington Refined Atomic Model Of Wh...eat Serine Carboxypeptidase Ii At 2.2-Angstroms Resolution Biochemistry V. 31 979...e=Cbp2; Triticum Aestivum Serine Carboxypeptidase Ii (E.C.3.4.16.1) (Cpdw-Ii) Hydrolase(Carboxypeptidase) D.-*I.Liao, S.J.Remingto...r_estrs.|PRINTS; PR00724; CRBOXYPTASEC.|ProDom; PD001189; Peptidase_S10; 1.|PROSITE; PS00560; CARBOXYPEPT_SE

  2. Dicty_cDB: Contig-U16107-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available AP004094_2( AP004094 |pid:none) Oryza sativa Japonica Group genomi... 99 7e-19 ( Q09991 ) RecName: Full=Uncharacterized serine... SSB412. 329 3e-85 1 ( EF589783 ) Triatoma brasiliensis serine carboxypeptidase SCP... 52... 1e-04 2 ( EF589782 ) Triatoma brasiliensis serine carboxypeptidase SCP... 52 1e-04 2 ( EF589781 ) Triatoma brasiliensis serine... carboxypeptidase CPVL; ... 206 9e-92 DQ104699_1( DQ104699 |pid:none) Triatoma infestans serine... carboxyp... 187 3e-83 EF589781_1( EF589781 |pid:none) Triatoma brasiliensis serine carbo... 186 4e-8

  3. Enzymatic digestibility of peptides cross-linked by ionizing radiation

    International Nuclear Information System (INIS)

    Digestibility by proteolytic enzymes of peptides cross-linked by ionizing radiation was investigated. Small peptides of alanine and phenylalanine were chosen as model compounds and aminopeptidases and carboxypeptidases were used as proteolytic enzymes. Peptides exposed to γ-radiation in aqueous solution were analysed by high-performance liquid chromatography before and after hydrolysis by aminopeptidase M, leucine aminopeptidase carboxypeptidase A and carboxypeptidase Y. The results obtained clearly demonstrate the different actions of these enzymes on cross-linked aliphatic and aromatic peptides. Peptide bonds of cross-linked dipeptides of alanine were completely resistant to enzymatic hydrolysis whereas the enzymes, except for carboxypeptidase Y, cleaved all peptide bonds of cross-linked peptides of phenylalanine. The actions of the enzymes on these particular compounds are discussed in detail. (author)

  4. Main: 1BCS [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available 1BCS 小麦 Bread Wheat ... Triticum aestivum Serine Carboxypeptidase Ii Chains A And B Name=Cbp2; Tri ... .Remington 1bcs 25 Peptide Aldehyde Complexes With Wheat ... Serine 1bcs 26 Carboxypeptidase Ii 1bcs 27to Be Pu ... Microbial Peptide Aldehyde Inhibitor 1bcs 21 CBP2_WHEAT :260,6|CBP2_WHEAT :418,266|PIR; A29639; A29639.|PDB; ...

  5. Main: 1BCR [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available 1BCR 小麦 Bread Wheat ... Triticum aestivum Serine Carboxypeptidase Ii Chains A And B Name=Cbp2; Tri ... .Remington 1bcr 25 Peptide Aldehyde Complexes With Wheat ... Serine 1bcr 26 Carboxypeptidase Ii 1bcr 27to Be Pu ... Microbial Peptide Aldehyde Inhibitor 1bcr 21 CBP2_WHEAT :259,6|CBP2_WHEAT :417,266|PIR; A29639; A29639.|PDB; ...

  6. Biochemical characterization of bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI)

    DEFF Research Database (Denmark)

    Valnickova, Zuzana; Thaysen-Andersen, Morten; Højrup, Peter;

    2009-01-01

    BACKGROUND: TAFI is a plasma protein assumed to be an important link between coagulation and fibrinolysis. The three-dimensional crystal structures of authentic mature bovine TAFI (TAFIa) in complex with tick carboxypeptidase inhibitor, authentic full lenght bovine plasma thrombin-activatable fib......BACKGROUND: TAFI is a plasma protein assumed to be an important link between coagulation and fibrinolysis. The three-dimensional crystal structures of authentic mature bovine TAFI (TAFIa) in complex with tick carboxypeptidase inhibitor, authentic full lenght bovine plasma thrombin...

  7. Different mouse mast cell populations express various combinations of at least six distinct mast cell serine proteases.

    OpenAIRE

    Reynolds, D. S.; Stevens, R L; Lane, W S; Carr, M H; Austen, K. F.; Serafin, W E

    1990-01-01

    Mouse serosal mast cells (SMCs) and Kirsten sarcoma virus-immortalized mast cells store large amounts of mast cell carboxypeptidase A and serine proteases in their secretory granules. Secretory granule proteins from 2.6 x 10(6) purified SMCs were separated by NaDodSO4/PAGE, trans-blotted to poly(vinylidine difluoride) membranes, and subjected to amino-terminal amino acid sequencing. Four distinct mast cell serine proteases were identified. With mast cell carboxypeptidase A, these serine prote...

  8. Gene : CBRC-ETEL-01-1421 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ETEL-01-1421 Novel UN D UNKNOWN NUOH1_GEOMG 1.1 37% ref|XP_001392987.1| carboxypeptidase Y ... cpy from patent ... WO9609397-A1-Aspergillus niger emb|CAK96655.1| car ... boxypeptidase Y cpy from patent ... WO9609397-A1-Aspergillus niger 0.10 34% METCICFNHH ...

  9. CYTOPLASMIC HIGH-LEVEL EXPRESSION OF A SOLUBLE, ENZYMATICALLY ACTIVE FORM OF THE ESCHERICHIA-COLI PENICILLIN-BINDING PROTEIN-5 AND PURIFICATION BY DYE CHROMATOGRAPHY

    NARCIS (Netherlands)

    VANDERLINDEN, MPG; MOTTL, H; KECK, W

    1992-01-01

    High-level expression of a soluble form of pencillin-binding protein 5 (PBP5), called PBP5s, and translocation across the cytoplasmic membrane results in lysis of Escherichia coli cells. The detrimental effect of increased amounts of this D,D-carboxypeptidase on the stability of the murein polymer c

  10. Sequence Classification: 894452 [

    Lifescience Database Archive (English)

    Full Text Available Y, and defects in other essential Pdi1p functions, caused by PDI1 deletion; Mpd1p || http://www.ncbi.nlm.nih.gov/protein/6324862 ... ...the protein disulfide isomerase (PDI) family; overexpression suppresses the defect in maturation of carboxypeptidase

  11. Prostate-specific membrane antigen and its truncated form PSM'

    Czech Academy of Sciences Publication Activity Database

    Mlčochová, Petra; Bařinka, Cyril; Tykvart, Jan; Šácha, Pavel; Konvalinka, Jan

    2009-01-01

    Roč. 69, č. 5 (2009), s. 471-479. ISSN 0270-4137 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : prostate specific membrane antigen * glutamate carboxypeptidase II * prostate cancer Subject RIV: CE - Biochemistry Impact factor: 3.081, year: 2009

  12. Multiple pathways for vacuolar sorting of yeast proteinase A

    DEFF Research Database (Denmark)

    Westphal, V; Marcusson, E G; Winther, Jakob R.; Emr, S D; van den Hazel, H B

    1996-01-01

    The sorting of the yeast proteases proteinase A and carboxypeptidase Y to the vacuole is a saturable, receptor-mediated process. Information sufficient for vacuolar sorting of the normally secreted protein invertase has in fusion constructs previously been found to reside in the propeptide of...

  13. Nerve injury evoked loss of latexin expression in spinal cord neurons contributes to the development of neuropathic pain.

    Directory of Open Access Journals (Sweden)

    Hilmar Nils Kühlein

    Full Text Available Nerve injury leads to sensitization mechanisms in the peripheral and central nervous system which involve transcriptional and post-transcriptional modifications in sensory nerves. To assess protein regulations in the spinal cord after injury of the sciatic nerve in the Spared Nerve Injury model (SNI we performed a proteomic analysis using 2D-difference gel electrophoresis (DIGE technology. Among approximately 2300 protein spots separated on each gel we detected 55 significantly regulated proteins after SNI whereof 41 were successfully identified by MALDI-TOF MS. Out of the proteins which were regulated in the DIGE analyses after SNI we focused on the carboxypeptidase A inhibitor latexin because protease dysfunctions contribute to the development of neuropathic pain. Latexin protein expression was reduced after SNI which could be confirmed by Western Blot analysis, quantitative RT-PCR and in-situ hybridisation. The decrease of latexin was associated with an increase of the activity of carboxypeptidase A indicating that the balance between latexin and carboxypeptidase A was impaired in the spinal cord after peripheral nerve injury due to a loss of latexin expression in spinal cord neurons. This may contribute to the development of cold allodynia because normalization of neuronal latexin expression in the spinal cord by AAV-mediated latexin transduction or administration of a small molecule carboxypeptidase A inhibitor significantly reduced acetone-evoked nociceptive behavior after SNI. Our results show the usefulness of proteomics as a screening tool to identify novel mechanisms of nerve injury evoked hypernociception and suggest that carboxypeptidase A inhibition might be useful to reduce cold allodynia.

  14. Digestive enzymatic patterns as possible biomarkers of endocrine disruption in the red mullet (Mullus barbatus): A preliminary investigation.

    Science.gov (United States)

    Caruso, Gabriella; De Pasquale, Francesca; Mita, Damiano Gustavo; Micale, Valeria

    2016-04-15

    During two seasonal trawl surveys (April and October, 2012), red mullet specimens were caught from two sites of the northern Sicilian coast (Western Mediterranean), characterized by different degrees of pollution, to assess whether their digestive enzymes could be cost-effective diagnostic tools for endocrine disruption. Pepsin, chymotrypsin, carboxypeptidases A and B, amylase and lipase were measured in the digestive tract of each fish. During both samplings, significant differences in the digestive enzymatic patterns of fish collected from the two sites were found. In April, pepsin and lipase contents were significantly lower in fish from the most impacted site than in those from the reference site. In October, the enzymatic patterns showed trends different from spring, with controversial results for carboxypeptidases A and B and amylase. Pepsin and lipase patterns suggest a detrimental effect played by organic pollutants and the use of these enzymes as possible biomarkers of exposure to endocrine disruptors. PMID:26971230

  15. Exocrine pancreatic secretion is stimulated in piglets fed Fish oil compared with those fed Coconut Oil or Lard

    DEFF Research Database (Denmark)

    Hedemann, Mette Skou; Pedersen, Asger Roer; Engberg, Ricarda M.

    2001-01-01

    An experiment was conducted to study the effect of feeding diets containing fat sources with different fatty acid composition (fish oil, coconut oil or lard, 10 g/100 g diet) on exocrine pancreatic secretion in piglets after weaning. A total of 16 barrows were weaned at 4 wk of age; 3 d later...... the coconut oil or lard diets. The output [U/(h. kg(0.75))] of lipase was higher in piglets fed fish oil than in piglets fed lard or coconut oil. The output of colipase was greater in piglets fed fish oil and coconut oil than in those fed lard. The dietary treatments did not affect the output of carboxylester...... hydrolase. The output of trypsin was significantly lower in piglets fed lard than in piglets fed fish oil or coconut oil diets and the output of carboxypeptidase B was greater in those fed the fish oil diet. Protein, chymotrypsin, carboxypeptidase A, elastase and amylase outputs did not differ among...

  16. Anaphylatoxin-mediated regulation of the immune response. I. C3a- mediated suppression of human and murine humoral immune responses

    OpenAIRE

    1982-01-01

    The C3a fragment of the third component of complement was found to have immunosuppressive properties. C3a is capable of suppressing both specific and polyclonal antibody responses. In contrast, C3a had no effect on antigen- or mitogen-induced B or T cell proliferative responses. The carboxy-terminal arginine is essential for C3a to exhibit its immunosuppressive properties. The serum carboxypeptidase inhibitor 2-mercaptomethyl-5-guanodinopentanoic acid, which prevents cleavage of the terminal ...

  17. Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates breast cancer cell metastatic behaviors through inhibition of plasminogen activation and extracellular proteolysis

    OpenAIRE

    Bazzi, Zainab A.; Lanoue, Danielle; El-Youssef, Mouhanned; Romagnuolo, Rocco; Tubman, Janice; Cavallo-Medved, Dora; Porter, Lisa A.; Boffa, Michael B.

    2016-01-01

    Background Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen, which can be converted to activated TAFI (TAFIa) through proteolytic cleavage by thrombin, plasmin, and most effectively thrombin in complex with the endothelial cofactor thrombomodulin (TM). TAFIa is a carboxypeptidase that cleaves carboxyl terminal lysine and arginine residues from protein and peptide substrates, including plasminogen-binding sites on cell surface receptors. Carboxyl terminal lysine residues ...

  18. Vps10p Transport from the trans-Golgi Network to the Endosome Is Mediated by Clathrin-coated Vesicles

    OpenAIRE

    Deloche, Olivier; Yeung, Bonny G.; Payne, Gregory S.; Schekman, Randy

    2001-01-01

    A native immunoisolation procedure has been used to investigate the role of clathrin-coated vesicles (CCVs) in the transport of vacuolar proteins between the trans-Golgi network (TGN) and the prevacuolar/endosome compartments in the yeast Saccharomyces cerevisiae. We find that Apl2p, one large subunit of the adaptor protein-1 complex, and Vps10p, the carboxypeptidase Y vacuolar protein receptor, are associated with clathrin molecules. Vps10p packaging in CCVs is re...

  19. Increased secretory demand rather than a defect in the proinsulin conversion mechanism causes hyperproinsulinemia in a glucose-infusion rat model of non-insulin-dependent diabetes mellitus.

    OpenAIRE

    Alarcón, C; Leahy, J L; Schuppin, G T; Rhodes, C. J.

    1995-01-01

    Hyperproinsulinemia in non-insulin-dependent diabetes mellitus (NIDDM) is due to an increased release of proinsulin from pancreatic beta cells. This could reside in increased secretory demand placed on the beta cell by hyperglycemia or in the proinsulin conversion mechanism. In this study, biosynthesis of the proinsulin conversion enzymes (PC2, PC3, and carboxypeptidase-H [CP-H]) and proinsulin, were examined in islets isolated from 48-h infused rats with 50% (wt/vol) glucose (hyperglycemic, ...

  20. Biochemical and clinical implications of proinsulin conversion intermediates.

    OpenAIRE

    Given, B D; Cohen, R M; Shoelson, S E; Frank, B H; Rubenstein, A H; Tager, H S

    1985-01-01

    Since a complete map of insulin-related peptides in humans requires consideration of proinsulin, Arg32/Glu33-split proinsulin, Arg65/Gly66-split proinsulin, des-Arg31,Arg32-proinsulin, des-Lys64, Arg65-proinsulin, and insulin, we applied high performance liquid chromatography coupled with radioimmunoassay to investigate the formation of proinsulin conversion intermediates in vitro and in vivo. Kinetic analysis of proinsulin processing by a mixture of trypsin and carboxypeptidase B (to stimula...

  1. Proinsulin Endoproteolysis Confers Enhanced Targeting of Processed Insulin to the Regulated Secretory Pathway

    OpenAIRE

    Kuliawat, Regina; Prabakaran, Daniel; Arvan, Peter

    2000-01-01

    Recently, two different prohormone-processing enzymes, prohormone convertase 1 (PC1) and carboxypeptidase E, have been implicated in enhancing the storage of peptide hormones in endocrine secretory granules. It is important to know the extent to which such molecules may act as “sorting receptors” to allow the selective trafficking of cargo proteins from the trans-Golgi network into forming granules, versus acting as enzymes that may indirectly facilitate intraluminal storage of processed horm...

  2. Peptidoglycan Synthesis in the Absence of Class A Penicillin-Binding Proteins in Bacillus subtilis

    OpenAIRE

    McPherson, Derrell C.; Popham, David L.

    2003-01-01

    Penicillin-binding proteins (PBPs) catalyze the final, essential reactions of peptidoglycan synthesis. Three classes of PBPs catalyze either trans-, endo-, or carboxypeptidase activities on the peptidoglycan peptide side chains. Only the class A high-molecular-weight PBPs have clearly demonstrated glycosyltransferase activities that polymerize the glycan strands, and in some species these proteins have been shown to be essential. The Bacillus subtilis genome sequence contains four genes encod...

  3. GenBank blastx search result: AK111921 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK111921 001-013-A10 U35369.1 Enterococcus faecalis vancomycin resistance genes, response regulator (van...RB), protein histidine kinase (vanSB), D,D-carboxypeptidase (vanYB), putative D-2-hydroxyacid dehydrogenase (van...HB), D-Ala:D-Lac ligase (vanB), and putative D,D-dipeptidase (vanXB) genes, complete cds.|BCT BCT 7e-11 +1 ...

  4. GenBank blastx search result: AK104788 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK104788 001-039-E04 U35369.1 Enterococcus faecalis vancomycin resistance genes, response regulator (van...RB), protein histidine kinase (vanSB), D,D-carboxypeptidase (vanYB), putative D-2-hydroxyacid dehydrogenase (van...HB), D-Ala:D-Lac ligase (vanB), and putative D,D-dipeptidase (vanXB) genes, complete cds.|BCT BCT 3e-20 +2 ...

  5. Isolation and Characterization of Aspergillus oryzae Vacuolar Protein Sorting Mutants

    OpenAIRE

    Ohneda, Mamoru; Arioka, Manabu; Kitamoto, Katsuhiko

    2005-01-01

    The vacuolar protein sorting (vps) system in the filamentous fungus Aspergillus oryzae, which has unique cell polarity and the ability to secrete large amounts of proteins, was evaluated by using mutants that missort vacuolar proteins into the medium. Vacuolar carboxypeptidase Y (CPY) fused with enhanced green fluorescent protein (EGFP) was used as a vacuolar marker. Twenty dfc (dim EGFP fluorescence in conidia) mutants with reduced intracellular EGFP fluorescence in conidia were isolated by ...

  6. Rare allelic variants determine folate status in an unsupplemented European population

    Czech Academy of Sciences Publication Activity Database

    Pavlíková, Markéta; Sokolová, J.; Janošíková, B.; Melenovská, P.; Krupková, L.; Zvárová, Jana; Kožich, V.

    2012-01-01

    Roč. 142, č. 8 (2012), s. 1403-1409. ISSN 0022-3166 R&D Projects: GA MZd(CZ) NS10036 Institutional research plan: CEZ:AV0Z10300504 Keywords : glutamate-carboxypeptidase-ii * coronary-artery-disease * one-carbon metabolism * methylenetetrahydrofolate reductase * homocysteine concentrations * genetic-determinants * common mutation * serum folate * polymorphisms * prevalence Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 4.196, year: 2012

  7. Mutants defective in secretory/vacuolar pathways in the EUROFAN collection of yeast disruptants.

    Science.gov (United States)

    Avaro, Sandrine; Belgareh-Touzé, Naïma; Sibella-Argüelles, Carla; Volland, Christiane; Haguenauer-Tsapis, Rosine

    2002-03-15

    We have screened the EUROFAN (European Functional Analysis Network) deletion strain collection for yeast mutants defective in secretory/vacuolar pathways and/or associated biochemical modifications. We used systematic Western immunoblotting to analyse the electrophoretic pattern of several markers of the secretory/vacuolar pathways, the soluble alpha-factor, the periplasmic glycoprotein invertase, the plasma membrane GPI-anchored protein Gas1p, and two vacuolar proteins, the soluble carboxypeptidase Y and the membrane-bound alkaline phosphatase, which are targeted to the vacuole by different pathways. We also used colony immunoblotting to monitor the secretion of carboxypeptidase Y into the medium, to identify disruptants impaired in vacuolar targeting. We identified 25 mutants among the 631 deletion strains. Nine of these mutants were disrupted in genes identified in recent years on the basis of their involvement in trafficking (VPS53, VAC7, VAM6, APM3, SYS1), or glycosylation (ALG12, ALG9, OST4, ROT2). Three of these genes were identified on the basis of trafficking defects by ourselves and others within the EUROFAN project (TLG2, RCY1, MON2). The deletion of ERV29, which encodes a COPII vesicle protein, impaired carboxypeptidase Y trafficking from the endoplasmic reticulum to the Golgi apparatus. We also identified eight unknown ORFs, the deletion of which reduced Golgi glycosylation or impaired the Golgi to vacuole trafficking of carboxypeptidase Y. YJR044c, which we identified as a new VPS gene, encodes a protein with numerous homologues of unknown function in sequence databases. PMID:11870858

  8. Biosynthesis of peptidoglycan in Gaffkya homari: processing of nascent glycan by reactivated membranes.

    OpenAIRE

    Bardin, C.; Sinha, R. K.; Kalomiris, E; Neuhaus, F C

    1984-01-01

    Membranes from Gaffkya homari reactivated by freezing and thawing were used to study the processing events involved in the assembly of both sodium dodecyl sulfate (SDS)-insoluble peptidoglycan (PG) and SDS-soluble PG. The ability to reactivate membranes for the synthesis of these polymers provided an opportunity to monitor those events that are not influenced by wall-linked PG. In G. homari, processing for the formation of cross-links requires the selective actions of DD-carboxypeptidase, LD-...

  9. A vital role of tubulin-tyrosine-ligase for neuronal organization

    OpenAIRE

    Erck, Christian; Peris, Leticia; Andrieux, Annie; Meissirel, Claire; Gruber, Achim; Vernet, Muriel; Schweitzer, Annie; Saoudi, Yasmina; Pointu, Hervé; Bosc, Christophe; Salin, Paul; Job, Didier; Wehland, Juergen

    2005-01-01

    http://www.pnas.org/content/102/22/7853.long International audience Tubulin is subject to a special cycle of detyrosination/tyrosination in which the C-terminal tyrosine of alpha-tubulin is cyclically removed by a carboxypeptidase and readded by a tubulin-tyrosine-ligase (TTL). This tyrosination cycle is conserved in evolution, yet its physiological importance is unknown. Here, we find that TTL suppression in mice causes perinatal death. A minor pool of tyrosinated (Tyr-)tubulin persist...

  10. Tissue expression and enzymologic characterization of human prostate specific membrane antigen and its rat and pig orthologs

    Czech Academy of Sciences Publication Activity Database

    Rovenská, Miroslava; Hlouchová, Klára; Šácha, Pavel; Mlčochová, Petra; Horák, Vratislav; Zámečník, J.; Bařinka, C.; Konvalinka, Jan

    2008-01-01

    Roč. 68, č. 2 (2008), s. 171-182. ISSN 0270-4137 R&D Projects: GA MŠk 1M0508; GA ČR GA524/04/0102 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50450515 Keywords : prostate specific membrane antigen * glutamate carboxypeptidase II * animal orthologs * prostate cancer * animal model Subject RIV: CE - Biochemistry Impact factor: 3.069, year: 2008

  11. HYDROLYSIS OF CHEESEWHEY PROTEINSWITH TRYPSIN, CHYMOTRYPSINAND CARBOXYPEPTIDASEA

    OpenAIRE

    M. F. CUSTÓDIO; A. J. GOULART; D. P. MARQUES; R.C. Giordano; R. L. C. Giordano; R. MONTI

    2009-01-01

    This work presents a method for adding value to cheese whey residues by whey proteins hydrolysis, using trypsin, chymotrypsin and carboxypeptidase A as catalysts. Sweet cheese whey was dialyzed and filtered in kaolin. Lactose and protein contents were analyzed after each step. The activities of bovine pancreas trypsin and chymotrypsin were measured at different pHs and temperatures. The optimal pH for the hydrolysis of whey proteins was 9.0 for both enzymes. Optima te...

  12. Intracellular proteinases Apr1p and CPz1p from Candida albicans

    Czech Academy of Sciences Publication Activity Database

    Bauerová, Václava; Dolejší, Elena; Hrušková-Heidingsfeldová, Olga; Pichová, Iva

    France : -, 2009. s. 96-96. [FEBS Advanced Lecture Course Human Fungal Pathogens Molecular Mechanism of Host-Pathogen /3./. 02.05.2009-08.05.2009, La Colle sur Loup] R&D Projects: GA ČR(CZ) GA310/09/1945; GA MŠk(CZ) LC531 Institutional research plan: CEZ:AV0Z40550506 Keywords : aspartic protease * Candida parapsilosis * carboxypeptidase Subject RIV: CE - Biochemistry

  13. Luv1p/Rki1p/Tcs3p/Vps54p, a Yeast Protein That Localizes to the Late Golgi and Early Endosome, Is Required for Normal Vacuolar Morphology.

    OpenAIRE

    Conboy, Michael J; Martha S Cyert

    2000-01-01

    We have characterized LUV1/RKI1/TCS3/VPS54, a novel yeast gene required to maintain normal vacuolar morphology. The luv1 mutant was identified in a genetic screen for mutants requiring the phosphatase calcineurin for vegetative growth. luv1 mutants lack a morphologically intact vacuole and instead accumulate small vesicles that are acidified and contain the vacuolar proteins alkaline phosphatase and carboxypeptidase Y and the vacuolar membrane H+-ATPase. Endocytosis appears qualitatively norm...

  14. Structural and Biochemical Characterization of a Novel Aminopeptidase from Human Intestine

    Czech Academy of Sciences Publication Activity Database

    Tykvart, Jan; Bařinka, Cyril; Svoboda, Michal; Navrátil, Václav; Souček, Radko; Hubálek, Martin; Hradilek, Martin; Šácha, Pavel; Lubkowski, J.; Konvalinka, Jan

    2015-01-01

    Roč. 290, č. 18 (2015), s. 11321-11336. ISSN 0021-9258 R&D Projects: GA ČR GAP304/12/0847; GA MŠk LO1302; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388963 ; RVO:86652036 Keywords : glutamate carboxypeptidase II * reaction mechanism * expression Subject RIV: CE - Biochemistry Impact factor: 4.573, year: 2014

  15. Qualitative changes in human γ-secretase underlie familial Alzheimer's disease.

    Science.gov (United States)

    Szaruga, Maria; Veugelen, Sarah; Benurwar, Manasi; Lismont, Sam; Sepulveda-Falla, Diego; Lleo, Alberto; Ryan, Natalie S; Lashley, Tammaryn; Fox, Nick C; Murayama, Shigeo; Gijsen, Harrie; De Strooper, Bart; Chávez-Gutiérrez, Lucía

    2015-11-16

    Presenilin (PSEN) pathogenic mutations cause familial Alzheimer's disease (AD [FAD]) in an autosomal-dominant manner. The extent to which the healthy and diseased alleles influence each other to cause neurodegeneration remains unclear. In this study, we assessed γ-secretase activity in brain samples from 15 nondemented subjects, 22 FAD patients harboring nine different mutations in PSEN1, and 11 sporadic AD (SAD) patients. FAD and control brain samples had similar overall γ-secretase activity levels, and therefore, loss of overall (endopeptidase) γ-secretase function cannot be an essential part of the pathogenic mechanism. In contrast, impaired carboxypeptidase-like activity (γ-secretase dysfunction) is a constant feature in all FAD brains. Significantly, we demonstrate that pharmacological activation of the carboxypeptidase-like γ-secretase activity with γ-secretase modulators alleviates the mutant PSEN pathogenic effects. Most SAD cases display normal endo- and carboxypeptidase-like γ-secretase activities. However and interestingly, a few SAD patient samples display γ-secretase dysfunction, suggesting that γ-secretase may play a role in some SAD cases. In conclusion, our study highlights qualitative shifts in amyloid-β (Aβ) profiles as the common denominator in FAD and supports a model in which the healthy allele contributes with normal Aβ products and the diseased allele generates longer aggregation-prone peptides that act as seeds inducing toxic amyloid conformations. PMID:26481686

  16. Degradation of ochratoxin A by Bacillus amyloliquefaciens ASAG1.

    Science.gov (United States)

    Chang, Xiaojiao; Wu, Zidan; Wu, Songling; Dai, Yanshi; Sun, Changpo

    2015-01-01

    Ochratoxin A (OTA) is widely found in food and feed products as a mycotoxin contaminant. It is produced by Penicillium species and several Aspergillus species. The identification OTA detoxification microorganisms is believed to be the best approach for decontamination. In this study, we isolated ASAG1, a bacterium with the ability to degrade OTA effectively, from grain depot-stored maize. A 16S rDNA sequencing approach was used to identify this strain as Bacillus amyloliquefaciens ASAG1. The degradation of OTA was detected in both medium and cell-free extracts after incubation with a culture of B. amyloliquefaciens ASAG1 cells. Subsequently, a hydrolysed enzyme (carboxypeptidase) related to the enzymatic conversion of OTA was cloned from the B. amyloliquefaciens ASAG1 genome. Using the Escherichia coli Expression System, we successfully expressed and purified this carboxypeptidase. When this enzyme was incubated with the engineered recombinant E. coli cells, the concentration of OTA was dramatically degraded. Our data therefore indicate that the carboxypeptidase produced by B. amyloliquefaciens ASAG1 is likely responsible for the biodegradation of OTA. PMID:25517039

  17. Qualitative changes in human γ-secretase underlie familial Alzheimer’s disease

    Science.gov (United States)

    Szaruga, Maria; Veugelen, Sarah; Benurwar, Manasi; Lismont, Sam; Sepulveda-Falla, Diego; Lleo, Alberto; Ryan, Natalie S.; Lashley, Tammaryn; Fox, Nick C.; Murayama, Shigeo; Gijsen, Harrie

    2015-01-01

    Presenilin (PSEN) pathogenic mutations cause familial Alzheimer’s disease (AD [FAD]) in an autosomal-dominant manner. The extent to which the healthy and diseased alleles influence each other to cause neurodegeneration remains unclear. In this study, we assessed γ-secretase activity in brain samples from 15 nondemented subjects, 22 FAD patients harboring nine different mutations in PSEN1, and 11 sporadic AD (SAD) patients. FAD and control brain samples had similar overall γ-secretase activity levels, and therefore, loss of overall (endopeptidase) γ-secretase function cannot be an essential part of the pathogenic mechanism. In contrast, impaired carboxypeptidase-like activity (γ-secretase dysfunction) is a constant feature in all FAD brains. Significantly, we demonstrate that pharmacological activation of the carboxypeptidase-like γ-secretase activity with γ-secretase modulators alleviates the mutant PSEN pathogenic effects. Most SAD cases display normal endo- and carboxypeptidase-like γ-secretase activities. However and interestingly, a few SAD patient samples display γ-secretase dysfunction, suggesting that γ-secretase may play a role in some SAD cases. In conclusion, our study highlights qualitative shifts in amyloid-β (Aβ) profiles as the common denominator in FAD and supports a model in which the healthy allele contributes with normal Aβ products and the diseased allele generates longer aggregation-prone peptides that act as seeds inducing toxic amyloid conformations. PMID:26481686

  18. dcp gene of Escherichia coli: cloning, sequencing, transcript mapping, and characterization of the gene product.

    Science.gov (United States)

    Henrich, B; Becker, S; Schroeder, U; Plapp, R

    1993-01-01

    Dipeptidyl carboxypeptidase is a C-terminal exopeptidase of Escherichia coli. We have isolated the respective gene, dcp, from a low-copy-number plasmid library by its ability to complement a dcp mutation preventing the utilization of the unique substrate N-benzoyl-L-glycyl-L-histidyl-L-leucine. Sequence analysis of a 2.9-kb DNA fragment revealed an open reading frame of 2,043 nucleotides which was assigned to the dcp gene by N-terminal amino acid sequencing and electrophoretic molecular mass determination of the purified dcp product. Transcript mapping by primer extension and S1 protection experiments verified the physiological significance of potential initiation and termination signals for dcp transcription and allowed the identification of a single species of monocistronic dcp mRNA. The codon usage pattern and the effects of elevated gene copy number indicated a relatively low level of dcp expression. The predicted amino acid sequence of dipeptidyl carboxypeptidase, containing a potential zinc-binding site, is highly homologous (78.8%) to the corresponding enzyme from Salmonella typhimurium. It also displays significant homology to the products of the S. typhimurium opdA and the E. coli prlC genes and to some metalloproteases from rats and Saccharomyces cerevisiae. No potential export signals could be inferred from the amino acid sequence. Dipeptidyl carboxypeptidase was enriched 80-fold from crude extracts of E. coli and used to investigate some of its biochemical and biophysical properties. Images PMID:8226676

  19. A defect in cell wall recycling triggers autolysis during the stationary growth phase of Escherichia coli.

    OpenAIRE

    Templin, M F; Ursinus, A; Höltje, J V

    1999-01-01

    The first gene of a family of prokaryotic proteases with a specificity for L,D-configured peptide bonds has been identified in Escherichia coli. The gene named ldcA encodes a cytoplasmic L, D-carboxypeptidase, which releases the terminal D-alanine from L-alanyl-D-glutamyl-meso-diaminopimelyl-D-alanine containing turnover products of the cell wall polymer murein. This reaction turned out to be essential for survival, since disruption of the gene results in bacteriolysis during the stationary g...

  20. Functional differences in yeast protein disulfide isomerases

    DEFF Research Database (Denmark)

    Nørgaard, P; Westphal, V; Tachibana, C;

    2001-01-01

    PDI1 is the essential gene encoding protein disulfide isomerase in yeast. The Saccharomyces cerevisiae genome, however, contains four other nonessential genes with homology to PDI1: MPD1, MPD2, EUG1, and EPS1. We have investigated the effects of simultaneous deletions of these genes. In several...... essentiality of protein disulfide isomerase-catalyzed oxidation. Most mutant combinations show defects in carboxypeptidase Y folding as well as in glycan modification. There are, however, no significant effects on ER-associated protein degradation in the various protein disulfide isomerase-deleted strains....

  1. GenBank blastx search result: AK104788 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK104788 001-039-E04 AF310956.2 Enterococcus faecium response regulator (vanRB2), p...rotein histidine kinase (vanSB2), D,D-carboxypeptidase (vanYB2), VanWB2 (vanWB2), D-2-hydroxyacid dehydrogenase (van...HB2), D-alanine:D-lactate ligase (vanB2), and D,D-dipeptidase (vanXB2) genes, complete cds.|BCT BCT 5e-22 +2 ...

  2. GenBank blastx search result: AK104725 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK104725 001-038-B08 AF310956.2 Enterococcus faecium response regulator (vanRB2), p...rotein histidine kinase (vanSB2), D,D-carboxypeptidase (vanYB2), VanWB2 (vanWB2), D-2-hydroxyacid dehydrogenase (van...HB2), D-alanine:D-lactate ligase (vanB2), and D,D-dipeptidase (vanXB2) genes, complete cds.|BCT BCT 3e-16 +1 ...

  3. GenBank blastx search result: AK111921 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK111921 001-013-A10 AF310956.2 Enterococcus faecium response regulator (vanRB2), p...rotein histidine kinase (vanSB2), D,D-carboxypeptidase (vanYB2), VanWB2 (vanWB2), D-2-hydroxyacid dehydrogenase (van...HB2), D-alanine:D-lactate ligase (vanB2), and D,D-dipeptidase (vanXB2) genes, complete cds.|BCT BCT 6e-12 +1 ...

  4. Regulation of glycoprotein synthesis in yeast by mating pheromones

    International Nuclear Information System (INIS)

    In Saccharomyces cerevisiae, glycosylated proteins amount to less than 2% of the cell protein. Two intensively studied examples of yeast glycoproteins are the external cell wall - associated invertase and the vacuolar carboxypeptidase Y. Recently, it was shown that the mating pheromone, alpha factor, specifically and strongly inhibits the synthesis of N-glycosylated proteins in haploid a cells, whereas O-glycosylated proteins are not affected. In this paper, the pathways of glycoprotein biosynthesis are summarized briefly, and evidence is presented that mating pheomones have a regulatory function in glycoprotein synthesis

  5. Comparative analysis of monoclonal antibodies against prostate-specific membrane antigen (PSMA)

    Czech Academy of Sciences Publication Activity Database

    Tykvart, Jan; Navrátil, Václav; Sedlák, František; Corey, E.; Colombatti, M.; Fracasso, G.; Koukolík, F.; Bařinka, Cyril; Šácha, Pavel; Konvalinka, Jan

    2014-01-01

    Roč. 74, č. 16 (2014), s. 1674-1690. ISSN 0270-4137 R&D Projects: GA ČR GAP304/12/0847; GA MŠk LO1302; GA ČR GAP301/12/1513; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388963 ; RVO:86652036 Keywords : glutamate carboxypeptidase II * prostate-specific membrane antigen * folate hydrolase * NAALADase * Western blot * immunohistochemistry * ELISA * flow cytometry * surface plasmon resonance Subject RIV: CE - Biochemistry Impact factor: 3.565, year: 2014

  6. Analysis of peptidoglycan precursors in vancomycin-resistant enterococci.

    OpenAIRE

    Billot-Klein, D; Gutmann, L; Collatz, E; van Heijenoort, J

    1992-01-01

    Analysis by high-pressure liquid chromatography of the cytoplasmic peptidoglycan precursors of a high- and a low-level vancomycin-resistant Enterococcus spp. was performed before and after induction of resistance. This analysis showed a decrease of the D-Ala-D-Ala and UDP-MurNac-pentapeptide pools, an increase of the UDP-MurNac-tripeptide pool, and the appearance of new UDP-MurNac-containing material. These results lead us to suggest that the vancomycin-induced carboxypeptidase activity cleav...

  7. SUT1 suppresses sec14-1 through upregulation of CSR1 in Saccharomyces cerevisiae.

    Science.gov (United States)

    Régnacq, Matthieu; Ferreira, Thierry; Puard, Julien; Bergès, Thierry

    2002-11-01

    SUT1 constitutive expression in aerobiosis suppressed the ts phenotype of the sec14-1 mutation, restored growth of the sec14-null mutant and corrected the translocation defect of the vacuolar carboxypeptidase Y. Therefore SUT1 was shown to be a novel potent sec14-1 suppressor. Further, the hypoxic gene CSR1 (YLR380W), a Sec14 homolog, was upregulated upon SUT1 constitutive expression. In addition, SUT1 effects on both sec14-1 suppression and on free sterol composition were abolished in a csr1-null background, showing that this gene acts downstream of SUT1. PMID:12435498

  8. Screening of Highly Expressed CPEΔN Lung Cancer H1299 Cells

    OpenAIRE

    Sun, Jing; Zhang, Guirong; Wang, Hongyue; Shen, Hui

    2015-01-01

    Background and objective The N-terminal truncated carboxypeptidase E (CPEΔN) protein is a novel biomarker of tumor metastasis. This study screened the H1299 cell line with a highly expressed CPEΔN gene for in vivo imaging experiment. Methods Human CPEΔN gene was cloned into the luciferase lentiviral vector. H1299 cells transduced with CPEΔN or control lentiviral vectors were selected with 2 µg/mL puromycin. The expression of CPEΔN was identified through Western blot analysis, and luciferase a...

  9. Engineering a prostate-specific membrane antigen-activated tumor endothelial cell prodrug for cancer therapy

    DEFF Research Database (Denmark)

    Denmeade, Samuel R; Mhaka, Annastasiah M; Rosen, D Marc;

    2012-01-01

    Heterogeneous expression of drug target proteins within tumor sites is a major mechanism of resistance to anticancer therapies. We describe a strategy to selectively inhibit, within tumor sites, the function of a critical intracellular protein, the sarcoplasmic/endoplasmic reticulum calcium...... adenosine triphosphatase (SERCA) pump, whose proper function is required by all cell types for viability. To achieve targeted inhibition, we took advantage of the unique expression of the carboxypeptidase prostate-specific membrane antigen (PSMA) by tumor endothelial cells within the microenvironment of...

  10. dcp gene of Escherichia coli: cloning, sequencing, transcript mapping, and characterization of the gene product.

    OpenAIRE

    Henrich, B; S. Becker; Schroeder, U; Plapp, R.

    1993-01-01

    Dipeptidyl carboxypeptidase is a C-terminal exopeptidase of Escherichia coli. We have isolated the respective gene, dcp, from a low-copy-number plasmid library by its ability to complement a dcp mutation preventing the utilization of the unique substrate N-benzoyl-L-glycyl-L-histidyl-L-leucine. Sequence analysis of a 2.9-kb DNA fragment revealed an open reading frame of 2,043 nucleotides which was assigned to the dcp gene by N-terminal amino acid sequencing and electrophoretic molecular mass ...

  11. HYDROLYSIS OF CHEESEWHEY PROTEINSWITH TRYPSIN, CHYMOTRYPSINAND CARBOXYPEPTIDASEA

    Directory of Open Access Journals (Sweden)

    M. F. CUSTÓDIO

    2009-01-01

    Full Text Available

    This work presents a method for adding value to cheese whey residues by whey proteins hydrolysis, using trypsin, chymotrypsin and carboxypeptidase A as catalysts. Sweet cheese whey was dialyzed and filtered in kaolin. Lactose and protein contents were analyzed after each step. The activities of bovine pancreas trypsin and chymotrypsin were measured at different pHs and temperatures. The optimal pH for the hydrolysis of whey proteins was 9.0 for both enzymes. Optima temperatures were 60ºC for trypsin, and 50ºC for chymotrypsin. Trypsin exhibited typical Michaelis-Menten behavior, but chymotrypsin did not. Electrophoretic analysis showed that neither trypsin nor chymotrypsin alone hydrolyzed whey proteins in less than three hours. Hydrolysis rates of -lactalbumin by trypsin, and of bovine serum albumin by chymotrypsin were low. When these enzymes were combined, however, all protein fractions were attacked and rates of hydrolysis were enhanced by one order of magnitude. The addition of carboxypeptidase A to the others enzymes did not improve the process yield.

  12. The spectrum of cobalt bovine procarboxypeptidase A, an index of catalytic function.

    Science.gov (United States)

    Behnke, W D; Vallee, B L

    1972-09-01

    The spectra of functionally essential, chromophoric metal atoms of metalloenzymes are thought to reflect catalytic potential [Vallee and Williams (1968) Proc. Nat. Acad. Sci. USA 59, 498]. The spectra of cobalt procarboxypeptidase and their perturbations by substrates and inhibitors are virtually the same as those of cobalt carboxypeptidase; both are consistent with a distorted tetrahedral geometry about the cobalt atom, suggesting irregular coordination geometries and low symmetries of metal-binding sites. We have, therefore, examined the enzymatic properties of cobalt procarboxypeptidase and found that it catalyzes the hydrolysis of a series of haloacylated amino acids at rates equal to or greater than those of native zinc carboxypeptidase. These observations demonstrate the existence both of the catalytic and of the binding sites for haloacylated amino acids of the zymogen, even before activation. Hence, the magnitude of conformational changes at the catalytic site thought to accompany its activation must be very small, no matter what their magnitude might be elsewhere in the molecule. The data support the entatic state hypothesis and suggest that it may profitably guide the exploration of catalytic potential of metalloproteins and metal-protein complexes. PMID:4506766

  13. Age-related changes in protein metabolism of beech (Fagus sylvatica L.) seeds during alleviation of dormancy and in the early stage of germination.

    Science.gov (United States)

    Ratajczak, Ewelina; Kalemba, Ewa M; Pukacka, Stanislawa

    2015-09-01

    The long-term storage of seeds generally reduces their viability and vigour. The aim of this work was to evaluate the effect of long-term storage on beech (Fagus sylvatica L.) seeds at optimal conditions, over 9 years, on the total and soluble protein levels and activity of proteolytic enzymes, including endopeptidases, carboxypeptidases and aminopeptidases, as well as free amino acid levels and protein synthesis, in dry seeds, after imbibition and during cold stratification leading to dormancy release and germination. The same analyses were conducted in parallel on seeds gathered from the same tree in the running growing season and stored under the same conditions for only 3 months. The results showed that germination capacity decreased from 100% in freshly harvested seeds to 75% in seeds stored for 9 years. The levels of total and soluble proteins were highest in freshly harvested seeds and decreased significantly during storage, these proportions were retained during cold stratification and germination of seeds. Significant differences between freshly harvested and stored seeds were observed in the activities of proteolytic enzymes, including endopeptidases, aminopeptidases and carboxypeptidases, and in the levels of free amino acids. The neosynthesis of proteins during dormancy release and in the early stage of seed germination was significantly weaker in stored seeds. These results confirm the importance of protein metabolism for seed viability and the consequences of its reduction during seed ageing. PMID:26071872

  14. An experimental test of dietary enzyme modulation in pine warblers Dendroica pinus.

    Science.gov (United States)

    Levey, D J; Place, A R; Rey, P J; Martínez Del Rio, C

    1999-01-01

    Modulation of gut function is important in an ecological and evolutionary context because it likely determines what food items an animal can and cannot eat. We examined how diet affects activity of digestive enzymes in an omnivorous bird, the pine warbler (Dendroica pinus). Pine warblers were fed insect-based, fruit-based, and seed-based diets for approximately 54 d. We then measured activity of amylase, maltase, sucrase, aminopeptidase-N, trypsin, chymotrypsin, carboxypeptidase A, carboxypeptidase B, pancreatic lipase, and carboxyl ester lipase. We predicted that carbohydrase activities would be highest in birds fed the diet highest in carbohydrates (fruit based), protease activities would be highest in those fed the diet highest in protein (insect based), and lipase activities would be highest in those fed the diets highest in lipid (insect based and seed based). Also, we predicted that pine warblers would exhibit greater dietary modulation of enzyme activity than reported for a less omnivorous congener, the yellow-rumped warbler (Dendroica coronata). All predictions were upheld, supporting the hypothesis that pine warblers modulate the activity of digestive enzymes in proportion to demand from substrates in the diet. PMID:10521325

  15. Chitosan enhances parasitism of Meloidogyne javanica eggs by the nematophagous fungus Pochonia chlamydosporia.

    Science.gov (United States)

    Escudero, Nuria; Ferreira, Sebastião R; Lopez-Moya, Federico; Naranjo-Ortiz, Miguel A; Marin-Ortiz, Ana I; Thornton, Christopher R; Lopez-Llorca, Luis V

    2016-04-01

    Pochonia chlamydosporia (Pc), a nematophagous fungus and root endophyte, uses appressoria and extracellular enzymes, principally proteases, to infect the eggs of plant parasitic nematodes (PPN). Unlike other fungi, Pc is resistant to chitosan, a deacetylated form of chitin, used in agriculture as a biopesticide to control plant pathogens. In the present work, we show that chitosan increases Meloidogyne javanica egg parasitism by P. chlamydosporia. Using antibodies specific to the Pc enzymes VCP1 (a subtilisin), and SCP1 (a serine carboxypeptidase), we demonstrate chitosan elicitation of the fungal proteases during the parasitic process. Chitosan increases VCP1 immuno-labelling in the cell wall of Pc conidia, hyphal tips of germinating spores, and in appressoria on infected M. javanica eggs. These results support the role of proteases in egg parasitism by the fungus and their activation by chitosan. Phylogenetic analysis of the Pc genome reveals a large diversity of subtilisins (S8) and serine carboxypeptidases (S10). The VCP1 group in the S8 tree shows evidence of gene duplication indicating recent adaptations to nutrient sources. Our results demonstrate that chitosan enhances Pc infectivity of nematode eggs through increased proteolytic activities and appressoria formation and might be used to improve the efficacy of M. javanica biocontrol. PMID:27020158

  16. The Proteome of Mesenteric Lymph During Acute Pancreatitis and Implications for Treatment

    Directory of Open Access Journals (Sweden)

    Anubhav Mittal

    2009-03-01

    Full Text Available The protein fraction of mesenteric lymph during acute pancreatitis and other critical illness is thought to contain toxic factors. However, we do not have a complete description of the mesenteric lymph proteome during acute pancreatitis. Objective The aim of this study was to define the proteomic changes in mesenteric lymph during acute pancreatitis. Setting Animal Laboratory, University of Auckland, New Zealand. Design Mesenteric lymph was collected from sixteen male Wistar rats randomised to Group 1 (n=8 with taurocholate induced acute pancreatitis and Group 2 (n=8 sham control. The lymph was subjected to proteomic analysis using iTRAQTM (Applied Biosystems, Foster City, CA, USA and liquid chromatography-tandem mass spectrometry. Results Two hundred and forty-five proteins including 35 hypothetical proteins were identified in mesenteric lymph. Eight of the 245 proteins had a significant increase in their relative abundance in acute pancreatitis conditioned mesenteric lymph, and 7 of these were pancreatic catabolic enzymes (pancreatic amylase 2, pancreatic lipase, carboxypeptidase A2, chymotrypsinogen B, carboxypeptidase B1, cationic trypsinogen, ribonuclease 1. Conclusions This is the first comprehensive description of the proteome of mesenteric lymph during acute pancreatitis and has demonstrated a significantly increased relative abundance of 7 secreted pancreatic catabolic enzymes in acute pancreatitis conditioned mesenteric lymph. This study provides a clear rationale for further research to investigate the efficacy of enteral protease inhibitors in the treatment of acute pancreatitis.

  17. Small peptides hydrolysis in dry-cured meats.

    Science.gov (United States)

    Mora, Leticia; Gallego, Marta; Escudero, Elizabeth; Reig, Milagro; Aristoy, M-Concepción; Toldrá, Fidel

    2015-11-01

    Large amounts of different peptides are naturally generated in dry-cured meats as a consequence of the intense proteolysis mechanisms which take place during their processing. In fact, meat proteins are extensively hydrolysed by muscle endo-peptidases (mainly calpains and cathepsins) followed by exo-peptidases (mainly, tri- and di-peptidyl peptidases, dipeptidases, aminopeptidases and carboxypeptidases). The result is a large amount of released free amino acids and a pool of numerous peptides with different sequences and lengths, some of them with interesting sequences for bioactivity. This manuscript is presenting the proteomic identification of small peptides resulting from the hydrolysis of four target proteins (glyceraldehyde-3-phosphate dehydrogenase, beta-enolase, myozenin-1 and troponin T) and discusses the enzymatic routes for their generation during the dry-curing process. The results indicate that the hydrolysis of peptides follows similar exo-peptidase mechanisms. In the case of dry-fermented sausages, most of the observed hydrolysis is the result of the combined action of muscle and microbial exo-peptidases except for the hydrolysis of di- and tri-peptides, mostly due to microbial di- and tri-peptidases, and the release of amino acids at the C-terminal that appears to be mostly due to muscle carboxypeptidases. PMID:25944374

  18. Isolation, Co-Crystallization and Structure-Based Characterization of Anabaenopeptins as Highly Potent Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa).

    Science.gov (United States)

    Schreuder, Herman; Liesum, Alexander; Lönze, Petra; Stump, Heike; Hoffmann, Holger; Schiell, Matthias; Kurz, Michael; Toti, Luigi; Bauer, Armin; Kallus, Christopher; Klemke-Jahn, Christine; Czech, Jörg; Kramer, Dan; Enke, Heike; Niedermeyer, Timo H J; Morrison, Vincent; Kumar, Vasant; Brönstrup, Mark

    2016-01-01

    Mature thrombin activatable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase that stabilizes fibrin clots by removing C-terminal arginines and lysines from partially degraded fibrin. Inhibition of TAFIa stimulates the degradation of fibrin clots and may help to prevent thrombosis. Applying a lead finding approach based on literature-mining, we discovered that anabaenopeptins, cyclic peptides produced by cyanobacteria, were potent inhibitors of TAFIa with IC50 values as low as 1.5 nM. We describe the isolation and structure elucidation of 20 anabaenopeptins, including 13 novel congeners, as well as their pronounced structure-activity relationships (SAR) with respect to inhibition of TAFIa. Crystal structures of the anabaenopeptins B, C and F bound to the surrogate protease carboxypeptidase B revealed the binding modes of these large (~850 Da) compounds in detail and explained the observed SAR, i.e. the strong dependence of the potency on a basic (Arg, Lys) exocyclic residue that addressed the S1' binding pocket, and a broad tolerance towards substitutions in the pentacyclic ring that acted as a plug of the active site. PMID:27604544

  19. Radiation-induced products of peptides and their enzymatic digestibility

    International Nuclear Information System (INIS)

    Chemical characterization of radiation-induced products of peptides and proteins is essential for understanding the effect of ionizing radiation on peptides and proteins. Furthermore, peptides containing radiation-altered amino acid residues might not be completely digestible by proteolytic enzymes. In this work, small homopeptides of Ala, Phe and Met were chosen as model peptides. Lysozyme was used to investigate the effect of ionizing radiation on a small protein. All peptides and lysozyme were irradiated in diluted, oxygen free, N2O-saturated aqueous solutions, using a 60Co-γ-source. HPLC, capillary GC and GC-MS were applied to isolate and characterize the radiation-induced products. The enzymatic digestibility of the products was investigated using aminopeptidase M, leucine aminopeptidase, carboxypeptidase A and carboxypeptidase Y. It was found that irradiation of peptides examined in this work leads to racemization and alteration of amino acid residues and crosslinks between the peptide chains. In addition, it was established that exopeptidases act differently on radiation-induced dimers of peptides composed of aliphatic, aromatic and sulfur-containing amino acids

  20. FOLH1/GCPII is elevated in IBD patients, and its inhibition ameliorates murine IBD abnormalities

    Science.gov (United States)

    Wozniak, Krystyna M.; Stathis, Marigo; Hollinger, Kristen R.; Thomas, Ajit G.; Rojas, Camilo; Vornov, James J.; Marohn, Michael; Li, Xuhang; Slusher, Barbara S.

    2016-01-01

    Recent gene-profiling analyses showed significant upregulation of the folate hydrolase (FOLH1) gene in the affected intestinal mucosa of patients with inflammatory bowel disease (IBD). The FOLH1 gene encodes a type II transmembrane glycoprotein termed glutamate carboxypeptidase II (GCPII). To establish that the previously reported increased gene expression was functional, we quantified the glutamate carboxypeptidase enzymatic activity in 31 surgical specimens and report a robust 2.8- to 41-fold increase in enzymatic activity in the affected intestinal mucosa of IBD patients compared with an uninvolved area in the same patients or intestinal mucosa from healthy controls. Using a human-to-mouse approach, we next showed a similar enzymatic increase in two well-validated IBD murine models and evaluated the therapeutic effect of the potent FOLH1/ GCPII inhibitor 2-phosphonomethyl pentanedioic acid (2-PMPA) (IC50 = 300 pM). In the dextran sodium sulfate (DSS) colitis model, 2-PMPA inhibited the GCPII activity in the colonic mucosa by over 90% and substantially reduced the disease activity. The significance of the target was confirmed in FOLH1−/− mice who exhibited resistance to DSS treatment. In the murine IL-10−/− model of spontaneous colitis, daily 2-PMPA treatment also significantly reduced both macroscopic and microscopic disease severity. These results provide the first evidence of FOLH1/GCPII enzymatic inhibition as a therapeutic option for IBD. PMID:27536732

  1. In Vivo Effects of Bradykinin B2 Receptor Agonists with Varying Susceptibility to Peptidases

    Science.gov (United States)

    Jean, Mélissa; Gera, Lajos; Charest-Morin, Xavier; Marceau, François; Bachelard, Hélène

    2016-01-01

    We reported evidence of bradykinin (BK) regeneration from C-terminal extended BK sequences that behave as peptidase-activated B2 receptor (B2R) agonists. Further to these in vitro studies, we carried out in vivo experiments to verify hemodynamic effects of BK analogs exhibiting variable susceptibility toward vascular and blood plasma peptidases. Rats were anesthetized and instrumented to record blood pressure and heart rate responses to bolus intravenous (i.v.) injection of increasing doses of BK, B-9972 (D-Arg-[Hyp3,Igl5,Oic7,Igl8]-BK), BK-Arg, BK-His-Leu or BK-Ala-Pro, in the absence or presence of specific inhibitors. In some experiments, pulsed Doppler flow probes measured hindquarter Doppler shift in response to i.v. injections of kinins. BK caused rapid, transient and dose-related hypotensive effects. These effects were potentiated ∼15-fold by the angiotensin converting enzyme (ACE) inhibitor, enalaprilat, but extensively inhibited by icatibant (a B2R antagonist) and not influenced by the Arg-carboxypeptidase (CP) inhibitor (Plummer’s inhibitor). The hypotensive responses elicited by the peptidase-resistant B2R agonist, B-9972, were not affected by enalaprilat, but were inhibited by icatibant. The hypotensive responses to BK-Arg were abolished by pre-treatment with either the Arg-CP inhibitor or icatibant, pharmacologically evidencing BK regeneration. The hypotensive effects of BK-His-Leu and BK-Ala-Pro, previously reported as ACE-activated substrates, were abolished by icatibant, but not by enalaprilat. In vivo regeneration of BK from these two C-terminally extended analogs with no affinity for the B2R must follow alternative cleavage rules involving unidentified carboxypeptidase(s) when ACE is blocked. The transient hypotensive responses to BK and three tested analogs coincided with concomitant vasodilation (increased Doppler shift signal). Together, these results provide in vivo evidence that interesting hypotensive and vasodilator effects can be

  2. Proteolytic processing and activation of Clostridium perfringens epsilon toxin by caprine small intestinal contents.

    Science.gov (United States)

    Freedman, John C; Li, Jihong; Uzal, Francisco A; McClane, Bruce A

    2014-01-01

    Epsilon toxin (ETX), a pore-forming toxin produced by type B and D strains of Clostridium perfringens, mediates severe enterotoxemia in livestock and possibly plays a role in human disease. During enterotoxemia, the nearly inactive ETX prototoxin is produced in the intestines but then must be activated by proteolytic processing. The current study sought to examine ETX prototoxin processing and activation ex vivo using the intestinal contents of a goat, a natural host species for ETX-mediated disease. First, this study showed that the prototoxin has a KEIS N-terminal sequence with a molecular mass of 33,054 Da. When the activation of ETX prototoxin ex vivo by goat small intestinal contents was assessed by SDS-PAGE, the prototoxin was processed in a stepwise fashion into an ~27-kDa band or higher-molecular-mass material that could be toxin oligomers. Purified ETX corresponding to the ~27-kDa band was cytotoxic. When it was biochemically characterized by mass spectrometry, the copresence of three ETX species, each with different C-terminal residues, was identified in the purified ~27-kDa ETX preparation. Cytotoxicity of each of the three ETX species was then demonstrated using recombinant DNA approaches. Serine protease inhibitors blocked the initial proteotoxin processing, while carboxypeptidase inhibitors blocked further processing events. Taken together, this study provides important new insights indicating that, in the intestinal lumen, serine protease (including trypsin and possibly chymotrypsin) initiates the processing of the prototoxin but other proteases, including carboxypeptidases, then process the prototoxin into multiple active and stable species. Importance: Processing and activation by intestinal proteases is a prerequisite for ETX-induced toxicity. Previous studies had characterized the activation of ETX using only arbitrarily chosen amounts of purified trypsin and/or chymotrypsin. Therefore, the current study examined ETX activation ex vivo by natural

  3. Influence of dietary zinc and copper on digestive enzyme activity and intestinal morphology in weaned pigs.

    Science.gov (United States)

    Hedemann, M S; Jensen, B B; Poulsen, H D

    2006-12-01

    The current study was conducted to investigate the effects of high dietary concentrations of Zn as zinc oxide and Cu as copper sulfate on the activity of digestive enzymes in the pancreas and the intestinal mucosa, intestinal morphology, and mucin histochemistry in pigs after weaning. Thirty-two pigs were weaned at 4 wk of age. The pigs were fed standard weaning diets supplemented with Zn (100 or 2,500 ppm) and Cu (0 or 175 ppm) in a 2 x 2 factorial arrangement of treatments for a 14-d period. In pancreatic tissue, the activity of amylase, carboxypeptidase A, chymotrypsin, trypsin, and lipase increased (P pigs fed 2,500 ppm of Zn, whereas the activity of carboxypeptidase B and carboxylester hydrolase was unaffected. Copper had no effect on the activity of pancreatic enzymes. In small intestinal contents, the total activity of amylase and carboxypeptidase A was greater in pigs fed 100 ppm of Zn (P pigs fed 100 ppm of Zn than in pigs fed 2,500 ppm of Zn, but otherwise there were no clear effects of Zn and Cu supplementation on intestinal morphology. In the cranial small intestine, the activity of maltase (P pigs fed 100 ppm of Zn, even though there was a Zn x Cu interaction (P pigs fed 100 ppm of Zn, the activity of aminopeptidase N was greater in the caudal small intestine, but dietary Zn or Cu had no effect on aminopeptidase N in the cranial and middle small intestine. No effect of dietary Zn or Cu supplementation was found on carbohydrate histochemistry in the caudal small intestine, whereas high dietary Zn increased the area of neutral, acidic, and sulfomucins in the cecum (P < 0.01) and in the colon (P < 0.001). In summary, high dietary Zn increased the activity of several enzymes in the pancreatic tissue and increased the mucin staining area in the large intestine, whereas Cu had no clear effect on these variables. However, no definite answers were found as to how the growth promoting and diarrhea reducing effects of excess dietary Zn are exerted. PMID:17093223

  4. HB Les Andelys [alpha83(F4)LEU-->PRO]: a new moderately unstable variant.

    Science.gov (United States)

    Wajcman, H; Promé, D; Préhu, C; Déon, C; Riou, J; Bouanga, J C; Papassotiriou, I; Lahary, A; Galactéros, F

    1998-03-01

    Hb Les Andelys [alpha83(F4)Leu-->Pro] is a mildly unstable variant that was found during glycated hemoglobin measurement in a French family. In this hemoglobin molecule the affected site, in the alpha chain, and the amino acid substitution are identical to those of Hb Santa Ana, an unstable beta chain variant. The structural abnormality was demonstrated by protein chemistry methods, involving, in addition to the classical techniques, a selective precipitation of the abnormal hemoglobin by isopropanol and a mass spectrometry analysis of the alphaT-9 peptide following carboxypeptidase digestion. DNA sequencing demonstrated that the mutation was CTG-->CCG at codon 83 of the alpha2 gene. PMID:9576330

  5. [Effect of proteolysis inhibitors on the incorporation of labelled amino acids into proteins].

    Science.gov (United States)

    Konikova, A S; Korotkina, R N

    1975-01-01

    Role of peptide bond breaks in the incorporation of amino acids into proteins in a "protein--amino acid" system is investigated. For this purpose the incorporation of labelled amino acids into trypsin under the inhibition of its autolysis by a specific inhibitor from soybean and epsilon-amino-caproic acid is studied. The trypsin inhibitor from soybean is found to suppress considerably the incorporation of 14C-glycine, 14C-lysine and 14C-methionine into crystal trypsin and not to affect the incorporation of labelled amino acids into chomotrypsin, papain and carboxypeptidase. Epsilon-Aminocaproic acid inhibited 14C-glycine incorporation into crystal trypsin by 40% and did not change its incorporation level into serum albumin. The dependency of amino acid incorporation level into trypsin on the activity of autolysis in the "protein--amino acid" system is demonstrated. PMID:1212456

  6. ECUT (Energy Conversion and Utilization Technologies) program: Biocatalysis Project

    Science.gov (United States)

    1988-03-01

    Fiscal year 1987 research activities and accomplishments for the Biocatalysis Project of the U.S. Department of Energy, Energy Conversion and Utilization Technologies (ECUT) Division are presented. The project's technical activities were organized into three work elements. The Molecular Modeling and Applied Genetics work element includes modeling and simulation studies to verify a dynamic model of the enzyme carboxypeptidase; plasmid stabilization by chromosomal integration; growth and stability characteristics of plasmid-containing cells; and determination of optional production parameters for hyper-production of polyphenol oxidase. The Bioprocess Engineering work element supports efforts in novel bioreactor concepts that are likely to lead to substantially higher levels of reactor productivity, product yields, and lower separation energetics. The Bioprocess Design and Assessment work element attempts to develop procedures (via user-friendly computer software) for assessing the economics and energetics of a given biocatalyst process.

  7. Dicty_cDB: Contig-U10502-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 2 9 ( CR488583 ) mth2-157H4RM1 BAC end, cultivar Jemalong A17 of M... 40 2.2 2 ( CP000942 ) Ureaplasma... carboxypeptidase-like 32; ... 132 8e-30 AM502236_45( AM502236 |pid:none) Leishma... thaliana DNA chromoso... 106 6e-22 EU481040_1( EU481040 |pid:none) Caenorhabditis remanei strain PB20... |pid:none) Aspergillus fumigatus carboxypepti... 103 5e-21 EU481045_1( EU481045 |pid:none) Caenorhabditis remanei...ntig VV78X264453.... 102 7e-21 EU481041_1( EU481041 |pid:none) Caenorhabditis remanei strain PB21... 102 9e-

  8. Preparation of Tyr-C-peptide from genetically altered human insulin presursor

    Energy Technology Data Exchange (ETDEWEB)

    Huaiyu Sun; Jianguo Tang; Meihao Hu [Peking Univ., Beijing (China)

    1995-12-01

    C-peptide radiommunoassay (C-peptide RIA) is widely used in determination of pancreatic B-cell secretion activity. {sup 125}I labeled Tyr-C-peptide is indispensable in C-peptide RIA kit. Herein we discuss a way of obtaining recombinant Tyr-C-peptide. Arg32Tyr human proinsulin mutant (R32Y-proinsulin) gene was constructed by site-directed mutagenesis and overexpressed in Escherichia coli. Purified R32Y-proinsulin was converted to insulin and Tyr-C-peptide by trypsin and carboxypeptidase B codigestion. Tyr-C-peptide was isolated through reverse-phase HPLC (RP-HPLC) and identified by C-peptide RIA and amino acid analysis. 12 refs., 5 figs., 1 tab.

  9. Purification and characterization of cathepsin B from the skeletal muscles of agama stellio stellio

    International Nuclear Information System (INIS)

    1. Cathepsin b from the muscles of Jordanian lizard Agama stellio stellio was purified to homogeneity by a series of column chromatography on DEAE-sephadex, thio propyl sepharose and sephadex G-100 2. The molecular weight of cathepsin B isolated was to be 31800 dalton by using SDS-PAGE, and 33000 dalton by gel filtration, and its isoelectric point was measured to be 4.2 by isoelectric focusing. 3. Cathepsin B had ph optimum of 5.5, required a thiol-reducing reagent for activation and was inhibited by thiol-protease inhibitors. 4. The Km and K cat values for Z-Phe-Arg-Mca were determined to be 0.161mM and 238 S-1. 5. Cathepsin B acted on oligopeptide substrates mainly as di peptidyl carboxypeptidase. (authors). 22 refs., 3 tabs., 2 figs

  10. Effects of in vitro ozone treatment on proteolysis of purified rubisco from two hybrid poplar clones. [Populus maximowizii x trichocarpa

    Energy Technology Data Exchange (ETDEWEB)

    Landry, L.G.; Pell, E.J. (Pennsylvania State Univ., University Park (USA))

    1989-04-01

    Plants exposed to ozone (O{sub 3}) exhibited symptoms of premature senescence, including early decline in quantity of rubisco. O{sub 3}-induced oxidation may cause changes in protein conformation of rubisco, resulting in enhanced proteolysis. To test this hypothesis, rubisco was purified from two hybrid clones of Populus maximowizii x trichocarpa, clones 388 and 245, and treated in vitro with O{sub 3} or air. Rubisco was then challenged with bromelain, papain, chymotrypsin, carboxypeptidase A, or endoproteinase Glu-C and percent degradation measured by SDS-PAGE and densitometric scanning of the gels. Degree of rubisco sensitivity to oxidation may be related to available sulfhydryl (SH) groups on the protein. The number of SH groups in native and denatured rubisco was measured for purified rubisco of both clones by DTNB titration method. The relationship between sensitivity to proteolysis and number and availability of SH groups is discussed.

  11. Serglycin proteoglycan is not implicated in localizing exocrine pancreas enzymes to zymogen granules

    DEFF Research Database (Denmark)

    Niemann, Carsten U; Cowland, Jack B; Ralfkiaer, Elisabeth;

    2009-01-01

    Storage and release of proteins from granules forms the basis of cellular functions as diverse as cell mediated cytotoxicity, neuronal communication, activation of muscle fibres, and release of hormones or digestive enzymes from endocrine and exocrine glands, such as the pancreas. Serglycin...... is the major intracellular proteoglycan of haematopoietic cells. Serglycin is important for localization of proteins in granules of different haematopoietic cell types. Previous reports have indicated a role for serglycin in granule formation and localization of zymogens in granules of the exocrine pancreas...... in rat. We here present data showing that serglycin is not present at the protein level in human or murine pancreas. Furthermore, the amount and localization of three exocrine pancreas zymogens (amylase, trypsinogen, and carboxypeptidase A) is not affected by the absence of serglycin in a serglycin knock...

  12. Aloe vera

    International Nuclear Information System (INIS)

    We review the scientific literature regarding the aloe vera plant and its products. Aloe vera is known to contain several pharmacologically active ingredients, including a carboxypeptidase that inactivates bradykinin in vitro, salicylates, and a substance(s) that inhibits thromboxane formation in vivo. Scientific studies exist that support an antibacterial and antifungal effect for substance(s) in aloe vera. Studies and case reports provide support for the use of aloe vera in the treatment of radiation ulcers and stasis ulcers in man and burn and frostbite injuries in animals. The evidence for a potential beneficial effect associated with the use of aloe vera is sufficient to warrant the design and implementation of well-controlled clinical trials. 27 references

  13. Active site mutations in yeast protein disulfide isomerase cause dithiothreitol sensitivity and a reduced rate of protein folding in the endoplasmic reticulum

    DEFF Research Database (Denmark)

    Holst, B; Tachibana, C; Winther, Jakob R.

    1997-01-01

    . Such mutations had no significant effect on growth. The domains however, were not equivalent since the rate of folding of carboxypeptidase Y (CPY) in vivo was reduced by inactivation of the a domain but not the a' domain. To investigate the relevance of PDI redox potential, the G and H positions of...... each CGHC active site were randomly mutagenized. The resulting mutant PDIs were ranked by their growth phenotype on medium containing increasing concentrations of DTT. The rate of CPY folding in the mutants showed the same ranking as the DTT sensitivity, suggesting that the oxidative power of PDI is an...... important factor in folding in vivo. Mutants with a PDI that cannot perform oxidation reactions on its own (CGHS) had a strongly reduced growth rate. The growth rates, however, did not correlate with CPY folding, suggesting that the protein(s) required for optimal growth are dependent on PDI for oxidation...

  14. Competition between folding and glycosylation in the endoplasmic reticulum

    DEFF Research Database (Denmark)

    Holst, B; Bruun, A W; Kielland-Brandt, Morten; Winther, Jakob R.

    1996-01-01

    Using carboxypeptidase Y in Saccharomyces cerevisiae as a model system, the in vivo relationship between protein folding and N-glycosylation was studied. Seven new sites for N-glycosylation were introduced at positions buried in the folded protein structure. The level of glycosylation of such new...... acceptor sites. In some cases, all the newly synthesized mutant protein was modified at the novel site while in others no modification took place. In the most interesting category of mutants, the level of glycosylation was dependent on the conditions for folding. This shows that folding and glycosylation...... reactions can compete in vivo and that glycosylation does not necessarily precede folding. The approach described may be generally applicable for the analysis of protein folding in vivo....

  15. AcEST: DK953624 [AcEST

    Lifescience Database Archive (English)

    Full Text Available ype prothallia with plantlets Developmental stage gametophytes with sporophytes C...Q2HVX5|Q2HVX5_MEDTR Biotin/lipoyl attachment OS=Medicago trun... 39 0.18 tr|Q5GMI...X 2.2.19 [Nov-02-2008] Reference: Altschul, Stephen F., Thomas L. Madden, Alejandro A. Sch...6.2 E-value 1.0e-09 Report BLASTX 2.2.19 [Nov-02-2008] Reference: Altschul, Stephen F., Thomas L. Madden, Alejandro A. Sch...D-alanyl-D-alanine carboxypeptidase dacF OS... 32 2.5 sp|Q5F829|ISPD_NEIG1 2-C-methyl-D-erythritol 4-phospha

  16. Temperature dependence of protein hydration hydrodynamics by molecular dynamics simulations.

    Energy Technology Data Exchange (ETDEWEB)

    Lau, E Y; Krishnan, V V

    2007-07-18

    The dynamics of water molecules near the protein surface are different from those of bulk water and influence the structure and dynamics of the protein itself. To elucidate the temperature dependence hydration dynamics of water molecules, we present results from the molecular dynamic simulation of the water molecules surrounding two proteins (Carboxypeptidase inhibitor and Ovomucoid) at seven different temperatures (T=273 to 303 K, in increments of 5 K). Translational diffusion coefficients of the surface water and bulk water molecules were estimated from 2 ns molecular dynamics simulation trajectories. Temperature dependence of the estimated bulk water diffusion closely reflects the experimental values, while hydration water diffusion is retarded significantly due to the protein. Protein surface induced scaling of translational dynamics of the hydration waters is uniform over the temperature range studied, suggesting the importance protein-water interactions.

  17. Extracellular proteases as targets for drug development.

    Science.gov (United States)

    Cudic, Mare; Fields, Gregg B

    2009-08-01

    Proteases constitute one of the primary targets in drug discovery. In the present review, we focus on extracellular proteases (ECPs) because of their differential expression in many pathophysiological processes, including cancer, cardiovascular conditions, and inflammatory, pulmonary, and periodontal diseases. Many new ECP inhibitors are currently under clinical investigation and a significant increase in new therapies based on protease inhibition can be expected in the coming years. In addition to directly blocking the activity of a targeted protease, one can take advantage of differential expression in disease states to selectively deliver therapeutic or imaging agents. Recent studies in targeted drug development for the metalloproteases (matrix metalloproteinases, adamalysins, pappalysins, neprilysin, angiotensin-converting enzyme, metallocarboxypeptidases, and glutamate carboxypeptidase II), serine proteases (elastase, coagulation factors, tissue/urokinase plasminogen activator system, kallikreins, tryptase, dipeptidyl peptidase IV) and cysteine proteases (cathepsin B) are discussed herein. PMID:19689354

  18. Aloe vera

    Energy Technology Data Exchange (ETDEWEB)

    Klein, A.D.; Penneys, N.S.

    1988-04-01

    We review the scientific literature regarding the aloe vera plant and its products. Aloe vera is known to contain several pharmacologically active ingredients, including a carboxypeptidase that inactivates bradykinin in vitro, salicylates, and a substance(s) that inhibits thromboxane formation in vivo. Scientific studies exist that support an antibacterial and antifungal effect for substance(s) in aloe vera. Studies and case reports provide support for the use of aloe vera in the treatment of radiation ulcers and stasis ulcers in man and burn and frostbite injuries in animals. The evidence for a potential beneficial effect associated with the use of aloe vera is sufficient to warrant the design and implementation of well-controlled clinical trials. 27 references.

  19. Examination of the Mechanism of Human Brain Aspartoacylase through the Binding of an Intermediate Analogue†‡

    Science.gov (United States)

    Le Coq, Johanne; Pavlovsky, Alexander; Malik, Radhika; Sanishvili, Ruslan; Xu, Chengfu; Viola, Ronald E.

    2009-01-01

    Canavan disease is a fatal neurological disorder caused by the malfunctioning of a single metabolic enzyme, aspartoacylase, that catalyzes the deacetylation of N-acetyl-l-aspartate to produce l-aspartate and acetate. The structure of human brain aspartoacylase has been determined in complex with a stable tetrahedral intermediate analogue, N-phosphonomethyl-l-aspartate. This potent inhibitor forms multiple interactions between each of its heteroatoms and the substrate binding groups arrayed within the active site. The binding of the catalytic intermediate analogue induces the conformational ordering of several substrate binding groups, thereby setting up the active site for catalysis. The highly ordered binding of this inhibitor has allowed assignments to be made for substrate binding groups and provides strong support for a carboxypeptidase-type mechanism for the hydrolysis of the amide bond of the substrate, N-acetyl-l-aspartate. PMID:18293939

  20. Subclinical zinc deficiency impairs pancreatic digestive enzyme activity and digestive capacity of weaned piglets.

    Science.gov (United States)

    Brugger, Daniel; Windisch, Wilhelm M

    2016-08-01

    This study investigated the effects of short-term subclinical Zn deficiency on exocrine pancreatic activity and changes in digestive capacity. A total of forty-eight weaned piglets were fed ad libitum a basal diet (maize and soyabean meal) with adequate Zn supply (88 mg Zn/kg diet) during a 2-week acclimatisation phase. Animals were then assigned to eight dietary treatment groups (n 6) according to a complete randomised block design considering litter, live weight and sex. All pigs were fed restrictively (450 g diet/d) the basal diet but with varying ZnSO4.7H2O additions, resulting in 28·1, 33·6, 38·8, 42·7, 47·5, 58·2, 67·8 and 88·0 mg Zn/kg diet for a total experimental period of 8 d. Pancreatic Zn concentrations and pancreatic activities of trypsin, chymotrypsin, carboxypeptidase A and B, elastase and α-amylase exhibited a broken-line response to stepwise reduction in dietary Zn by declining beneath thresholds of 39·0, 58·0, 58·0, 41·2, 47·5, 57·7 and 58·0 mg Zn/kg diet, respectively. Furthermore, carboxypeptidase B and α-amylase activities were significantly lower in samples with reduced pancreatic Zn contents. Coefficients of faecal digestibility of DM, crude protein, total lipids and crude ash responded similarly to pancreatic enzyme activities by declining below dietary thresholds of 54·7, 45·0, 46·9 and 58·2 mg Zn/kg diet, respectively. In conclusion, (1) subclinical Zn deficiency impaired pancreatic exocrine enzymes, (2) this response was connected to pancreatic Zn metabolism and (3) the decline in catalytic activity impaired faecal digestibility already after 1 week of insufficient alimentary Zn supply and very early before clinical deficiency symptoms arise. PMID:27230230

  1. Specialized peptidoglycan hydrolases sculpt the intra-bacterial niche of predatory Bdellovibrio and increase population fitness.

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    Thomas R Lerner

    2012-02-01

    Full Text Available Bdellovibrio are predatory bacteria that have evolved to invade virtually all gram-negative bacteria, including many prominent pathogens. Upon invasion, prey bacteria become rounded up into an osmotically stable niche for the Bdellovibrio, preventing further superinfection and allowing Bdellovibrio to replicate inside without competition, killing the prey bacterium and degrading its contents. Historically, prey rounding was hypothesized to be associated with peptidoglycan (PG metabolism; we found two Bdellovibrio genes, bd0816 and bd3459, expressed at prey entry and encoding proteins with limited homologies to conventional dacB/PBP4 DD-endo/carboxypeptidases (responsible for peptidoglycan maintenance during growth and division. We tested possible links between Bd0816/3459 activity and predation. Bd3459, but not an active site serine mutant protein, bound β-lactam, exhibited DD-endo/carboxypeptidase activity against purified peptidoglycan and, importantly, rounded up E. coli cells upon periplasmic expression. A ΔBd0816 ΔBd3459 double mutant invaded prey more slowly than the wild type (with negligible prey cell rounding and double invasions of single prey by more than one Bdellovibrio became more frequent. We solved the crystal structure of Bd3459 to 1.45 Å and this revealed predation-associated domain differences to conventional PBP4 housekeeping enzymes (loss of the regulatory domain III, alteration of domain II and a more exposed active site. The Bd3459 active site (and by similarity the Bd0816 active site can thus accommodate and remodel the various bacterial PGs that Bdellovibrio may encounter across its diverse prey range, compared to the more closed active site that "regular" PBP4s have for self cell wall maintenance. Therefore, during evolution, Bdellovibrio peptidoglycan endopeptidases have adapted into secreted predation-specific proteins, preventing wasteful double invasion, and allowing activity upon the diverse prey peptidoglycan

  2. Cross genome comparisons of serine proteases in Arabidopsis and rice

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    Sowdhamini R

    2006-08-01

    Full Text Available Abstract Background Serine proteases are one of the largest groups of proteolytic enzymes found across all kingdoms of life and are associated with several essential physiological pathways. The availability of Arabidopsis thaliana and rice (Oryza sativa genome sequences has permitted the identification and comparison of the repertoire of serine protease-like proteins in the two plant species. Results Despite the differences in genome sizes between Arabidopsis and rice, we identified a very similar number of serine protease-like proteins in the two plant species (206 and 222, respectively. Nearly 40% of the above sequences were identified as potential orthologues. Atypical members could be identified in the plant genomes for Deg, Clp, Lon, rhomboid proteases and species-specific members were observed for the highly populated subtilisin and serine carboxypeptidase families suggesting multiple lateral gene transfers. DegP proteases, prolyl oligopeptidases, Clp proteases and rhomboids share a significantly higher percentage orthology between the two genomes indicating substantial evolutionary divergence was set prior to speciation. Single domain architectures and paralogues for several putative subtilisins, serine carboxypeptidases and rhomboids suggest they may have been recruited for additional roles in secondary metabolism with spatial and temporal regulation. The analysis reveals some domain architectures unique to either or both of the plant species and some inactive proteases, like in rhomboids and Clp proteases, which could be involved in chaperone function. Conclusion The systematic analysis of the serine protease-like proteins in the two plant species has provided some insight into the possible functional associations of previously uncharacterised serine protease-like proteins. Further investigation of these aspects may prove beneficial in our understanding of similar processes in commercially significant crop plant species.

  3. Mast cell proteases as pharmacological targets.

    Science.gov (United States)

    Caughey, George H

    2016-05-01

    Mast cells are rich in proteases, which are the major proteins of intracellular granules and are released with histamine and heparin by activated cells. Most of these proteases are active in the granule as well as outside of the mast cell when secreted, and can cleave targets near degranulating mast cells and in adjoining tissue compartments. Some proteases released from mast cells reach the bloodstream and may have far-reaching actions. In terms of relative amounts, the major mast cell proteases include the tryptases, chymases, cathepsin G, carboxypeptidase A3, dipeptidylpeptidase I/cathepsin C, and cathepsins L and S. Some mast cells also produce granzyme B, plasminogen activators, and matrix metalloproteinases. Tryptases and chymases are almost entirely mast cell-specific, whereas other proteases, such as cathepsins G, C, and L are expressed by a variety of inflammatory cells. Carboxypeptidase A3 expression is a property shared by basophils and mast cells. Other proteases, such as mastins, are largely basophil-specific, although human basophils are protease-deficient compared with their murine counterparts. The major classes of mast cell proteases have been targeted for development of therapeutic inhibitors. Also, a human β-tryptase has been proposed as a potential drug itself, to inactivate of snake venins. Diseases linked to mast cell proteases include allergic diseases, such as asthma, eczema, and anaphylaxis, but also include non-allergic diseases such as inflammatory bowel disease, autoimmune arthritis, atherosclerosis, aortic aneurysms, hypertension, myocardial infarction, heart failure, pulmonary hypertension and scarring diseases of lungs and other organs. In some cases, studies performed in mouse models suggest protective or homeostatic roles for specific proteases (or groups of proteases) in infections by bacteria, worms and other parasites, and even in allergic inflammation. At the same time, a clearer picture has emerged of differences in the

  4. Suspicion of drug-drug interaction between high-dose methotrexate and proton pump inhibitors: a case report - should the practice be changed?

    Science.gov (United States)

    Ranchon, F; Vantard, N; Gouraud, A; Schwiertz, V; Franchon, E; Pham, B N; Vial, T; You, B; Bouafia, F; Salles, G; Rioufol, C

    2011-01-01

    We report a case of a potential drug-drug interaction in a woman treated by a first injection of high-dose methotrexate for a T-lymphoblastic lymphoma. Valaciclovir, fluoxetine and pantoprazole were given concomitantly. A methotrexate overdosage was shown at 36 h after infusion associated with a severe renal failure. Alkaline hyperhydration, folinic acid and carboxypeptidase G2 were given. Prescription analyses by pharmacists and literature research have permitted us to suggest that a drug-drug interaction between methotrexate and proton pump inhibitors (PPI) was responsible for this renal failure. Several mechanisms of interaction were suggested and might be related to the inhibition of renal methotrexate transporters by PPI, an increase in the methotrexate efflux to the blood by an upregulation of multidrug resistance protein 3 by PPI or genetic polymorphisms. This case shows that pharmacists can help physicians to optimize patient treatment: they consensually decided on the systematic discontinuation of PPI or a switch to ranitidine when patients were treated by high-dose methotrexate. PMID:21597286

  5. Generation of Alveolar Epithelial Spheroids via Isolated Progenitor Cells from Human Pluripotent Stem Cells

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    Shimpei Gotoh

    2014-09-01

    Full Text Available No methods for isolating induced alveolar epithelial progenitor cells (AEPCs from human embryonic stem cells (hESCs and induced pluripotent stem cells (hiPSCs have been reported. Based on a study of the stepwise induction of alveolar epithelial cells (AECs, we identified carboxypeptidase M (CPM as a surface marker of NKX2-1+ “ventralized” anterior foregut endoderm cells (VAFECs in vitro and in fetal human and murine lungs. Using SFTPC-GFP reporter hPSCs and a 3D coculture system with fetal human lung fibroblasts, we showed that CPM+ cells isolated from VAFECs differentiate into AECs, demonstrating that CPM is a marker of AEPCs. Moreover, 3D coculture differentiation of CPM+ cells formed spheroids with lamellar-body-like structures and an increased expression of surfactant proteins compared with 2D differentiation. Methods to induce and isolate AEPCs using CPM and consequently generate alveolar epithelial spheroids would aid human pulmonary disease modeling and regenerative medicine.

  6. Bacterial chemotactic oligopeptides and the intestinal mucosal barrier

    International Nuclear Information System (INIS)

    Intestinal absorption and enterohepatic circulation of N-formyl-methionyl-leucyl-125I-tyrosine, a bioactive synthetic analog of the bacterial chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine has been investigated in the rat. In ileum and proximal and distal colon, dithiothreitol, which increases mucosal permeability, increased peptide absorption and biliary recovery fourfold, 70-fold, and 20-fold over control values, respectively. When dithiothreitol was combined with d-l-benzyl succinate, a potent inhibitor of intestinal carboxypeptidase, absorption and biliary recovery from ileal loops increased markedly to 40-fold over control, whereas there was no further increase in absorption from colon loops. There was a strong correlation between biliary N-formyl-methionyl-leucyl-125I-tyrosine recovery and intestinal absorption of 51Cr-ethylenediaminetetraacetate, a marker of passive mucosal permeability (r = 0.97). We conclude that in the ileum both enzymic degradation and restricted mucosal permeability contribute to the intestinal barrier to luminal bacterial formyl oligopeptides. In the colon, however, enzymic mechanisms are less active and restricted mucosal permeability is the major factor. Abnormalities of the intestinal mucosal barrier to proinflammatory bacterial peptides could play a role in inflammatory disorders of the gut

  7. Characterization and Optimization of Biosynthesis of Bioactive Secondary Metabolites Produced by Streptomyces sp. 8812.

    Science.gov (United States)

    Rajnisz, Aleksandra; Guśpiel, Adam; Postek, Magdalena; Ziemska, Joanna; Laskowska, Anna; Rabczenko, Daniel; Solecka, Jolanta

    2016-01-01

    The nutritional requirements and environmental conditions for a submerged culture of Streptomyces sp. 8812 were determined. Batch and fed-batch Streptomyces sp. 8812 fermentations were conducted to obtain high activity of secondary metabolites. In the study several factors were examined for their influence on the biosynthesis of the active metabolites-7-hydroxy-6-oxo-2,3,4,6-tetrahydroisoquinoline-3-carboxy acid (C10H9NO4) and N-acetyl-3,4-dihydroxy-L-phenylalanine (C11H13NO5): changes in medium composition, pH of production medium, various growth phases of seed culture, amino acid supplementation and addition of anion exchange resin to the submerged culture. Biological activities of secondary metabolites were examined with the use of DD-carboxypeptidase 64-575 and horseradish peroxidase. Streptomyces sp. 8812 mycelium was evaluated under fluorescent microscopy and respiratory activity of the strain was analyzed. Moreover, the enzymatic profiles of the strain with the use of Api ZYM test were analyzed and genetic analysis made. Phylogenetic analysis of Streptomyces sp. 8812 revealed that its closest relative is Streptomyces capoamus JCM 4734 (98%), whereas sequence analysis for 16S rRNA gene using NCBI BLAST algorithm showed 100% homology between these two strains. Biosynthetic processes, mycelium growth and enzyme inhibitory activities of these two strains were also compared. PMID:27281994

  8. Comparative Proteomic Analysis of Labellum and Inner Lateral Petals in Cymbidium ensifolium Flowers

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    Xiaobai Li

    2014-10-01

    Full Text Available The labellum in orchids shares homology with the inner lateral petals of the flower. The labellum is a modified petal and often distinguished from other petals and sepals due to its large size and irregular shape. Herein, we combined two-dimensional gel electrophoresis (2-DE and matrix assisted laser desorption/ionization time of flight/time of flight (MALDI-TOF/TOF approaches to identify the differentially expressed proteome between labellum and inner lateral petal in one of Orchid species (C. ensifolium. A total of 30 protein spots were identified, which showed more than a two-fold significant difference (p < 0.05 in their expression. Compared with C. ensifolium transcriptome (sequenced in house, 21 proteins matched the translated nucleotide. The proteins identified were classified into 48 categories according to gene ontology (GO. Additionally, these proteins were involved in 18 pathways and 9 possible protein-protein interactions. Serine carboxypeptidase and beta-glucosidase were involved in the phenylpropanoid pathway, which could regulate biosynthesis of floral scent components. Malate dehydrogenase (maeB and triosephosphate isomerase (TPI in carbon fixation pathway could regulate the energy metabolism. Xyloglucan endotransglucosylase/hydrolase (XET/XTH could promote cell wall formation and aid the petal’s morphogenesis. The identification of such differentially expressed proteins provides new targets for future studies; these will assess the proteins’ physiological roles and significance in labellum and inner lateral petals.

  9. Latexin is involved in bone morphogenetic protein-2-induced chondrocyte differentiation

    International Nuclear Information System (INIS)

    Latexin is the only known carboxypeptidase A inhibitor in mammals. We previously demonstrated that BMP-2 significantly induced latexin expression in Runx2-deficient mesenchymal cells (RD-C6 cells), during chondrocyte and osteoblast differentiation. In this study, we investigated latexin expression in the skeleton and its role in chondrocyte differentiation. Immunohistochemical studies revealed that proliferating and prehypertrophic chondrocytes expressed latexin during skeletogenesis and bone fracture repair. In the early phase of bone fracture, latexin mRNA expression was dramatically upregulated. BMP-2 upregulated the expression of the mRNAs of latexin, Col2a1, and the gene encoding aggrecan (Agc1) in a micromass culture of C3H10T1/2 cells. Overexpression of latexin additively stimulated the BMP-2-induced expression of the mRNAs of Col2a, Agc1, and Col10a1. BMP-2 treatment upregulated Sox9 expression, and Sox9 stimulated the promoter activity of latexin. These results indicate that latexin is involved in BMP-2-induced chondrocyte differentiation and plays an important role in skeletogenesis and skeletal regeneration.

  10. Genetics of acute and chronic pancreatitis: An update

    Institute of Scientific and Technical Information of China (English)

    VV; Ravi; Kanth; D; Nageshwar; Reddy

    2014-01-01

    Progress made in identifying the genetic susceptibility underlying acute and chronic pancreatitis has benefitted the clinicians in understanding the pathogenesis of the disease in a better way. The identification of mutations in cationic trypsinogen gene(PRSS1 gene; functional gain mutations) and serine protease inhibitor kazal type 1(SPINK1 gene; functional loss mutations) and other potential susceptibility factors in genes that play an important role in the pancreatic secretory functions or response to inflammation during pancreatic injury has changed the current concepts and understanding of a complex multifactorial disease like pancreatitis. An indi-vidual’s susceptibility to the disease is governed by ge-netic factors in combination with environmental factors. Candidate gene and genetic linkage studies have iden-tified polymorphisms in cationic trypsinogen(PRSS1), SPINK1, cystic fibrosis trans-membrane conductance regulator(CFTR), Chymotrypsinogen C(CTRC), Ca-thepsin B(CTSB) and calcium sensing receptor(CASR). Individuals with polymorphisms in the mentioned genes and other as yet identified genes are at an enhanced risk for the disease. Recently, polymorphisms in genes other than those involved in "intra-pancreatic trypsin regulatory mechanism" namely Claudin-2(CLDN2) andCarboxypeptidase A1(CPA1) gene have also been iden-tified for their association with pancreatitis. With ever growing number of studies trying to identify the genetic susceptibility in the form of single nucleotide polymor-phisms, this review is an attempt to compile the avail-able information on the topic.

  11. Buffalo Cheese Whey Proteins, Identification of a 24 kDa Protein and Characterization of Their Hydrolysates: In Vitro Gastrointestinal Digestion

    Science.gov (United States)

    Bassan, Juliana C.; Goulart, Antonio J.; Nasser, Ana L. M.; Bezerra, Thaís M. S.; Garrido, Saulo S.; Rustiguel, Cynthia B.; Guimarães, Luis H. S.; Monti, Rubens

    2015-01-01

    Milk whey proteins are well known for their high biological value and versatile functional properties, characteristics that allow its wide use in the food and pharmaceutical industries. In this work, a 24 kDa protein from buffalo cheese whey was analyzed by mass spectrometry and presented homology with Bos taurus beta-lactoglobulin. In addition, the proteins present in buffalo cheese whey were hydrolyzed with pepsin and with different combinations of trypsin, chymotrypsin and carboxypeptidase-A. When the TNBS method was used the obtained hydrolysates presented DH of 55 and 62% for H1 and H2, respectively. Otherwise for the OPA method the DH was 27 and 43% for H1 and H2, respectively. The total antioxidant activities of the H1 and H2 samples with and without previous enzymatic hydrolysis, determined by DPPH using diphenyl-p-picrylhydrazyl radical, was 4.9 and 12 mM of Trolox equivalents (TE) for H2 and H2Dint, respectively. The increased concentrations for H1 and H2 samples were approximately 99% and 75%, respectively. The in vitro gastrointestinal digestion efficiency for the samples that were first hydrolyzed was higher compared with samples not submitted to previous hydrolysis. After in vitro gastrointestinal digestion, several amino acids were released in higher concentrations, and most of which were essential amino acids. These results suggest that buffalo cheese whey is a better source of bioavailable amino acids than bovine cheese whey. PMID:26465145

  12. CPB1 of Aedes aegypti Interacts with DENV2 E Protein and Regulates Intracellular Viral Accumulation and Release from Midgut Cells

    Directory of Open Access Journals (Sweden)

    Hong-Wai Tham

    2014-12-01

    Full Text Available Aedes aegypti is a principal vector responsible for the transmission of dengue viruses (DENV. To date, vector control remains the key option for dengue disease management. To develop new vector control strategies, a more comprehensive understanding of the biological interactions between DENV and Ae. aegypti is required. In this study, a cDNA library derived from the midgut of female adult Ae. aegypti was used in yeast two-hybrid (Y2H screenings against DENV2 envelope (E protein. Among the many interacting proteins identified, carboxypeptidase B1 (CPB1 was selected, and its biological interaction with E protein in Ae. aegypti primary midgut cells was further validated. Our double immunofluorescent assay showed that CPB1-E interaction occurred in the endoplasmic reticulum (ER of the Ae. aegypti primary midgut cells. Overexpression of CPB1 in mosquito cells resulted in intracellular DENV2 genomic RNA or virus particle accumulation, with a lower amount of virus release. Therefore, we postulated that in Ae. aegypti midgut cells, CPB1 binds to the E protein deposited on the ER intraluminal membranes and inhibits DENV2 RNA encapsulation, thus inhibiting budding from the ER, and may interfere with immature virus transportation to the trans-Golgi network.

  13. Developmental patterns for pancreatic opioids in the rat.

    Science.gov (United States)

    Powell, A M; Voyles, N R; Wilkins, S D; Zalenski, C M; Timmers, K I; Recant, L

    1989-01-01

    Developmental patterns for rat pancreatic opioid peptides and islet hormones were studied from gestational day 20 through adulthood. Fetal tissue was obtained as well as pancreas at birth (day 0), and postnatal days 3, 7, 14, and 21, and 7 weeks. The hormones measured included insulin, glucagon, and somatostatin. The opioids measured were beta-endorphin, Met- and Leu-enkephalins, and the high molecular weight enkephalin precursors. Pancreata were pooled as necessary and extracted (acid alcohol, or hot acetic acid), and opioids were further purified on reversed-phase C-18 (Sep-pak) cartridges. In all instances measurements were made by radioimmunoassays. Precursor peptides were first digested (with trypsin and carboxypeptidase B) prior to immunoassay. All opioids and hormones except the precursors for enkephalins showed a well-defined surge in pancreatic concentration during the first postnatal week. In contrast, the precursors had the highest concentration in the fetus, and by the seventh day of life had decreased by greater than 50%. This progressive decrease may represent maturation of the enkephalin convertase and trypsin-like enzymes in the islets. The opioid and hormonal surges that we have described are similar to the surge in islet concentration of thyroid-releasing hormone (TRH) previously described in neonatal rat islets. It is suggested that these postnatal alterations in opioid and hormone concentration relate to a specific function in the development of the endocrine pancreas. PMID:2530576

  14. Copper deficiency in rats increases pancreatic enkephalin-containing peptides and insulin.

    Science.gov (United States)

    Recant, L; Voyles, N R; Timmers, K I; Zalenski, C; Fields, M; Bhathena, S J

    1986-01-01

    Free enkephalins (enk) and higher molecular weight enkephalin-containing peptides (enk-c-p) are present in the endocrine pancreas of rats, presumably in B cells. To determine whether these opioid peptides show dynamic alterations as insulin content of pancreas changes, we utilized a copper deficient rat model, in which the exocrine pancreas atrophies and the endocrine pancreas is "intact" and insulin (IRI) content increases. Dietary copper deficiency (-C) was produced in weanling male rats for 4 and 7 weeks. The deficient and copper supplemented (+C) groups were further subdivided to receive all dietary carbohydrate as either 62% fructose (F) or 62% starch (S). -CF rats showed the most severe deficiency. After 7 weeks, total units of pancreatic IRI in -CF were 7.5 +CF 2.1, -CS 7.9 and in +CS 2.8 (p less than 0.001). Pancreatic content of Met5- and Leu5-enk was measured in extracts which were purified on C-18 Seppaks with and without prior treatment with trypsin and carboxypeptidase B. -C animals showed progressive, significant increases in pancreatic content of Leu-enk-c-p, with a decrease in free Leu- and Met-enk (p less than 0.02-0.01). The pancreatic findings are compatible with a co-localization of enkephalins and insulin in the endocrine pancreas and are suggestive of co-regulation. PMID:3550724

  15. Using cornstarch in microparticulate diets for larvicultured tropical gar (Atractosteus tropicus).

    Science.gov (United States)

    Frías-Quintana, C A; Domínguez-Lorenzo, J; Álvarez-González, C A; Tovar-Ramírez, D; Martínez-García, R

    2016-04-01

    Aquaculture in Mexico has been developed by the cultivation of commercial species. In Tabasco, the cultivation of native species is mainly limited by the lack of nutrition studies to support its crop profitability. Among these species is the tropical gar (Atractosteus tropicus), which has great potential for cultivation. However, the nutritional value of carbohydrates in diets for this species which contribute to improved growth and survival, have not been evalulated,. Thus, in the present investigation, isoprotein and isolipid diets have been designed based on the substitution of cellulose by corn starch (D1: 0% starch-15% cellulose, D2: 7.5% starch-7.5% cellulose and D3: 15% starch-0% cellulose) and compared with a commercial trout diet (45% protein and 16% lipids). A total of 1800 larvae (0.008 ± 0.002 g and 10.5 ± LT 0.126 mm) were used, distributed in a recirculation system in order to evaluate growth and survival for 30 days. The results show higher growth and survival of 97% of larvae fed the D3 diet, while cannibalism in the species was mitigated. Major digestive enzyme activities occurred (acid protease, alkaline protease, trypsin, chymotrypsin, leucine aminopeptidase, carboxypeptidase A, lipase, α-glucosidase and amylase) for larvae fed D3. It is concluded that the contribution of corn starch (15%) replacing cellulose in the diet improves growth and survival of this species. PMID:26573856

  16. A Peptidoglycan-Remodeling Enzyme Is Critical for Bacteroid Differentiation in Bradyrhizobium spp. During Legume Symbiosis.

    Science.gov (United States)

    Gully, Djamel; Gargani, Daniel; Bonaldi, Katia; Grangeteau, Cédric; Chaintreuil, Clémence; Fardoux, Joël; Nguyen, Phuong; Marchetti, Roberta; Nouwen, Nico; Molinaro, Antonio; Mergaert, Peter; Giraud, Eric

    2016-06-01

    In response to the presence of compatible rhizobium bacteria, legumes form symbiotic organs called nodules on their roots. These nodules house nitrogen-fixing bacteroids that are a differentiated form of the rhizobium bacteria. In some legumes, the bacteroid differentiation comprises a dramatic cell enlargement, polyploidization, and other morphological changes. Here, we demonstrate that a peptidoglycan-modifying enzyme in Bradyrhizobium strains, a DD-carboxypeptidase that contains a peptidoglycan-binding SPOR domain, is essential for normal bacteroid differentiation in Aeschynomene species. The corresponding mutants formed bacteroids that are malformed and hypertrophied. However, in soybean, a plant that does not induce morphological differentiation of its symbiont, the mutation does not affect the bacteroids. Remarkably, the mutation also leads to necrosis in a large fraction of the Aeschynomene nodules, indicating that a normally formed peptidoglycan layer is essential for avoiding the induction of plant immune responses by the invading bacteria. In addition to exopolysaccharides, capsular polysaccharides, and lipopolysaccharides, whose role during symbiosis is well defined, our work demonstrates an essential role in symbiosis for yet another rhizobial envelope component, the peptidoglycan layer. PMID:26959836

  17. High and Low Doses of Ionizing Radiation Induce Different Secretome Profiles in a Human Skin Model

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Qibin; Matzke, Melissa M.; Schepmoes, Athena A.; Moore, Ronald J.; Webb-Robertson, Bobbie-Jo M.; Hu, Zeping; Monroe, Matthew E.; Qian, Weijun; Smith, Richard D.; Morgan, William F.

    2014-03-18

    It is postulated that secreted soluble factors are important contributors of bystander effect and adaptive responses observed in low dose ionizing radiation. Using multidimensional liquid chromatography-mass spectrometry based proteomics, we quantified the changes of skin tissue secretome – the proteins secreted from a full thickness, reconstituted 3-dimensional skin tissue model 48 hr after exposure to 3, 10 and 200 cGy of X-rays. Overall, 135 proteins showed statistical significant difference between the sham (0 cGy) and any of the irradiated groups (3, 10 or 200 cGy) on the basis of Dunnett adjusted t-test; among these, 97 proteins showed a trend of downregulation and 9 proteins showed a trend of upregulation with increasing radiation dose. In addition, there were 21 and 8 proteins observed to have irregular trends with the 10 cGy irradiated group either having the highest or the lowest level among all three radiated doses. Moreover, two proteins, carboxypeptidase E and ubiquitin carboxyl-terminal hydrolase isozyme L1 were sensitive to ionizing radiation, but relatively independent of radiation dose. Conversely, proteasome activator complex subunit 2 protein appeared to be sensitive to the dose of radiation, as rapid upregulation of this protein was observed when radiation doses were increased from 3, to 10 or 200 cGy. These results suggest that different mechanisms of action exist at the secretome level for low and high doses of ionizing radiation.

  18. Yeast Gga coat proteins function with clathrin in Golgi to endosome transport.

    Science.gov (United States)

    Costaguta, G; Stefan, C J; Bensen, E S; Emr, S D; Payne, G S

    2001-06-01

    Gga proteins represent a newly recognized, evolutionarily conserved protein family with homology to the "ear" domain of the clathrin adaptor AP-1 gamma subunit. Yeast cells contain two Gga proteins, Gga1p and Gga2p, that have been proposed to act in transport between the trans-Golgi network and endosomes. Here we provide genetic and physical evidence that yeast Gga proteins function in trans-Golgi network clathrin coats. Deletion of Gga2p (gga2Delta), the major Gga protein, accentuates growth and alpha-factor maturation defects in cells carrying a temperature-sensitive allele of the clathrin heavy chain gene. Cells carrying either gga2Delta or a deletion of the AP-1 beta subunit gene (apl2Delta) alone are phenotypically normal, but cells carrying both gga2Delta and apl2Delta are defective in growth, alpha-factor maturation, and transport of carboxypeptidase S to the vacuole. Disruption of both GGA genes and APL2 results in cells so severely compromised in growth that they form only microcolonies. Gga proteins can bind clathrin in vitro and cofractionate with clathrin-coated vesicles. Our results indicate that yeast Gga proteins play an important role in cargo-selective clathrin-mediated protein traffic from the trans-Golgi network to endosomes. PMID:11408593

  19. Primary structure of a coagulant enzyme, bilineobin, from Agkistrodon bilineatus venom.

    Science.gov (United States)

    Nikai, T; Ohara, A; Komori, Y; Fox, J W; Sugihara, H

    1995-04-01

    The amino acid sequence and disulfide bridge location of the coagulant enzyme, named bilineobin, isolated from the venom of Agkistrodon bilineatus was determined by Edman sequencing of the peptides derived from digests with cyanogen bromide, clostripain, Staphylococcus aureus V8 protease, trypsin, and chymotrypsin. This enzyme has a molecular weight of 57,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; however, bilineobin consists of 235 amino acids and has a calculated molecular weight of 26,481. The enzyme contains fucose, GlcNAc, galactose, mannose and NeuAc and six N-linked glycosylation consensus sites. The carboxyterminal amino acid, proline, was determined using carboxypeptidase Y. The six disulfide bonds of bilineobin link Cys78 to Cys234, Cys120 to Cys188, Cys178 to Cys203, Cys7 to Cys141, Cys152 to Cys167, and Cys28 to Cys44. The amino acid sequence similarity to flavoxobin (T.C. Shieh et al., 1988, J. Biochem (Tokyo) 103, 596-605) and batroxobin (N. Itoh et al., 1987, J. Biol. Chem. 262, 3132-3135) was 67%. The deglycosylated enzyme more rapidly generated fibrinopeptide A than native bilineobin. PMID:7726578

  20. Proteolytic activities in yeast after UV irradiation. Pt. 1

    International Nuclear Information System (INIS)

    Specific proteolytic activities are known to be induced in Escherichia coli following irradiation. Consequently it seemed of interest to investigate whether variations in proteinase activities occur in yeast. Among the five most well known proteinases of Saccharomyces cerevisiae, we have found that proteinase B activity increases up to three times in wild-type RAD+ yeast cells after a dose of 50 Jm-2 of 254 nm ultraviolet light (40% survival). Carboxypeptidase Y and aminopeptidase I (leucin aminopeptidase) activities were only moderately increased. Proteinase A activity was only slightly enhanced, while aminopeptidase II (lysin aminopeptidase) was unaffected in both RAD+ strains studied. The observed post UV-increase in proteinase B activity was inhibited by cycloheximide and was dose dependent. Increases in proteinase B levels were independent of the activation method used to destroy the proteinase B-inhibitor complex present in the crude yeast extracts. A standard method for comparison of the postirradiation levels among different proteinases, strains and methods of activation is presented. (orig.)

  1. Participation of neutral endopeptidase 24.11 (NEP;enkephalinase A) in kinin metabolism in vitro and in vivo

    International Nuclear Information System (INIS)

    Studies were done in 2 phases in rats. (1) Bradykinin was added catheter-collected urine, and its hydrolysis was determined by RIA. Three different kiniases were found by application of specific inhibitors. Kininase I-type carboxypeptidase was inhibited by 2-mercaptomethyl-3-guanidinoethyl-thiopropanoic acid, kiniase II by captopril and NEP by phosphoramidon (PA). Surprisingly, NEP was responsible for 68% of total kininase, while kininase I and II contributed only 9 and 23%. (2) To study the effects of inhibition of NEP on renal function, rats were infused with PA (330 μg/hr/kg, n=6). Urinary kinin level, kininase, GFR, RBF, U/sub Na/V, U/sub K/V and UV were measured. PA decreased total urinary kininase activity from 284 to 58 ng/min/kg (77%, p 125I-Tyr-bradykinin infused into the aorta did not appear in urine intact during PA administration. In conclusion, this is the first demonstration of NEP catabolizing kinins in vivo; its inhibition increased the excretion of intrarenally generated kinins. Changes in water and electrolyte excretion may be caused by kinins generated in the distal nephron

  2. Disruption of KEX1 gene reduces the proteolytic degradation of secreted two-chain Insulin glargine in Pichia pastoris.

    Science.gov (United States)

    Sreenivas, Suma; Krishnaiah, Sateesh M; Shyam Mohan, Anil H; Mallikarjun, Niveditha; Govindappa, Nagaraja; Chatterjee, Amarnath; Sastry, Kedarnath N

    2016-02-01

    Insulin glargine is a slow acting analog of insulin used in diabetes therapy. It is produced by recombinant DNA technology in different hosts namely E. coli and Pichia pastoris. In our previous study, we have described the secretion of fully folded two-chain Insulin glargine into the medium by over-expression of Kex2 protease. The enhanced levels of the Kex2 protease was responsible for the processing of the glargine precursor with in the host. Apart from the two-chain glargine product we observed a small proportion of arginine clipped species. This might be due to the clipping of arginine present at the C-terminus of the B-chain as it is exposed upon Kex2 cleavage. The carboxypeptidase precursor Kex1 is known to be responsible for clipping of C-terminal lysine or arginine of the proteins or peptides. In order to address this issue we created a Kex1 knock out in the host using Cre/loxP mechanism of targeted gene deletion. When two-chain glargine was expressed in the Kex1 knock out host of P. pastoris GS115 the C-terminal clipped species reduced by ∼80%. This modification further improved the process by reducing the levels of product related impurities. PMID:26470649

  3. A simple, economical method of converting gene expression products of insulin into recombinant insulin and its application

    Institute of Scientific and Technical Information of China (English)

    ZHANG Zhou; CHEN Hui; TANG Yuehua; FENG Youmin

    2003-01-01

    A method, by which the gene expression product of recombinant single chain insulin can be converted into insulin by directly digesting with trypsin, has been established. This method has been used in process of porcine insulin precursor (PIP), [B16Ala]PIP and [B26Ala]PIP into (desB30)insulin, (desB30)[B16Ala]insulin and (desB30)[B26Ala]insulin, respectively, and all of them retain full biological activity of that of their corresponding parent, recombinant human insulin, [B16Ala]insulin and [B26Ala]insulin. The results further demonstrate that the C-terminal residue of B chain is not necessary for insulin's biological activity. Compared with the method of transpeptidation, this method is simple, with a high yield, and avoids the use of organic reagents, and in comparison with the trypsin/carboxypeptidase method, it omits the use of carboxylpeptidase. Besides, (desB30)[B16Ala]insulin and (desB30)[B26Ala]insulin still remained without self-association property as that of their parents, which demonstrate that they are monomeric insulin. So they can be used for substituting for monomeric insulin, [B16Ala]insulin and [B26Ala]insulin, in clinical applications.

  4. Non-invasive detection of candidate pregnancy protein biomarkers in the feces of captive polar bears (Ursus maritimus).

    Science.gov (United States)

    Curry, E; Stoops, M A; Roth, T L

    2012-07-15

    Currently, there is no method of accurately and non-invasively diagnosing pregnancy in polar bears. Specific proteins may exhibit altered profiles in the feces of pregnant bears, but predicting appropriate candidate proteins to investigate is speculative at best. The objective of this study was to identify potential pregnancy biomarker proteins based on their increased abundance in the feces of pregnant polar bears compared to pseudopregnant females (controls) using two-dimensional in-gel electrophoresis (2D-DIGE) and mass spectrometry (MS). Three 2D-DIGE gels were performed to evaluate fecal protein profiles from controls (n=3) and pregnant polar bears (n=3). There were 2224.67±52.39 (mean±SEM) spots resolved per gel. Of these, only five proteins were elevated in the pregnant group (P99.9% confidence interval. The 11 spots represented seven distinct proteins, five of which were significantly more abundant in the pregnant group: IgGFc-binding protein, filamin-C, carboxypeptidase B, transthyretin, and immunoglobulin heavy chain variable region. To our knowledge, this was the first study that employed 2D-DIGE to identify differentially expressed proteins in fecal samples to characterize a physiological condition other than those related to gastrointestinal disorders. These promising results provided a strong foundation for ensuing efforts to develop a non-invasive pregnancy assay for use in both captive and wild polar bears. PMID:22538002

  5. Antimicrobial Protein Candidates from the Thermophilic Geobacillus sp. Strain ZGt-1: Production, Proteomics, and Bioinformatics Analysis

    Science.gov (United States)

    Alkhalili, Rawana N.; Bernfur, Katja; Dishisha, Tarek; Mamo, Gashaw; Schelin, Jenny; Canbäck, Björn; Emanuelsson, Cecilia; Hatti-Kaul, Rajni

    2016-01-01

    A thermophilic bacterial strain, Geobacillus sp. ZGt-1, isolated from Zara hot spring in Jordan, was capable of inhibiting the growth of the thermophilic G. stearothermophilus and the mesophilic Bacillus subtilis and Salmonella typhimurium on a solid cultivation medium. Antibacterial activity was not observed when ZGt-1 was cultivated in a liquid medium; however, immobilization of the cells in agar beads that were subjected to sequential batch cultivation in the liquid medium at 60 °C showed increasing antibacterial activity up to 14 cycles. The antibacterial activity was lost on protease treatment of the culture supernatant. Concentration of the protein fraction by ammonium sulphate precipitation followed by denaturing polyacrylamide gel electrophoresis separation and analysis of the gel for antibacterial activity against G. stearothermophilus showed a distinct inhibition zone in 15–20 kDa range, suggesting that the active molecule(s) are resistant to denaturation by SDS. Mass spectrometric analysis of the protein bands around the active region resulted in identification of 22 proteins with molecular weight in the range of interest, three of which were new and are here proposed as potential antimicrobial protein candidates by in silico analysis of their amino acid sequences. Mass spectrometric analysis also indicated the presence of partial sequences of antimicrobial enzymes, amidase and dd-carboxypeptidase. PMID:27548162

  6. Human Proinsulin C-peptide from a Precursor Overexpressed in Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Yang-Bin HUANG; Jun REN; You-Shang ZHANG; Jiang LI; Xin GAO; Jiu-Ru SUN; Yi LU; Tao FENG; Jian FEI; Da-Fu CUI; Qi-Chang XIA

    2006-01-01

    In this article we report the production of human proinsulin C-peptide with 31 amino acid residues from a precursor overexpressed in Pichia pastoris. A C-peptide precursor expression plasmid containing nine C-peptide genes in tandem was constructed and used to transform P. pastoris. Transformants with a high copy number of the C-peptide precursor gene integrated into the chromosome of P. pastoris were selected. In high-density fermentation in a 300 liter fermentor using a simple culture medium composed mainly of salt and methanol, the C-peptide precursor was overexpressed to a level of 2.28 g per liter. A simple procedure was established to purify the expression product from the culture medium. The purified C-peptide precursor was converted into C-peptide by trypsin and carboxypeptidase B joint digestion. The yield of C-peptide with a purity of 96% was 730 mg per liter of culture. The purified C-peptide was characterized by mass spectrometry, N- and C-terminal amino acid sequencing, and sodium dodecylsulfate-polyacrylamide gel electrophoresis.

  7. Quantitative variation in obesity-related traits and insulin precursors linked to the OB gene region on human chromosome 7

    Energy Technology Data Exchange (ETDEWEB)

    Duggirala, R.; Stern, M.P.; Reinhart, L.J. [Univ. of Texas Health Science Center, San Antonio, TX (United States)] [and others

    1996-09-01

    Despite the evidence that human obesity has strong genetic determinants, efforts at identifying specific genes that influence human obesity have largely been unsuccessful. Using the sibship data obtained from 32 low-income Mexican American pedigrees ascertained on a type II diabetic proband and a multipoint variance-components method, we tested for linkage between various obesity-related traits plus associated metabolic traits and 15 markers on human chromosome 7. We found evidence for linkage between markers in the OB gene region and various traits, as follows: D7S514 and extremity skinfolds (LOD = 3.1), human carboxypeptidase A1 (HCPA1) and 32,33-split proinsulin level (LOD = 4.2), and HCPA1 and proinsulin level (LOD = 3.2). A putative susceptibility locus linked to the marker D7S514 explained 56% of the total phenotypic variation in extremity skinfolds. Variation at the HCPA1 locus explained 64% of phenotypic variation in proinsulin level and {approximately}73% of phenotypic variation in split proinsulin concentration, respectively. Weaker evidence for linkage to several other obesity-related traits (e.g., waist circumference, body-mass index, fat mass by bioimpedance, etc.) was observed for a genetic location, which is {approximately}15 cM telomeric to OB. In conclusion, our study reveals that the OB region plays a significant role in determining the phenotypic variation of both insulin precursors and obesity-related traits, at least in Mexican Americans. 66 refs., 3 figs., 4 tabs.

  8. Production of human insulin in an E. coli system with Met-Lys-human proinsulin as the expressed precursor

    Energy Technology Data Exchange (ETDEWEB)

    Jin-Qiu Chen; Hong-Tao Zhang; Mei-Hao Hu; Jian-Guo Tang [Peking Univ., Beijing (China)

    1995-10-01

    The construction of a gene encoding Lys-human proinsulin, its direct expression in E.coli, and the simple purification procedure are described here. The temperature inducible promotor was employed for induction in a very short time. The expression level could reach 20-30%. After a simple downstream processing and only one step of Sephadex G50 purification, 150 mg recombinant Lys-human proinsulin with a purity of up to 90% could be obtained easily from 1 L of high density fermentation medium. The obtained product is in the form of Met-Lys-human proinsulin because of the failure of the bacterial host to remove the initiator methionine residue. The Lys-human proinsulin could be changed into human insulin by tryspin and carboxypeptidase B treatment in later steps. After separation with DEAE Sephadex A25, human insulin with expected amino acid composition and full native biological activity could be obtained with a yield of 50 mg/L fermentation medium. 20 refs., 5 figs., 4 tabs.

  9. Profilin 1 as a target for cathepsin X activity in tumor cells.

    Directory of Open Access Journals (Sweden)

    Urša Pečar Fonović

    Full Text Available Cathepsin X has been reported to be a tumor promotion factor in various types of cancer; however, the molecular mechanisms linking its activity with malignant processes are not understood. Here we present profilin 1, a known tumor suppressor, as a target for cathepsin X carboxypeptidase activity in prostate cancer PC-3 cells. Profilin 1 co-localizes strongly with cathepsin X intracellularly in the perinuclear area as well as at the plasma membrane. Selective cleavage of C-terminal amino acids was demonstrated on a synthetic octapeptide representing the profilin C-terminal region, and on recombinant profilin 1. Further, intact profilin 1 binds its poly-L-proline ligand clathrin significantly better than it does the truncated one, as shown using cathepsin X specific inhibitor AMS-36 and immunoprecipitation of the profilin 1/clathrin complex. Moreover, the polymerization of actin, which depends also on the binding of poly-L-proline ligands to profilin 1, was promoted by AMS-36 treatment of cells and by siRNA cathepsin X silencing. Our results demonstrate that increased adhesion, migration and invasiveness of tumor cells depend on the inactivation of the tumor suppressive function of profilin 1 by cathepsin X. The latter is thus designated as a target for development of new antitumor strategies.

  10. The structure of Plasmodium vivax phosphatidylethanolamine-binding protein suggests a functional motif containing a left-handed helix

    International Nuclear Information System (INIS)

    The crystal structure of a phosphatidylethanolamine-binding protein from P. vivax, a homolog of Raf-kinase inhibitor protein (RKIP), has been solved to a resolution of 1.3 Å. The inferred interaction surface near the anion-binding site is found to include a distinctive left-handed α-helix. The structure of a putative Raf kinase inhibitor protein (RKIP) homolog from the eukaryotic parasite Plasmodium vivax has been studied to a resolution of 1.3 Å using multiple-wavelength anomalous diffraction at the Se K edge. This protozoan protein is topologically similar to previously studied members of the phosphatidylethanolamine-binding protein (PEBP) sequence family, but exhibits a distinctive left-handed α-helical region at one side of the canonical phospholipid-binding site. Re-examination of previously determined PEBP structures suggests that the P. vivax protein and yeast carboxypeptidase Y inhibitor may represent a structurally distinct subfamily of the diverse PEBP-sequence family

  11. Quality Control and Stability Studies with the Monoclonal Antibody, Trastuzumab: Application of 1D- vs. 2D-Gel Electrophoresis

    Directory of Open Access Journals (Sweden)

    Dashnor Nebija

    2014-04-01

    Full Text Available Recombinant monoclonal antibodies (rmAbs are medicinal products obtained by rDNA technology. Consequently, like other biopharmaceuticals, they require the extensive and rigorous characterization of the quality attributes, such as identity, structural integrity, purity and stability. The aim of this work was to study the suitability of gel electrophoresis for the assessment of charge heterogeneity, post-translational modifications and the stability of the therapeutic, recombinant monoclonal antibody, trastuzumab. One-dimensional, SDS-PAGE, under reducing and non-reducing conditions, and two-dimensional gel electrophoresis were used for the determination of molecular mass (Mr, the isoelectric point (pI, charge-related isoform patterns and the stability of trastuzumab, subjected to stressed degradation and long-term conditions. For the assessment of the influence of glycosylation in the charge heterogeneity pattern of trastuzumab, an enzymatic deglycosylation study has been performed using N-glycosidase F and sialidase, whereas carboxypeptidase B was used for the lysine truncation study. Experimental data documented that 1D and 2D gel electrophoresis represent fast and easy methods to evaluate the quality of biological medicinal products. Important stability parameters, such as the protein aggregation, can be assessed, as well.

  12. In Escherichia coli, MreB and FtsZ direct the synthesis of lateral cell wall via independent pathways that require PBP 2.

    Science.gov (United States)

    Varma, Archana; Young, Kevin D

    2009-06-01

    In Escherichia coli, the cytoplasmic proteins MreB and FtsZ play crucial roles in ensuring that new muropeptide subunits are inserted into the cell wall in a spatially correct way during elongation and division. In particular, to retain a constant diameter and overall shape, new material must be inserted into the wall uniformly around the cell's perimeter. Current thinking is that MreB accomplishes this feat through intermediary proteins that tether peptidoglycan synthases to the outer face of the inner membrane. We tested this idea in E. coli by using a DD-carboxypeptidase mutant that accumulates pentapeptides in its peptidoglycan, allowing us to visualize new muropeptide incorporation. Surprisingly, inhibiting MreB with the antibiotic A22 did not result in uneven insertion of new wall, although the cells bulged and lost their rod shapes. Instead, uneven (clustered) incorporation occurred only if MreB and FtsZ were inactivated simultaneously, providing the first evidence in E. coli that FtsZ can direct murein incorporation into the lateral cell wall independently of MreB. Inhibiting penicillin binding protein 2 (PBP 2) alone produced the same clustered phenotype, implying that MreB and FtsZ tether peptidoglycan synthases via a common mechanism that includes PBP 2. However, cell shape was determined only by the presence or absence of MreB and not by the even distribution of new wall material as directed by FtsZ. PMID:19346310

  13. Characterization of a high affinity cocaine binding site in rat brain

    International Nuclear Information System (INIS)

    Binding of [3H]cocaine to synaptic membranes from whole rat brain was reversible and saturable. Nonlinear regression analysis of binding isotherms indicated two binding affinities: one with k/sub d/ = 16 nM, B/sub max/ = 0.65 pmoles/mg protein and the other with K/sub d/ = 660 nM, B/sub max/ = 5.1 pmoles/mg protein. The high-affinity binding of [3H]cocaine was sensitive to the actions of trypsin and chymotrypsin but not carboxypeptidase, and was eliminated by exposure of the membranes to 950C for 5 min. Specific binding at 2 nM was higher at pH 8.8 than at pH 7.0. Binding of [3H]cocaine (15 nM) was inhibited by increasing concentrations of Na+ ions. Several cocaine analogues, neurotransmitter uptake inhibitors and local anesthetics displaced specific [3H]cocaine binding at 2 nM with various potencies. The cocaine analogue (-)-norcocaine was the most potent (IC50 = 10 nM), while the local anesthetic tetracaine was the least potent in inhibiting [3H]cocaine binding. Several biogenic amine uptake inhibitors, including tricyclic antidepressants and phencyclidine, had IC50 values below μM concentrations

  14. Structure of Mycobacterium tuberculosis Rv2714, a representative of a duplicated gene family in Actinobacteria

    International Nuclear Information System (INIS)

    The crystal structure of Rv2714, a protein of unknown function from M. tuberculosis, has been determined at 2.6 Å resolution using single-wavelength anomalous diffraction methods. The gene Rv2714 from Mycobacterium tuberculosis, which codes for a hypothetical protein of unknown function, is a representative member of a gene family that is largely confined to the order Actinomycetales of Actinobacteria. Sequence analysis indicates the presence of two paralogous genes in most mycobacterial genomes and suggests that gene duplication was an ancient event in bacterial evolution. The crystal structure of Rv2714 has been determined at 2.6 Å resolution, revealing a trimer in which the topology of the protomer core is similar to that observed in a functionally diverse set of enzymes, including purine nucleoside phosphorylases, some carboxypeptidases, bacterial peptidyl-tRNA hydrolases and even the plastidic form of an intron splicing factor. However, some structural elements, such as a β-hairpin insertion involved in protein oligomerization and a C-terminal α-helical domain that serves as a lid to the putative substrate-binding (or ligand-binding) site, are only found in Rv2714 bacterial homologues and represent specific signatures of this protein family

  15. Yeast Vps55p, a functional homolog of human obesity receptor gene-related protein, is involved in late endosome to vacuole trafficking.

    Science.gov (United States)

    Belgareh-Touzé, Naïma; Avaro, Sandrine; Rouillé, Yves; Hoflack, Bernard; Haguenauer-Tsapis, Rosine

    2002-05-01

    The Saccharomyces cerevisiae VPS55 (YJR044c) gene encodes a small protein of 140 amino acids with four potential transmembrane domains. VPS55 belongs to a family of genes of unknown function, including the human gene encoding the obesity receptor gene-related protein (OB-RGRP). Yeast cells with a disrupted VPS55 present normal vacuolar morphology, but exhibit an abnormal secretion of the Golgi form of the soluble vacuolar carboxypeptidase Y. However, trafficking of the membrane-bound vacuolar alkaline phosphatase remains normal. The endocytosis of uracil permease, used as an endocytic marker, is normal in vps55Delta cells, but its degradation is delayed and this marker transiently accumulates in late endosomal compartments. We also found that Vps55p is mainly localized in the late endosomes. Collectively, these results indicate that Vps55p is involved in late endosome to vacuole trafficking. Finally, we show that human OB-RGRP displays the same distribution as Vps55p and corrects the phenotypic defects of the vps55Delta strain. Therefore, the function of Vps55p has been conserved throughout evolution. This study highlights the importance of the multispanning Vps55p and OB-RGRP in membrane trafficking to the vacuole/lysosome of eukaryotic cells. PMID:12006663

  16. Screening of Highly Expressed CPEΔN Lung Cancer H1299 Cells

    Directory of Open Access Journals (Sweden)

    Jing SUN

    2015-06-01

    Full Text Available Background and objective The N-terminal truncated carboxypeptidase E (CPEΔN protein is a novel biomarker of tumor metastasis. This study screened the H1299 cell line with a highly expressed CPEΔN gene for in vivo imaging experiment. Methods Human CPEΔN gene was cloned into the luciferase lentiviral vector. H1299 cells transduced with CPEΔN or control lentiviral vectors were selected with 2 µg/mL puromycin. The expression of CPEΔN was identified through Western blot analysis, and luciferase activity was measured using luciferase reporters. Results The human CPEΔN lentiviral expression vector was successfully constructed. The transfection rate of H1299 cells by the lentivirus achieved 80%, with an infection multiplicity of 20. The H1299 cell line with high CPEΔN (H1299-CPEΔN expression was established, with an increase in CPEΔN expression by four times compared with the control lentivirus-transfected H1299 cell line (H1299-control. As H1299-CPEΔN and H1299-control can effectively decompose luciferase substrates, they can be applied in in vivo imaging. Conclusion H1299-CPEΔN and H1299-control can be used in in vivo imaging experiment for further research on molecular mechanisms and signal transduction to elucidate the role of CPEΔN in lung cancer metastasis.

  17. Clinical Applications of Phage-Derived sFvs and sFv Fusion Proteins

    Directory of Open Access Journals (Sweden)

    K. A. Chester

    2000-01-01

    Full Text Available Single chain Fv antibodies (sFvs have been produced from filamentous bacteriophage libraries obtained from immunised mice. MFE-23, the most characterised of these sFvs, is reactive with carcinoembryonic antigen (CEA, a glycoprotein that is highly expressed in colorectal adenocarcinomas. MFE-23 has been expressed in bacteria and purified in our laboratory for two clinical trials; a gamma camera imaging trial using 123I-MFE-23 and a radioimmunoguided surgery trial using 125I-MFE-23, where tumour deposits are detected by a hand-held probe during surgery. Both these trials show MFE-23 is safe and effective in localising tumour deposits in patients with cancer. We are now developing fusion proteins which use MFE-23 to deliver a therapeutic moiety; MFE-23::CPG2 targets the enzyme carboxypeptidase G2 (CPG2 for use in the ADEPT (antibody directed enzyme prodrug therapy system and MFE::TNFα aims to reduce sequestration and increase tumor concentrations of systemically administered TNFα.

  18. Biochemical characterization of bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI

    Directory of Open Access Journals (Sweden)

    Kristensen Torsten

    2009-05-01

    Full Text Available Abstract Background TAFI is a plasma protein assumed to be an important link between coagulation and fibrinolysis. The three-dimensional crystal structures of authentic mature bovine TAFI (TAFIa in complex with tick carboxypeptidase inhibitor, authentic full lenght bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI, and recombinant human TAFI have recently been solved. In light of these recent advances, we have characterized authentic bovine TAFI biochemically and compared it to human TAFI. Results The four N-linked glycosylation sequons within the activation peptide were all occupied in bovine TAFI, similar to human TAFI, while the sequon located within the enzyme moiety of the bovine protein was non-glycosylated. The enzymatic stability and the kinetic constants of TAFIa differed somewhat between the two proteins, as did the isoelectric point of TAFI, but not TAFIa. Equivalent to human TAFI, bovine TAFI was a substrate for transglutaminases and could be proteolytically cleaved by trypsin or thrombin/solulin complex, although small differences in the fragmentation patterns were observed. Furthermore, bovine TAFI exhibited intrinsic activity and TAFIa attenuated tPA-mediated fibrinolysis similar to the human protein. Conclusion The findings presented here suggest that the properties of these two orthologous proteins are similar and that conclusions reached using the bovine TAFI may be extrapolated to the human protein.

  19. Proteomics reveals major components of oogenesis in the reproductive tract of sugar-fed Anopheles aquasalis.

    Science.gov (United States)

    Dias-Lopes, Geovane; Borges-Veloso, Andre; Saboia-Vahia, Leonardo; Padrón, Gabriel; de Faria Castro, Cássia Luana; Guimarães, Ana Carolina Ramos; Britto, Constança; Cuervo, Patricia; De Jesus, Jose Batista

    2016-05-01

    Anopheles (Nyssorhynchus) aquasalis is a malaria vector mainly distributed along the coastal regions of South and Central America. In the absence of an effective vaccine against malaria, strategies for controlling the vector are the main tool for interrupting parasite transmission. Mechanisms of oogenesis and embryogenesis in anautogenous mosquitoes are mainly modulated by blood feeding. However, the expression, at the protein level, of genes involved in such mechanisms in sugar-fed females is unknown. In this work, total protein extracts of the reproductive tract of female An. aquasalis that were fed sugar were analyzed using liquid chromatography followed by mass spectrometry for protein identification and bioinformatic tools for data mining. We identified 922 proteins expressed in the organ, and using several databases, we attributed biological meaning for several of them. Remarkably, nine proteins involved in oogenesis were identified in females fed sugar. Putative vitellogenins, vitellogenin receptor, lipid storage droplet, transferrin, ferritin, and apolipoprotein, identified here, are proteins involved in egg development. Proteins involved in embryonic development, such as paxillin, exuperantia, several growth factors, and dorsal switch protein, were identified. Interestingly, in this study, we identified 15 peptidases of various classes such as aminopeptidases, carboxypeptidases, serine protease, cathepsin, and metalloprotease that could potentially interact with male seminal components. Here, we demonstrated that the reproductive tract of female An. aquasalis fed on sugar expresses proteins involved in oogenesis and embryonic development. These findings reveal unknown aspects of the physiology of this organ under the given nutritional conditions. PMID:26850722

  20. Genome wide microarray based expression profiles associated with BmNPV resistance and susceptibility in Indian silkworm races of Bombyx mori.

    Science.gov (United States)

    Lekha, Govindaraj; Gupta, Tania; Awasthi, Arvind K; Murthy, Geetha N; Trivedy, Kanika; Ponnuvel, Kangayam M

    2015-12-01

    The molecular mechanism involved in BmNPV resistance was investigated using a genome wide microarray in midgut tissue of Indian silkworm Bombyx mori. In resistant race (Sarupat), 735 genes up-regulated and 589 genes down-regulated at 12 h post BmNPV infection. Similarly, in case of susceptible race (CSR-2), 2183 genes up-regulated and 2115 genes down-regulated. Among these, nine up-regulated and eight down-regulated genes were validated using real-time qPCR analysis. In Sarupat, vacuolar protein sorting associated, Xfin-like protein and carboxypeptidase E-like protein genes significantly up-regulated in infected midgut; prominently down-regulated genes were glutamate receptor ionotropic kainite 2-like, BTB/POZ domain and transferrin. Considerably up-regulated genes in the CSR-2 were peptidoglycan recognition protein S6 precursor and rapamycin while the conspicuous down-regulated genes were facilitated trehalose transporter and zinc transporter ZIP1-like gene. The up-regulation of genes in resistant race after BmNPV infection indicates their possible role in antiviral immune response. PMID:26376410

  1. Design, synthesis and biological evaluation of phosphorodiamidate prodrugs of antiviral and anticancer nucleosides

    Science.gov (United States)

    McGuigan, Christopher; Bourdin, Claire; Derudas, Marco; Hamon, Nadège; Hinsinger, Karen; Kandil, Sahar; Madela, Karolina; Meneghesso, Silvia; Pertusati, Fabrizio; Serpi, Michaela; Slusarczyk, Magdalena; Chamberlain, Stanley; Kolykhalov, Alexander; Vernachio, John; Vanpouille, Christophe; Introini, Andrea; Margolis, Leonid; Balzarini, Jan

    2014-01-01

    We herein report the application of the phosphorodiamidate phosphate prodrug approach to a series of thirteen nucleoside analogs with antiviral or anticancer activity. Twenty-five symmetrical phosphorodiamidates were synthesized, bearing esterified l-Alanine (and in one case d-alanine) in the prodrug moiety, each as single stereoisomer. The presence of an achiral phosphorus represents a potential advantage over the phosphoramidate ProTide approach, where diastereoisomeric mixtures are routinely obtained, and different biological profiles may be expected from the diastereoisomers. Optimization of the synthetic pathway allowed us to identify two general methods depending on the particular nucleoside analogs. All the compounds were biologically evaluated in antiviral and anticancer assays and several showed improvement of activity compared to their parent nucleosides, as in the case of ddA, d4T, abacavir and acyclovir against HIV-1 and/or HIV-2. The biological results were supported by metabolism studies with carboxypeptidase Y monitored by 31P NMR to investigate their bioactivation. This work further validates the phosphorodiamidate approach as a monophosphate prodrug motif with broad application in the antiviral and anticancer fields. PMID:24177359

  2. Antimicrobial Protein Candidates from the Thermophilic Geobacillus sp. Strain ZGt-1: Production, Proteomics, and Bioinformatics Analysis.

    Science.gov (United States)

    Alkhalili, Rawana N; Bernfur, Katja; Dishisha, Tarek; Mamo, Gashaw; Schelin, Jenny; Canbäck, Björn; Emanuelsson, Cecilia; Hatti-Kaul, Rajni

    2016-01-01

    A thermophilic bacterial strain, Geobacillus sp. ZGt-1, isolated from Zara hot spring in Jordan, was capable of inhibiting the growth of the thermophilic G. stearothermophilus and the mesophilic Bacillus subtilis and Salmonella typhimurium on a solid cultivation medium. Antibacterial activity was not observed when ZGt-1 was cultivated in a liquid medium; however, immobilization of the cells in agar beads that were subjected to sequential batch cultivation in the liquid medium at 60 °C showed increasing antibacterial activity up to 14 cycles. The antibacterial activity was lost on protease treatment of the culture supernatant. Concentration of the protein fraction by ammonium sulphate precipitation followed by denaturing polyacrylamide gel electrophoresis separation and analysis of the gel for antibacterial activity against G. stearothermophilus showed a distinct inhibition zone in 15-20 kDa range, suggesting that the active molecule(s) are resistant to denaturation by SDS. Mass spectrometric analysis of the protein bands around the active region resulted in identification of 22 proteins with molecular weight in the range of interest, three of which were new and are here proposed as potential antimicrobial protein candidates by in silico analysis of their amino acid sequences. Mass spectrometric analysis also indicated the presence of partial sequences of antimicrobial enzymes, amidase, and dd-carboxypeptidase. PMID:27548162

  3. Dietary protein deficiency reduces lysosomal and nonlysosomal ATP-dependent proteolysis in muscle

    Science.gov (United States)

    Tawa, N. E. Jr; Kettelhut, I. C.; Goldberg, A. L.

    1992-01-01

    When rats are fed a protein deficient (PD) diet for 7 days, rates of proteolysis in skeletal muscle decrease by 40-50% (N. E. Tawa, Jr., and A. L. Goldberg. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E317-325, 1992). To identify the underlying biochemical adaptations, we measured different proteolytic processes in incubated muscles. The capacity for intralysosomal proteolysis, as shown by sensitivity to methylamine or lysosomal protease inhibitors, fell 55-75% in muscles from PD rats. Furthermore, extracts of muscles of PD rats showed 30-70% lower activity of many lysosomal proteases, including cathepsins B, H, and C, and carboxypeptidases A and C, as well as other lysosomal hydrolases. The fall in cathepsin B and proteolysis was evident by 3 days on the PD diet, and both returned to control levels 3 days after refeeding of the normal diet. In muscles maintained under optimal conditions, 80-90% of protein breakdown occurs by nonlysosomal pathways. In muscles of PD rats, this ATP-dependent process was also 40-60% slower. Even though overall proteolysis decreased in muscles of PD rats, their capacity for Ca(2+)-dependent proteolysis increased (by 66%), as did the activity of the calpains (+150-250%). Thus the lysosomal and the ATP-dependent processes decrease coordinately and contribute to the fall in muscle proteolysis in PD animals.

  4. Subfamily-specific adaptations in the structures of two penicillin-binding proteins from Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Daniil M Prigozhin

    Full Text Available Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows that Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis.

  5. Transcriptome Analysis of the Carmine Spider Mite, Tetranychus cinnabarinus (Boisduval, 1867 (Acari: Tetranychidae, and Its Response to β-Sitosterol

    Directory of Open Access Journals (Sweden)

    Chunya Bu

    2015-01-01

    Full Text Available Tetranychus cinnabarinus (Acari: Tetranychidae is a worldwide polyphagous agricultural pest that has the title of resistance champion among arthropods. We reported previously the identification of the acaricidal compound β-sitosterol from Mentha piperita and Inula japonica. However, the acaricidal mechanism of β-sitosterol is unclear. Due to the limited genetic research carried out, we de novo assembled the transcriptome of T. cinnabarinus using Illumina sequencing and conducted a differential expression analysis of control and β-sitosterol-treated mites. In total, we obtained >5.4 G high-quality bases for each sample with unprecedented sequencing depth and assembled them into 22,941 unigenes. We identified 617 xenobiotic metabolism-related genes involved in detoxification, binding, and transporting of xenobiotics. A highly expanded xenobiotic metabolic system was found in mites. T. cinnabarinus detoxification genes—including carboxyl/cholinesterase and ABC transporter class C—were upregulated after β-sitosterol treatment. Defense-related proteins, such as Toll-like receptor, legumain, and serine proteases, were also activated. Furthermore, other important genes—such as the chloride channel protein, cytochrome b, carboxypeptidase, peritrophic membrane chitin binding protein, and calphostin—may also play important roles in mites’ response to β-sitosterol. Our results demonstrate that high-throughput-omics tool facilitates identification of xenobiotic metabolism-related genes and illustration of the acaricidal mechanisms of β-sitosterol.

  6. Detecting zinc metal enzyme syntheses after zinc supplementation to deficient animals by measuring 65Zn in individual organs

    International Nuclear Information System (INIS)

    For the purpose of detecting the synthesis of zinc metalloenzymes after zinc supplementation in an experiment using rats the animals were first depleted for 15 days and subsequently injected a labelled zinc salt solution (65Zn-ZnCl2) in a dosis of 0.4 mg (in terms of Zn) and with an activity of 3.0 μCi/100 μl. After a 5-day depletion period, the activity of the metallo-enzymes alkaline phosphatase in plasma and in the femur and carboxy-peptidase A and B in the pancreatic gland was found to rise at the same rate as the 65Zn-measuring rate in plasma, femur and pancreatic gland. By calculating correlations this interdependence was demonstrated. Thus the highly significant correlation coefficients prove for these metalloenzymes that a synthesis with the injected zinc salt has taken place whilst for carbo-anhydrase in blood this evidence was not furnished. As the zinc dosis is not exclusively used for the enzyme synthesis, but additional 65Zn is incorporated into the individual organs, it does not appear to be possible to draw conclusions from the 65Zn-measuring rate in the individual organs on the intermediary availability. (author)

  7. The Prediction of the Expected Current Selection Coefficient of Single Nucleotide Polymorphism Associated with Holstein Milk Yield, Fat and Protein Contents.

    Science.gov (United States)

    Lee, Young-Sup; Shin, Donghyun; Lee, Wonseok; Taye, Mengistie; Cho, Kwanghyun; Park, Kyoung-Do; Kim, Heebal

    2016-01-01

    Milk-related traits (milk yield, fat and protein) have been crucial to selection of Holstein. It is essential to find the current selection trends of Holstein. Despite this, uncovering the current trends of selection have been ignored in previous studies. We suggest a new formula to detect the current selection trends based on single nucleotide polymorphisms (SNP). This suggestion is based on the best linear unbiased prediction (BLUP) and the Fisher's fundamental theorem of natural selection both of which are trait-dependent. Fisher's theorem links the additive genetic variance to the selection coefficient. For Holstein milk production traits, we estimated the additive genetic variance using SNP effect from BLUP and selection coefficients based on genetic variance to search highly selective SNPs. Through these processes, we identified significantly selective SNPs. The number of genes containing highly selective SNPs with p-value spectrin, beta, non-erythrocytic 1 (SPTBN1), ADP-ribosylation factor interacting protein 1 (ARFIP1), mutL homolog 1 (MLH1), transmembrane channel-like 7 (TMC7), carboxypeptidase X, member 2 (CPXM2) and ADAM metallopeptidase domain 12 (ADAM12). These genes may be important for future artificial selection trends. Also, we found that the SNP effect predicted from BLUP was the key factor to determine the expected current selection coefficient of SNP. Under Hardy-Weinberg equilibrium of SNP markers in current generation, the selection coefficient is equivalent to 2*SNP effect. PMID:26732326

  8. The genome sequence of Leishmania (Leishmania) amazonensis: functional annotation and extended analysis of gene models.

    Science.gov (United States)

    Real, Fernando; Vidal, Ramon Oliveira; Carazzolle, Marcelo Falsarella; Mondego, Jorge Maurício Costa; Costa, Gustavo Gilson Lacerda; Herai, Roberto Hirochi; Würtele, Martin; de Carvalho, Lucas Miguel; Carmona e Ferreira, Renata; Mortara, Renato Arruda; Barbiéri, Clara Lucia; Mieczkowski, Piotr; da Silveira, José Franco; Briones, Marcelo Ribeiro da Silva; Pereira, Gonçalo Amarante Guimarães; Bahia, Diana

    2013-12-01

    We present the sequencing and annotation of the Leishmania (Leishmania) amazonensis genome, an etiological agent of human cutaneous leishmaniasis in the Amazon region of Brazil. L. (L.) amazonensis shares features with Leishmania (L.) mexicana but also exhibits unique characteristics regarding geographical distribution and clinical manifestations of cutaneous lesions (e.g. borderline disseminated cutaneous leishmaniasis). Predicted genes were scored for orthologous gene families and conserved domains in comparison with other human pathogenic Leishmania spp. Carboxypeptidase, aminotransferase, and 3'-nucleotidase genes and ATPase, thioredoxin, and chaperone-related domains were represented more abundantly in L. (L.) amazonensis and L. (L.) mexicana species. Phylogenetic analysis revealed that these two species share groups of amastin surface proteins unique to the genus that could be related to specific features of disease outcomes and host cell interactions. Additionally, we describe a hypothetical hybrid interactome of potentially secreted L. (L.) amazonensis proteins and host proteins under the assumption that parasite factors mimic their mammalian counterparts. The model predicts an interaction between an L. (L.) amazonensis heat-shock protein and mammalian Toll-like receptor 9, which is implicated in important immune responses such as cytokine and nitric oxide production. The analysis presented here represents valuable information for future studies of leishmaniasis pathogenicity and treatment. PMID:23857904

  9. Application of Proteomics to the Study of Pollination Drops

    Directory of Open Access Journals (Sweden)

    Natalie Prior

    2013-04-01

    Full Text Available Premise of the study: Pollination drops are a formative component in gymnosperm pollen-ovule interactions. Proteomics offers a direct method for the discovery of proteins associated with this early stage of sexual reproduction. Methods: Pollination drops were sampled from eight gymnosperm species: Chamaecyparis lawsoniana (Port Orford cedar, Ephedra monosperma, Ginkgo biloba, Juniperus oxycedrus (prickly juniper, Larix ×marschlinsii, Pseudotsuga menziesii (Douglas-fir, Taxus ×media, and Welwitschia mirabilis. Drops were collected by micropipette using techniques focused on preventing sample contamination. Drop proteins were separated using both gel and gel-free methods. Tandem mass spectrometric methods were used including a triple quadrupole and an Orbitrap. Results: Proteins are present in all pollination drops. Consistency in the protein complement over time was shown in L. ×marschlinsii. Representative mass spectra from W. mirabilis chitinase peptide and E. monosperma serine carboxypeptidase peptide demonstrated high quality results. We provide a summary of gymnosperm pollination drop proteins that have been discovered to date via proteomics. Discussion: Using proteomic methods, a dozen classes of proteins have been identified to date. Proteomics presents a way forward in deepening our understanding of the biological function of pollination drops.

  10. Crystal Structure of Homo Sapiens PTD012 Reveals a Zinc-Containing Hydrolase Fold

    Energy Technology Data Exchange (ETDEWEB)

    Manjasetty,B.; Bussow, K.; Fieber-ErdMan, M.; Roske, Y.; Gobam, J.; Scheich, C.; Gotz, F.; Niesen, F.; Heinemann, U.

    2006-01-01

    The human protein PTD012 is the longer product of an alternatively spliced gene and was described to be localized in the nucleus. The X-ray structure analysis at 1.7 Angstroms resolution of PTD012 through SAD phasing reveals a monomeric protein and a novel fold. The shorter splice form was also studied and appears to be unfolded and non-functional. The structure of PTD012 displays an {alpha}{beta}{beta}{alpha} four-layer topology. A metal ion residing between the central {beta}-sheets is partially coordinated by three histidine residues. X-ray absorption near-edge structure (XANES) analysis identifies the PTD012-bound ion as Zn{sup 2+}. Tetrahedral coordination of the ion is completed by the carboxylate oxygen atom of an acetate molecule taken up from the crystallization buffer. The binding of Zn{sup 2+} to PTD012 is reminiscent of zinc-containing enzymes such as carboxypeptidase, carbonic anhydrase, and {beta}-lactamase. Biochemical assays failed to demonstrate any of these enzyme activities in PTD012. However, PTD012 exhibits ester hydrolase activity on the substrate p-nitrophenyl acetate.

  11. 单核细胞增生性李斯特菌prfA缺失株及其亲本株胞外蛋白的比较蛋白质组学%Comparative proteomics analysis of extracellular proteins from Listeria monocytogenes and its isogenic prfA deletion mutant

    Institute of Scientific and Technical Information of China (English)

    殷月兰; 白春光; 王国梁; 贾艳艳; 渠瑾; 付红; 高云飞; 焦新安

    2013-01-01

    [目的]PrfA蛋白对单核细胞增生性李斯特菌致病过程中毒力基因的表达起着重要调控作用,本文从蛋白质水平上初步探讨了PrfA的调控功能.[方法]对LM4及LM4 AprfA的胞外蛋白采用双向凝胶电泳结合基质辅助激光解析电离飞行时间质谱鉴定技术,进行比较蛋白质组学研究.[结果]发现差异表达的有31个蛋白点,质谱鉴定成功19个点,对应12种蛋白,其中已知的毒力相关蛋白有:InlC、ActA、LLO,此外还发现丙氨酸丙氨酰羧肽酶、GW重复表面蛋白、假定转录调节因子、天冬氨酸半醛脱氢酶和一些假定蛋白.采用实时荧光定量PCR方法对蛋白质组学方法获得的结果进行了验证,结果显示hly、actA、inlC的基因表达量显著下降,丙氨酰氨羧肽酶、GW重复表面蛋白的mRNA转录水平降低.[结论]PrfA蛋白对毒力岛LIPI-Ⅰ和毒力岛LIPI-Ⅱ中毒力基因的表达具有重要的调控作用,新发现的转录调控因子和假定蛋白的功能有待于进一步深入研究.%[ Objective ] Positive regulatory factor A ( PrfA) protein plays a key role in the pathogenicity of Listeria monocytogenes by regulating the expression of virulence genes. We studied the regulation functions of PrfA and its role in Listeria monocytogenes (Lm) virulence. [Method] Extracellular proteins were obtained from the supernatants of parental strain LM4 and mutant strain LM4ΔprfA cultured in minimal medium. We used two-dimensional gel electrophoresis and matrix associated laser dissociation/ionization time of flight mass spectrometry ( MALDI- TOF-MS ) to analyze the differences of secreted proteins between LM4 and LM4ΔprfA. [Results] The electrophoresis results show that 31 different spots, 19 spots corresponding 12 proteins were identified by MALDI- TOF-MS. Some virulence related proteins were verified, such as InlC, ActA and LLO. Some new proteins that are regulated by PrfA include D-alanyl-D-alanine carboxypeptidase, dipeptide

  12. 筛选高表达CPEΔN的H1299肺癌细胞株%Screening of Highly Expressed CPEΔN Lung Cancer H1299 Cells

    Institute of Scientific and Technical Information of China (English)

    孙静; 张桂荣; 王虹月; 申慧

    2015-01-01

    Background and objective hTe N-terminal truncated carboxypeptidase E (CPEΔN) protein is a novel biomarker of tumor metastasis. hTis study screened the H1299 cell line with a highly expressed CPEΔN gene forin vivo imag-ing experiment.Methods Human CPEΔN gene was cloned into the luciferase lentiviral vector. H1299 cells transduced with CPEΔN or control lentiviral vectors were selected with 2 µg/mL puromycin. hTe expression of CPEΔN was identiifed through Western blot analysis, and luciferase activity was measured using luciferase reporters.Results hTe human CPEΔN lentiviral expression vector was successfully constructed. hTe transfection rate of H1299 cells by the lentivirus achieved 80%, with an infection multiplicity of 20. hTe H1299 cell line with high CPEΔN (H1299-CPEΔN) expression was established, with an in-crease in CPEΔN expression by four times compared with the control lentivirus-transfected H1299 cell line (H1299-control). As H1299-CPEΔN and H1299-control can effectively decompose luciferase substrates, they can be applied in in vivo imaging. Conclusion H1299-CPEΔN and H1299-control can be used inin vivo imaging experiment for further research on molecular mechanisms and signal transduction to elucidate the role of CPEΔN in lung cancer metastasis.%背景与目的 N端截短的羧肽酶E(N-terminal truncated carboxypeptidase E, CPEΔN)是一个新的肿瘤转移相关蛋白。本研究旨在筛选高表达CPEΔN的H1299肺癌细胞株,为完成小鼠活体成像实验创造条件。方法构建CPEΔN的慢病毒表达载体。分别用CPEΔN慢病毒表达载体或对照慢病毒空载体转染H1299细胞,2µg/mL的嘌呤霉素加压筛选。Western blot分析CPEΔN蛋白的表达,荧光素酶报告基因实验分析荧光素酶对底物的分解作用。结果当感染倍数(multiple of infection, MOI)是20时,慢病毒对H1299细胞的转染效率可以达到80%。CPEΔN高表达H1299细胞株(H1299-CPEΔN)和

  13. Sputum mast cell subtypes relate to eosinophilia and corticosteroid response in asthma.

    Science.gov (United States)

    Wang, Gang; Baines, Katherine J; Fu, Juan Juan; Wood, Lisa G; Simpson, Jodie L; McDonald, Vanessa M; Cowan, Douglas C; Taylor, D Robin; Cowan, Jan O; Gibson, Peter G

    2016-04-01

    Mast cells are a resident inflammatory cell of the airways, involved in both the innate and adaptive immune response. The relationship between mast cells and inflammatory phenotypes and treatment response of asthma is not clear.Clinical characteristics of subjects with stable asthma (n=55), inflammatory cell counts and gene expression microarrays in induced sputum were analysed. Sputum mast cell subtypes were determined by molecular phenotyping based on expression of mast cell biomarkers (tryptase (TPSAB1), chymase (CMA1) and carboxypeptidase A3 (CPA3)). Effects of mast cell subtypes on steroid response were observed in a prospective cohort study (n=50).MCT(n=18) and MCT/CPA3(mRNA expression ofTPSAB1andCPA3; n=29) subtypes were identified, as well as a group without mast cell gene expression (n=8). The MCT/CPA3subtype had elevated exhaled nitric oxide fraction, sputum eosinophils, bronchial sensitivity and reactivity, and poorer asthma control. This was accompanied by upregulation of 13 genes. Multivariable logistic regression identifiedCPA3(OR 1.21, p=0.004) rather thanTPSAB1(OR 0.92, p=0.502) as a determinant of eosinophilic asthma. The MCT/CPA3subtype had a better clinical response and reduced signature gene expression with corticosteroid treatment.Sputum mast cell subtypes of asthma can be defined by a molecular phenotyping approach. The MCT/CPA3subtype demonstrated increased bronchial sensitivity and reactivity, and signature gene expression, which was associated with airway eosinophilia and greater corticosteroid responsiveness. PMID:26699720

  14. Knockdown of midgut genes by dsRNA-transgenic plant-mediated RNA interference in the hemipteran insect Nilaparvata lugens.

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    Wenjun Zha

    Full Text Available BACKGROUND: RNA interference (RNAi is a powerful technique for functional genomics research in insects. Transgenic plants producing double-stranded RNA (dsRNA directed against insect genes have been reported for lepidopteran and coleopteran insects, showing potential for field-level control of insect pests, but this has not been reported for other insect orders. METHODOLOGY/PRINCIPAL FINDINGS: The Hemipteran insect brown planthopper (Nilaparvata lugens Stål is a typical phloem sap feeder specific to rice (Oryza sativa L.. To analyze the potential of exploiting RNAi-mediated effects in this insect, we identified genes (Nlsid-1 and Nlaub encoding proteins that might be involved in the RNAi pathway in N. lugens. Both genes are expressed ubiquitously in nymphs and adult insects. Three genes (the hexose transporter gene NlHT1, the carboxypeptidase gene Nlcar and the trypsin-like serine protease gene Nltry that are highly expressed in the N. lugens midgut were isolated and used to develop dsRNA constructs for transforming rice. RNA blot analysis showed that the dsRNAs were transcribed and some of them were processed to siRNAs in the transgenic lines. When nymphs were fed on rice plants expressing dsRNA, levels of transcripts of the targeted genes in the midgut were reduced; however, lethal phenotypic effects after dsRNA feeding were not observed. CONCLUSIONS: Our study shows that genes for the RNAi pathway (Nlsid-1 and Nlaub are present in N. lugens. When insects were fed on rice plant materials expressing dsRNAs, RNA interference was triggered and the target genes transcript levels were suppressed. The gene knockdown technique described here may prove to be a valuable tool for further investigations in N. lugens. The results demonstrate the potential of dsRNA-mediated RNAi for field-level control of planthoppers, but appropriate target genes must be selected when designing the dsRNA-transgenic plants.

  15. The inactivation of the sortilin gene leads to a partial disruption of prosaposin trafficking to the lysosomes

    International Nuclear Information System (INIS)

    Lysosomes are intracellular organelles which contain enzymes and activator proteins involved in the digestion and recycling of a variety of cellular and extracellular substances. We have identified a novel sorting receptor, sortilin, which is involved in the lysosomal trafficking of the sphingolipid activator proteins, prosaposin and GM2AP, and the soluble hydrolases cathepsin D, cathepsin H, and acid sphingomyelinase. Sortilin belongs to a growing family of receptors with homology to the yeast Vps10 protein, which acts as a lysosomal sorting receptor for carboxypeptidase Y. In this study we examined the effects of the sortilin gene inactivation in mice. The inactivation of this gene did not yield any noticeable lysosomal pathology. To determine the existence of an alternative receptor complementing the sorting function of sortilin, we quantified the concentration of prosaposin in the lysosomes of the nonciliated epithelial cells lining the efferent ducts. These cells were chosen because they express sortilin and have a large number of lysosomes containing prosaposin. In addition, the nonciliated cells are known to endocytose luminal prosaposin that is synthesized and secreted by Sertoli cells into the seminiferous luminal fluids. Consequently, the nonciliated cells are capable of targeting both exogenous and endogenous prosaposin to the lysosomes. Using electron microscope immunogold labeling and quantitative analysis, our results demonstrate that inactivation of the sortilin gene produces a significant decrease of prosaposin in the lysosomes. When luminal prosaposin was excluded from the efferent ducts, the level of prosaposin in lysosomes was even lower in the mutant mice. Nonetheless, a significant amount of prosaposin continues to reach the lysosomal compartment. These results strongly suggest the existence of an alternative receptor that complements the function of sortilin and explains the lack of lysosomal storage disorders in the sortilin-deficient mice.

  16. Unblocking the Sink: Improved CID-Based Analysis of Phosphorylated Peptides by Enzymatic Removal of the Basic C-Terminal Residue

    Science.gov (United States)

    Lanucara, Francesco; Chi Hoo Lee, Dave; Eyers, Claire E.

    2013-12-01

    A one-step enzymatic reaction for improving the collision-induced dissociation (CID)-based tandem mass spectrometry (MS/MS) analysis of phosphorylated peptides in an ion trap is presented. Carboxypeptidase-B (CBP-B) was used to selectively remove C-terminal arginine or lysine residues from phosphorylated tryptic/Lys-C peptides prior to their MS/MS analysis by CID with a Paul-type ion trap. Removal of this basic C-terminal residue served to limit the extent of gas-phase neutral loss of phosphoric acid (H3PO4), favoring the formation of diagnostic b and y ions as determined by an increase in both the number and relative intensities of the sequence-specific product ions. Such differential fragmentation is particularly valuable when the H3PO4 elimination is so predominant that localizing the phosphorylation site on the peptide sequence is hindered. Improvement in the quality of tandem mass spectral data generated by CID upon CBP-B treatment resulted in greater confidence both in assignment of the phosphopeptide primary sequence and for pinpointing the site of phosphorylation. Higher Mascot ion scores were also generated, combined with lower expectation values and higher delta scores for improved confidence in site assignment; Ascore values also improved. These results are rationalized in accordance with the accepted mechanisms for the elimination of H3PO4 upon low energy CID and insights into the factors dictating the observed dissociation pathways are presented. We anticipate this approach will be of utility in the MS analysis of phosphorylated peptides, especially when alternative electron-driven fragmentation techniques are not available.

  17. [Biochemical diagnostics in acute pancreatitis recognition and outcome predicition].

    Science.gov (United States)

    Olczyk, Paweł; Kozma, Ewa M; Olczyk, Krystyna; Komosińska-Vassev, Katarzyna

    2004-01-01

    Acute pancreatitis (AP) is a common disease associated with an improper activation of pancreatic zymogens leading to autodigestion of the gland and if excessive--to multiple organ dysfunction. Acute necrotizing pancreatitis manifested by 20% of patients with acute pancreatitis is a life threatening disorder requiring subsequent management in intensive care unit. Unfortunately, none of biochemical tests presently used for laboratory assessment of acute pancreatitis at the early stage of the disease is able to estimate accurately: diagnosis, etiology and severity. At present, diagnosis of acute pancreatitis is based on evaluation of serum amylase and lipase activity due to easy availability and simplicity of these enzymatic tests. Low specificity of the mentioned enzymes resulted in studies concerning pancreatic isoamylase, elastase-1, chymotrypsine, procarboxy-peptidase B, trypsinogen-2 and immunoreactive trypsinogen usefulness in the laboratory diagnosis of AP. The prediction of severity in acute pancreatitis using multifactorial scoring systems is cumbersome especially due to their complexity. On the other hand the biochemical method of choice, estimation of serum C reactive protein, is useless in the early phase of disease. Unfortunately, the computed tomography--the most accurate method in severity assessing--is not always available. Recent studies have brought some progress in severity predicting, such as phospholipase A2, cellular immunity markers, cytokines, activation peptides of trypsinogen and carboxypeptidase B, procalcitonine, pancreatitis associated protein and serum amyloid A. All these newly introduced biochemical methods allow to look optimistically into the future of laboratory diagnostics of the acute pancreatitis believing that the problem of diagnosing and predicting the AP severity will be solved. PMID:15850341

  18. Activation of autophagy by unfolded proteins during endoplasmic reticulum stress.

    Science.gov (United States)

    Yang, Xiaochen; Srivastava, Renu; Howell, Stephen H; Bassham, Diane C

    2016-01-01

    Endoplasmic reticulum stress is defined as the accumulation of unfolded proteins in the endoplasmic reticulum, and is caused by conditions such as heat or agents that cause endoplasmic reticulum stress, including tunicamycin and dithiothreitol. Autophagy, a major pathway for degradation of macromolecules in the vacuole, is activated by these stress agents in a manner dependent on inositol-requiring enzyme 1b (IRE1b), and delivers endoplasmic reticulum fragments to the vacuole for degradation. In this study, we examined the mechanism for activation of autophagy during endoplasmic reticulum stress in Arabidopsis thaliana. The chemical chaperones sodium 4-phenylbutyrate and tauroursodeoxycholic acid were found to reduce tunicamycin- or dithiothreitol-induced autophagy, but not autophagy caused by unrelated stresses. Similarly, over-expression of BINDING IMMUNOGLOBULIN PROTEIN (BIP), encoding a heat shock protein 70 (HSP70) molecular chaperone, reduced autophagy. Autophagy activated by heat stress was also found to be partially dependent on IRE1b and to be inhibited by sodium 4-phenylbutyrate, suggesting that heat-induced autophagy is due to accumulation of unfolded proteins in the endoplasmic reticulum. Expression in Arabidopsis of the misfolded protein mimics zeolin or a mutated form of carboxypeptidase Y (CPY*) also induced autophagy in an IRE1b-dependent manner. Moreover, zeolin and CPY* partially co-localized with the autophagic body marker GFP-ATG8e, indicating delivery to the vacuole by autophagy. We conclude that accumulation of unfolded proteins in the endoplasmic reticulum is a trigger for autophagy under conditions that cause endoplasmic reticulum stress. PMID:26616142

  19. Ineffective degradation of immunogenic gluten epitopes by currently available digestive enzyme supplements.

    Directory of Open Access Journals (Sweden)

    George Janssen

    Full Text Available Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP.Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1 enzyme assays and 2 mass spectrometric identification. Gluten epitope degradation was monitored by 1 R5 ELISA, 2 mass spectrometric analysis of the degradation products and 3 T cell proliferation assays.The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data.Currently available digestive enzyme supplements are ineffective in

  20. Structure of Csd3 from Helicobacter pylori, a cell shape-determining metallopeptidase

    International Nuclear Information System (INIS)

    H. pylori Csd3 (HP0506), together with other peptidoglycan hydrolases, plays an important role in determining cell shape. Its crystal structure in the latent state is reported. Helicobacter pylori is associated with various gastrointestinal diseases such as gastritis, ulcers and gastric cancer. Its colonization of the human gastric mucosa requires high motility, which depends on its helical cell shape. Seven cell shape-determining genes (csd1, csd2, csd3/hdpA, ccmA, csd4, csd5 and csd6) have been identified in H. pylori. Their proteins play key roles in determining the cell shape through modifications of the cell-wall peptidoglycan by the alteration of cross-linking or by the trimming of peptidoglycan muropeptides. Among them, Csd3 (also known as HdpA) is a bifunctional enzyme. Its d, d-endopeptidase activity cleaves the d-Ala4-mDAP3 peptide bond between cross-linked muramyl tetrapeptides and pentapeptides. It is also a d, d-carboxypeptidase that cleaves off the terminal d-Ala5 from the muramyl pentapeptide. Here, the crystal structure of this protein has been determined, revealing the organization of its three domains in a latent and inactive state. The N-terminal domain 1 and the core of domain 2 share the same fold despite a very low level of sequence identity, and their surface-charge distributions are different. The C-terminal LytM domain contains the catalytic site with a Zn2+ ion, like the similar domains of other M23 metallopeptidases. Domain 1 occludes the active site of the LytM domain. The core of domain 2 is held against the LytM domain by the C-terminal tail region that protrudes from the LytM domain

  1. Investigation of molecular mechanisms in photodynamic action and radiobiology with nanosecond flash photolysis and pulse radiolysis. Progress report, July 1, 1976--September 30, 1977

    Energy Technology Data Exchange (ETDEWEB)

    Grossweiner, L.I.

    1977-06-01

    Laser flash photolysis investigations on aromatic amino acids and proteins have demonstrated that monophotonic electron ejection is the major initial act, leading to e/sup -//sub aq/ and the corresponding aromatic radicals, followed by back reactions limited by available e/sup -//sub aq/ scavengers. Results with ribonuclease A, lysozyme and carboxypeptidase A have led to information about the relationship of the photoionization efficiency of aromatic residues to the microenvironment. Measurements on the decay kinetics of photoelectrons have shown that the lifetimes and their dependence on scavenger concentrations and dose are inconsistent with homogeneous reactions. A new theory is proposed in which the photoelectron diffuses through the medium as a quasi-free particle, where original pair-recombination competes with scavenging and pair-pair interactions. This theory is in good agreement with laser flash photolysis studies on I/sup -/, FE(CN)/sub 6//sup 4 -/, tryptophan and tyrosine and consistent with earlier photochemical scavenging measurements. The general analysis of radition sensitivity has been extended to suspensions of large biological targets, such as vesicles, viruses and cells, particularly where the radical diffusion length is smaller than or comparable to the collision radius. The development is exemplified with new work on inactivation of T7 bacteriophag by 25 MeV electrons and photodynamic inactivation of Saccharomyces cerevisiae. Detailed studies on yeast have shown that the sensitivity to singlet oxygen attack depends on the temperature and the culture growth phase. Spin label ESR measurements indicate that he conditions of low photosensitivity parallel low membrane fluidity.

  2. Effect of heparin on TAFI-dependent inhibition of fibrinolysis: relative importance of TAFIa generated by clot-bound and fluid phase thrombin.

    Science.gov (United States)

    Colucci, Mario; Pentimone, Anna; Binetti, Bianca M; Cramarossa, Marialisa; Piro, Donatella; Semeraro, Nicola

    2002-08-01

    Heparin has been proposed to enhance thrombolysis by inhibiting thrombin-dependent generation of activated TAFI (thrombin activatable fibrinolysis inhibitor), a carboxypeptidase that inhibits fibrinolysis. We evaluated the effect of heparin in an in vitro thrombolysis model consisting of a radiolabelled blood clot submerged in defibrinated plasma. Fibrinolysis was induced by adding t-PA (250 ng/ml) and calcium to the plasma bath. Control experiments indicated that thrombin generation induced by recalcification caused significant TAFI activation and inhibited clot lysis. Heparin (up to 1 U/ml), added to the plasma bath, failed to enhance clot lysis. Thrombin generation in the fluid phase was totally inhibited by heparin at concentrations > 0.5 U/ml. In contrast, thrombin generation on the clot surface was not inhibited by heparin (1 U/ml). TAFIa generation did occur in heparin-containing samples (1 U/ml) and amounted to about 10% of TAFIa formed in control samples. This low amount of TAFIa did exert antifibrinolytic activity as indicated by the observation that the addition of a specific TAFIa inhibitor (PTI) along with heparin enhanced clot lysis. Hirudin (10 micrograms/ml), at variance with heparin, inhibited clot-bound thrombin and enhanced clot lysis. These data show that heparin is unable to stimulate fibrinolysis through a TAFI-dependent mechanism, most likely because of its inefficiency in inhibiting thrombin generation on the clot surface. Moreover, they suggest that clot-bound thrombin plays a major role in TAFI-mediated inhibition of fibrinolysis through "localized" TAFIa generation. PMID:12195701

  3. Characterization of iodinated adrenomedullin derivatives suitable for lung nuclear medicine

    Energy Technology Data Exchange (ETDEWEB)

    Fu Yan; Letourneau, Myriam; Chatenet, David [Laboratoire d' etudes moleculaires et pharmacologiques des peptides, INRS-Institut Armand-Frappier, Ville de Laval, Qc, H7V 1B7 (Canada); Dupuis, Jocelyn [Research Center, Montreal Heart Institute, Montreal, Qc (Canada); Department of Medicine, University of Montreal, Montreal, Qc (Canada); Fournier, Alain, E-mail: alain.fournier@iaf.inrs.ca [Laboratoire d' etudes moleculaires et pharmacologiques des peptides, INRS-Institut Armand-Frappier, Ville de Laval, Qc, H7V 1B7 (Canada)

    2011-08-15

    Introduction: We have recently demonstrated the effectiveness of 99m-technetium adrenomedullin (AM) as a new molecular lung imaging agent that could provide significant advantages for the diagnosis and follow-up of disorders affecting the pulmonary circulation such as pulmonary embolism and pulmonary hypertension. Having the possibility to conjugate the targeting molecule with different radionuclides would offer more flexibility and potential advantages depending on clinical situations. Since various iodine isotopes are currently used in nuclear medicine and in pharmacological studies, we have evaluated which iodination method should be privileged in order to produce a good iodinated AM-derived nuclear medicine agent. Methods: Synthetic AM was labeled with iodine through chemical and lactoperoxidase oxidation methods. Position of the iodine atom on the peptide was determined by MALDI-TOF mass spectrometry analysis following cyanogen bromide cleavage and carboxypeptidase Y digestion. Binding affinity of iodinated AM analogues was evaluated by competition and saturation binding experiments on dog lung preparations. Results: In this study, we demonstrated that, upon lactoperoxidase oxidation, iodination occurred at Tyr{sup 1} and that this radioligand retained higher binding affinity and specificity over preparations obtained through chemical oxidation. Conclusions: These results emphasize the fact that even a small chemical modification, i.e. iodination, might deeply modify the pharmacological profile of a compound and support observations that the C-terminal tail of human AM plays an important role in the AM receptor binding process. Consequently, incorporation of a radionuclide to produce an AM-based nuclear medicine agent should privilege the N-terminus of the molecule.

  4. A crucial role in fertility for the oyster angiotensin-converting enzyme orthologue CgACE.

    Science.gov (United States)

    Riviere, Guillaume; Fellous, Alexandre; Franco, Alban; Bernay, Benoit; Favrel, Pascal

    2011-01-01

    Angiotensin-converting enzyme (ACE) is a highly conserved metallopeptidase. In mammals, the somatic isoform governs blood pressure whereas the germinal isoform (tACE) is required for fertility. In Ecdysozoans, ACE-like enzymes are implicated in reproduction. Despite ACE orthologues being present from bacteria to humans, their function(s) remain(s) unknown in distant organisms such as Lophotrochozoans. In silico analysis of an oyster (Crassostrea gigas) EST library suggested the presence of an ACE orthologue in molluscs. Primer walking and 5'-RACE revealed that the 1.9 kb cDNA encodes CgACE, a 632 amino acid protein displaying a conserved single active site and a putative C-terminal transmembrane anchor, thus resembling human tACE, as supported by molecular modelling. FRET activity assays and Maldi-TOF spectrometry indicated that CgACE is a functional dipeptidyl-carboxypeptidase which is active on Angiotensin I and sensitive to ACE inhibitors and chloride ion concentration. Immunocytochemistry revealed that, as its human counterpart, recombinant CgACE is synthesised as a transmembrane enzyme. RT-qPCR, in-situ hybridization and immunohistochemistry shed light on a tissue, and development stage, specific expression pattern for CgACE, which is increased in the gonad during spermatogenesis. The use of ACE inhibitors in vivo indicates that the dipeptidase activity of CgACE is crucial for the oyster fertilization. Our study demonstrates that a transmembrane active ACE is present in the oyster Crassostrea gigas, and for the first time ascribes a functional role for ACE in Lophotrochozoans. Its biological function in reproduction is conserved from molluscs to humans, a finding of particular evolutionary interest especially since oysters represent the most important aquaculture resource worldwide. PMID:22174750

  5. Plasma protein(s) yields met-enkephalin-related peptides in near-micromolar concentrations when treated with pepsin.

    Science.gov (United States)

    Singer, E A; Mitra, S P; Carraway, R E

    1986-10-01

    Treatment of animal and human plasmas with pepsin yielded large quantities of immunoreactive methionine5-enkephalin (i-met-ENK). The concentrations measured after pepsin treatment were 0.1-0.5 microM, about 1000 times the normal circulating level of i-met-ENK (0.03-0.3 nM). The reaction was shown to be time and pH dependent and to involve the action of pepsin on a protein(s) of about 65,000 mol wt. Pepsin-generated i-met-ENK from rat plasma gave three major peaks during reverse phase HPLC, one of which (approximately 25% of the total) coeluted with methionine5-enkephalin sulfoxide and also completed in a radioreceptor assay for opiate-related substances. In addition, this material produced met-ENK-like effects on vascular permeability in rat skin and inhibited electrically induced contractions of the isolated guinea pig ileum in a naloxone-sensitive manner. The plasma substrate(s) that yielded i-met-ENK was distinguished from adrenal proenkephalins, since partially purified plasma substrate(s) did not liberate i-met-ENK upon digestion with trypsin and carboxypeptidase B. Although it is possible that these peptides differ from met-ENK in amino acid sequence, the results presented here suggest that met-ENK-related substances might be formed physiologically by the action of a pepsin-related processing enzyme(s) on plasma substrate(s). Such a mechanism would be analogous to that used in the renin-angiotensin system. PMID:3093194

  6. Human bradykinin B(2) receptor is activated by kallikrein and other serine proteases.

    Science.gov (United States)

    Hecquet, C; Tan, F; Marcic, B M; Erdös, E G

    2000-10-01

    Bradykinin (BK) and kallidin (Lys-BK), liberated from kininogens by kallikreins, are ligands of the BK B(2) receptor. We investigated whether kallikreins, besides releasing peptide agonist, could also activate the receptor directly. We studied the effect of porcine and human recombinant tissue kallikrein and plasma kallikrein on [Ca(2+)](i) mobilization and [(3)H]arachidonic acid release from cultured cells stably transfected to express human BK B(2) receptor (CHO/B(2), MDCK/B(2), HEK/B(2)), and endothelial cells were used as control cells. As with BK, the actions of kallikrein were blocked by the B(2) antagonist, HOE 140. Kallikrein was inactive on cells lacking B(2) receptor. Kallikrein and BK desensitized the receptor homologously but there was no cross-desensitization. Furthermore, 50 nM human cathepsin G and 50 nM trypsin also activated the receptor; this also was blocked by HOE 140. Experiments excluded a putative kinin release by proteases. [(3)H]AA release by BK was reduced by 40% by added kininase I (carboxypeptidase M); however, receptor activation by tissue kallikrein, trypsin, or cathepsin G was not affected. Prokallikrein and inhibited kallikrein were inactive, suggesting cleavage of a peptide bond in the receptor. Kallikreins were active on mutated B(2) receptor missing the 19 N-terminal amino acids, suggesting a type of activation different from that of thrombin receptor. Paradoxically, tissue kallikreins decreased the [(3)H]BK binding to the receptor with a low K(D) (3 nM) and inhibited it 78%. Thus, kallikreins and some other proteases activate human BK B(2) receptor directly, independent of BK release. The BK B(2) receptor may belong to a new group of serine protease-activated receptors. PMID:10999954

  7. A genome-wide immunodetection screen in S. cerevisiae uncovers novel genes involved in lysosomal vacuole function and morphology.

    Directory of Open Access Journals (Sweden)

    Florante Ricarte

    Full Text Available Vacuoles of yeast Saccharomyces cerevisiae are functionally analogous to mammalian lysosomes. Both are cellular organelles responsible for macromolecular degradation, ion/pH homeostasis, and stress survival. We hypothesized that undefined gene functions remain at post-endosomal stage of vacuolar events and performed a genome-wide screen directed at such functions at the late endosome and vacuole interface - ENV genes. The immunodetection screen was designed to identify mutants that internally accumulate precursor form of the vacuolar hydrolase carboxypeptidase Y (CPY. Here, we report the uncovering and initial characterizations of twelve ENV genes. The small size of the collection and the lack of genes previously identified with vacuolar events are suggestive of the intended exclusive functional interface of the screen. Most notably, the collection includes four novel genes ENV7, ENV9, ENV10, and ENV11, and three genes previously linked to mitochondrial processes - MAM3, PCP1, PPE1. In all env mutants, vesicular trafficking stages were undisturbed in live cells as assessed by invertase and active α-factor secretion, as well as by localization of the endocytic fluorescent marker FM4-64 to the vacuole. Several mutants exhibit defects in stress survival functions associated with vacuoles. Confocal fluorescence microscopy revealed the collection to be significantly enriched in vacuolar morphologies suggestive of fusion and fission defects. These include the unique phenotype of lumenal vesicles within vacuoles in the novel env9Δ mutant and severely fragmented vacuoles upon deletion of GET4, a gene recently implicated in tail anchored membrane protein insertion. Thus, our results establish new gene functions in vacuolar function and morphology, and suggest a link between vacuolar and mitochondrial events.

  8. Structure of Csd3 from Helicobacter pylori, a cell shape-determining metallopeptidase

    Energy Technology Data Exchange (ETDEWEB)

    An, Doo Ri [Seoul National University, Seoul 151-742 (Korea, Republic of); Kim, Hyoun Sook [Seoul National University, Seoul 151-742 (Korea, Republic of); Seoul National University, Seoul 151 742 (Korea, Republic of); Kim, Jieun; Im, Ha Na; Yoon, Hye Jin; Yoon, Ji Young; Jang, Jun Young [Seoul National University, Seoul 151-742 (Korea, Republic of); Hesek, Dusan; Lee, Mijoon; Mobashery, Shahriar [University of Notre Dame, Notre Dame, IN 46556 (United States); Kim, Soon-Jong [Mokpo National University, Chonnam 534-729 (Korea, Republic of); Lee, Byung Il [National Cancer Center, Gyeonggi 410-769 (Korea, Republic of); Suh, Se Won, E-mail: sewonsuh@snu.ac.kr [Seoul National University, Seoul 151-742 (Korea, Republic of); Seoul National University, Seoul 151-742 (Korea, Republic of)

    2015-03-01

    H. pylori Csd3 (HP0506), together with other peptidoglycan hydrolases, plays an important role in determining cell shape. Its crystal structure in the latent state is reported. Helicobacter pylori is associated with various gastrointestinal diseases such as gastritis, ulcers and gastric cancer. Its colonization of the human gastric mucosa requires high motility, which depends on its helical cell shape. Seven cell shape-determining genes (csd1, csd2, csd3/hdpA, ccmA, csd4, csd5 and csd6) have been identified in H. pylori. Their proteins play key roles in determining the cell shape through modifications of the cell-wall peptidoglycan by the alteration of cross-linking or by the trimming of peptidoglycan muropeptides. Among them, Csd3 (also known as HdpA) is a bifunctional enzyme. Its d, d-endopeptidase activity cleaves the d-Ala{sup 4}-mDAP{sup 3} peptide bond between cross-linked muramyl tetrapeptides and pentapeptides. It is also a d, d-carboxypeptidase that cleaves off the terminal d-Ala{sup 5} from the muramyl pentapeptide. Here, the crystal structure of this protein has been determined, revealing the organization of its three domains in a latent and inactive state. The N-terminal domain 1 and the core of domain 2 share the same fold despite a very low level of sequence identity, and their surface-charge distributions are different. The C-terminal LytM domain contains the catalytic site with a Zn{sup 2+} ion, like the similar domains of other M23 metallopeptidases. Domain 1 occludes the active site of the LytM domain. The core of domain 2 is held against the LytM domain by the C-terminal tail region that protrudes from the LytM domain.

  9. Biochemical characterization of antigens of the blood group rhesus system

    International Nuclear Information System (INIS)

    Human red cells of various rhesus (rh) phenotypes were surface-labelled with 125I and rh-specific labelled polypeptides were isolated by preparative SDS-PAGE. The purified rh proteins when analysed by SDS-PAGE comigrated with immunoprecipitated rh proteins and showed the characteristic properties due to their hydrophobicity. Two-dimensional iso-electric focusing/SDS-PAGE of rh proteins resulted in the pI=6,8-7,0 for all investigated rh types and did not show any separation of presumably different rh-specific polypeptide chains. The purified 125I-labelled rh proteins were subjected to limited proteolysis and the resulting fragments were analysed by SDS-PAGE and autoradiography. Chymotryptic peptide maps of proteins obtained from rh(D)-positive and -negative types appeared to be identical, indicating a high degree of structural homology between these proteins. In contrast, the tryptic peptide maps revealed a characteristic difference: a fragment of Mr 17500 was associated with the rh(D) antigen and one of Mr 19000 with the rh(C/c, E/e) antigens. This result supports other evidence that the rh(D) protein is not identical with the rh(C/c, E/e) protein(s). Treatment of rh polypeptides with carboxypeptidase Y prior to tryptic digestion resulted in a shift of nearly all tryptic fragment, including a small fragment of Mr 8000, indicating that the surface label was incorporated into the C-terminal part of the molecules. Moreover, by chemical cleavage a cysteine-residue was identified within the C-terminal region. This region which contains both the surface label and the cysteine-residue is well suited for the location of the rh-specific epitopes. 40 refs., 35 figs., 8 tabs. (Author)

  10. Susceptibility and Immune Defence Mechanisms of Rhynchophorus ferrugineus (Olivier (Coleoptera: Curculionidae against Entomopathogenic Fungal Infections

    Directory of Open Access Journals (Sweden)

    Abid Hussain

    2016-09-01

    Full Text Available Insects infected with entomopathogenic fungi, experience physiological changes that influence their growth and immune defence. The potential of nine isolates of entomopathogenic fungi was evaluated after determining percent germination and relative conidial hydrophobicity. However, nutritional indices were evaluated after immersing eighth-instar Rhynchophorus ferrugineus larvae into each isolate suspension (1 × 107 conidia/mL. The results showed that isolates B6884 and M9374 had 44.51% and 39.02% higher conidial hydrophobicity compared with isolate I03011 (least virulent. The results of nutritional index assays revealed a significant reduction in growth indices after infection with different isolates. Compared with control, B6884 and M9374 greatly decreased larval growth by reducing the efficacy of conversion of ingested food (36%–47% and Efficacy of conversion of digested food (50%–63%. Furthermore, only isolate B6884 induced 100% mortality within 12 days. Compared with control, isolate I03011, possessing the lowest conidial hydrophobicity, only reduced 0.29% of the efficacy of conversion of ingested food (ECI and 0.48% of the efficacy of conversion of digested food (ECD. Similarly, transcriptomic analysis of genes related to the Red palm weevil (RPW immune response, including pathogen recognition receptors (C-type lectin and endo-beta-1,4-glucanse, signal modulator (Serine protease-like protein, signal transductors (Calmodulin-like protein and EF-hand domain containing protein and effectors (C-type lysozyme, Cathepsin L., Defensin-like protein, Serine carboxypeptidase, and Thaumatin-like protein, was significantly increased in larval samples infected with B6884 and M9374. These results suggest that for an isolate to be virulent, conidial hydrophobicity and germination should also be considered during pathogen selection, as these factors could significantly impact host growth and immune defence mechanisms.

  11. A crucial role in fertility for the oyster angiotensin-converting enzyme orthologue CgACE.

    Directory of Open Access Journals (Sweden)

    Guillaume Riviere

    Full Text Available Angiotensin-converting enzyme (ACE is a highly conserved metallopeptidase. In mammals, the somatic isoform governs blood pressure whereas the germinal isoform (tACE is required for fertility. In Ecdysozoans, ACE-like enzymes are implicated in reproduction. Despite ACE orthologues being present from bacteria to humans, their function(s remain(s unknown in distant organisms such as Lophotrochozoans. In silico analysis of an oyster (Crassostrea gigas EST library suggested the presence of an ACE orthologue in molluscs. Primer walking and 5'-RACE revealed that the 1.9 kb cDNA encodes CgACE, a 632 amino acid protein displaying a conserved single active site and a putative C-terminal transmembrane anchor, thus resembling human tACE, as supported by molecular modelling. FRET activity assays and Maldi-TOF spectrometry indicated that CgACE is a functional dipeptidyl-carboxypeptidase which is active on Angiotensin I and sensitive to ACE inhibitors and chloride ion concentration. Immunocytochemistry revealed that, as its human counterpart, recombinant CgACE is synthesised as a transmembrane enzyme. RT-qPCR, in-situ hybridization and immunohistochemistry shed light on a tissue, and development stage, specific expression pattern for CgACE, which is increased in the gonad during spermatogenesis. The use of ACE inhibitors in vivo indicates that the dipeptidase activity of CgACE is crucial for the oyster fertilization. Our study demonstrates that a transmembrane active ACE is present in the oyster Crassostrea gigas, and for the first time ascribes a functional role for ACE in Lophotrochozoans. Its biological function in reproduction is conserved from molluscs to humans, a finding of particular evolutionary interest especially since oysters represent the most important aquaculture resource worldwide.

  12. Chemical and organic fertilizers affect physiological performance and antioxidant

    Directory of Open Access Journals (Sweden)

    M Mardani-Talaee1

    2016-04-01

    Full Text Available Myzus persicae is a widespread and polyphagous insect that causes severe damages to hundreds of host plants. In the current study, zinc sulfate and vermicompost as chemical and organic fertilizers, were added into cultural soil of Capsicum annuum to determine their effects on physiology and antioxidant activities of M. persicae. The aphids reared on zinc sulfate-treated culture showed the highest activities of general protease, trypsin, cathepsins, carboxypeptidase and lipase but activities of chymotrypsin and aminopeptidase were the highest in vermicompost-treated culture. Although activities of α-amylase in the fertilizer-treated cultures were higher than control but activities of α- and β-glucosideases showed the highest values in zinc sulfate and vermicompost treatments, respectively. Aspartate aminotransferase and γ-glutamyl transferase showed the highest activity in the aphids reared on the vermicompost-treated culture but alanine aminotransferase activity got the lowest value in fertilizer-treated cultures. Activities of aldolase and lactate dehydrogenase in the fertilizer-treated aphids were higher than those of control and vermicompost-treated aphids, but alkaline phosphatase showed the lower activity although activity of acid phosphatase decreased in vermicompost- treated aphids compared to other treatments. Activities of antioxidant enzymes were found to be the highest in the aphids fed on vermicompost-treated culture including glucose-6-phosphate dehydrogenase, superoxide dismutase, peroxidase and ascorbate oxidase but catalase in vermicompost treatment had lower activity than control and zinc-sulfate treatments. Also, malondialdehyde and RSSR/RSH ratio demonstrated higher values in the aphids fed on zinc sulfate- and vermicompost-treated plants than control, respectively. Finally, the amounts of glycogen and triglyceride revealed the highest values in zinc sulfate-treated plants compared to other treatments. These results

  13. Radiolabelling of glycosylated MFE-23::CPG2 fusion protein (MFECP1) with 99mTc for quantitation of tumour antibody-enzyme localisation in antibody-directed enzyme pro-drug therapy (ADEPT)

    International Nuclear Information System (INIS)

    MFECP1 is a glycosylated recombinant fusion protein composed of MFE-23, a high-affinity anti-carcinoembryonic antigen (CEA) single chain Fv (scFv), fused to the enzyme carboxypeptidase G2 (CPG2), and has been constructed for use in antibody-directed enzyme pro-drug therapy (ADEPT). Radiolabelling of glycosylated MFECP1 with technetium-99m was developed for the purpose of determining tumour localisation of MFECP1 in a phase I ADEPT clinical study. The method used was 99mTc-carbonyl [99mTc(H2O)3(CO)3]+ (abbreviated to TcCO) mediated labelling of 99mTc to the hexahistidine (His) tag of MFECP1. MFECP1 fusion protein was labelled with TcCO under a variety of conditions, and this was shown to be a relatively simple and robust method. Tissue biodistribution was assessed in a CEA-expressing LS174T (human colon carcinoma) nude mouse xenograft model. Tissues were taken at 1, 4 and 6 h for assessment of distribution of radioactivity and for measurement of CPG2 enzyme levels. The amount of radioactivity retained by the tumour proved to be an accurate estimation of actual measured enzyme activity, indicating that this radiolabelling method does not appear to damage the antibody-antigen binding or the enzyme activity of MFECP1. However, correlation between CPG2 enzyme activity and measured radioactivity in liver, spleen and kidney was poor, indicating retention of radioactivity in non-tumour sites but loss of enzyme activity. The high retention of technetium radioisotope in normal tissues may limit the clinical applicability of this radiolabelling method for MFECP1; however, these results suggest that this technique does have applicability for measuring the biodistribution of His-tagged recombinant proteins. (orig.)

  14. Effect of chitosan and thiolated chitosan coating on the inhibition behaviour of PIBCA nanoparticles against intestinal metallopeptidases

    International Nuclear Information System (INIS)

    Surface modified nanoparticles composed of poly(isobutylcyanoacrylate) (PIBCA) cores surrounded by a chitosan and thiolated chitosan gel layer were prepared and characterized in previous works. The presence of such biopolymers on the nanoparticle surface conferred those nanosystems interesting characteristics that might partially overcome the gastrointestinal enzymatic barrier, improving the oral administration of pharmacologically active peptides. In the present work, the antiprotease behaviour of this family of core-shell nanoparticles was in vitro tested against two model metallopeptidases present in the gastrointestinal tract (GIT): Carboxypeptidase A -CP A- (luminal protease) and Leucine Aminopeptidase M -LAP M- (membrane protease). As previous step, the zinc-binding capacity of these nanoparticles was evaluated. Interestingly, an improvement of both the zinc-binding capacity and the antiprotease effect of chitosan was observed when the biopolymers (chitosan and thiolated chitosan) were used as coating component of the core-shell nanoparticles, in comparison with their behaviour in solution, thanks to the different biopolymer chains rearrangement. The presence of amino, hydroxyl and thiol groups on the nanoparticle surface promoted zinc binding and hence the inhibition of the metallopeptidases analysed. On the contrary, the occurrence of a cross-linked structure in the gel layer surrounding the PIBCA cores of thiolated formulations, due to the formation of interchain and intrachain disulphide bonds, partially limited the inhibition of the proteases. The low accessibility of cations to the active groups of the cross-linked polymeric shell was postulated as a possible explanation of this behaviour. Results obtained in this work make this family of surface-modified nanocarriers promising candidates for the successfull administration of pharmacologically active peptides and proteins by the oral route.

  15. Solvent exchange of buried water and hydrogen exchange of peptide NH groups hydrogen bonded to buried waters in bovine pancreatic trypsin inhibitor

    International Nuclear Information System (INIS)

    Solvent exchange of 18O-labeled buried water in bovine pancreatic trypsin inhibitor (BPTI), trypsin, and trypsin-BPTI complex is measured by high-precision isotope ratio mass spectroscopy. Buried water is labeled by equilibrium of the protein in 18O-enriched water. Protein samples are then rapidly dialyzed against water of normal isotope composition by gel filtration and stored. The exchangeable 18O label eluting with the protein in 10-300 s is determined by an H2O-CO2 equilibration technique. Exchange of buried waters with solvent water is complete before 10-15 s in BPTI, trypsin, and BPTI-trypsin, as well as in lysozyme and carboxypeptidase measured as controls. When in-exchange dialysis and storage are carried out at pH ≥ 2.5, trypsin-BPTI and trypsin, but not free BPTI, have the equivalent of one 18O atom that exchanges slowly (after 300 s and before several days). This oxygen is probably covalently bound to a specific site in trypsin. When in-exchange dialysis and storage are carried out at pH 1.1, the equivalent of three to seven 18O atoms per molecule is associated with the trypsin-BPTI complex, apparently due to nonspecific covalent 18O labeling of carboxyl groups at low pH. In addition to 18O exchange of buried waters, the hydrogen isotope exchange of buried NH groups H bonded to buried waters was also measured. Their base-catalyzed exchange rate constants are on the order of NH groups that in the crystal are exposed to solvent and hydrogen-bonded main chain O, and their pH/sub min/ is similar to that for model compounds. The pH dependence of their exchange rate constants suggests that direct exchange with water may significantly contribute to their observed exchange rate

  16. Use of anaerobic green fluorescent protein versus green fluorescent protein as reporter in lactic acid bacteria.

    Science.gov (United States)

    Landete, José M; Langa, Susana; Revilla, Concepción; Margolles, Abelardo; Medina, Margarita; Arqués, Juan L

    2015-08-01

    Lactic acid bacteria (LAB) are commonly used in the production of fermented and probiotic foods. Development of molecular tools to discriminate the strains of interest from the endogenous microbiota in complex environments like food or gut is of high interest. Green fluorescent protein (GFP)-like chromophores strictly requires molecular oxygen for maturation of fluorescence, which restrict the study of microorganisms in low-oxygen environments. In this work, we have developed a noninvasive cyan-green fluorescent based reporter system for real-time tracking of LAB that is functional under anoxic conditions. The evoglow-Pp1 was cloned downstream from the promoters D-alanyl-D-alanine carboxypeptidase and elongation factor Tu of Lactobacillus reuteri CECT925 using pNZ8048 and downstream of the lactococcal P1 promoter using pT1NX. The classical gfp was also cloned in pT1NX. These recombinant expression vectors were electroporated into Lactococccus, Lactobacillus, and Enterococcus strains with biotechnological and/or probiotic interests to assess and compare their functionality under different conditions of oxygen and pH. The expression was analyzed by imaging and fluorometric methods as well as by flow cytometry. We demonstrate that reporter systems pNZ:TuR-aFP and pT1-aFP are two versatile molecular markers for monitoring LAB in food and fecal environments without the potential problems caused by oxygen and pH limitations, which could be exploited for in vivo studies. Production of the fluorescent protein did not disturb any important physiological properties of the parental strains, such as growth rate, reuterin, or bacteriocin production. PMID:26129953

  17. SpeB proteolysis with imaged capillary isoelectric focusing for the characterization of domain-specific charge heterogeneities of reference and biosimilar Rituximab.

    Science.gov (United States)

    Zhang, Zichuan; Perrault, Ronel; Zhao, Yun; Ding, Julia

    2016-05-01

    The charge variations of therapeutic monoclonal antibody reveal important information of the post-translational modifications that may potentially impact the potency and safety of pharmaceutical products, especially during the evaluation of biosimilarity of therapeutic proteins. In this work, a novel SpeB-based proteolysis strategy coupling with imaged capillary isoelectric focusing was developed for the determination of domain-specific charge heterogeneities of innovator and generic Rituximab drug products from United States, European and Indian markets. It was observed that innovator Rituximab from the United States and Europe share highly similar peak distributions and charge heterogeneities with 26.2-26.6% Fc/2, 28.9-29.3% LC and 44.4-44.5% Fd peak areas detected, respectively, while multiple basic variations of Fc/2 and less acidic LC and Fd species were found from generic Rituximab from India with 20.9% Fc/2, 32.3% LC and 46.9% Fd peak areas detected. It was also demonstrated that structural changes caused by Carboxypeptidase B treatment and deamidation study at pH extremes could be sensitively captured with the established method, with the results further indicating that the generic product's basic variations of Fc/2 were un-cleaved Lysine residues, while the lack of certain acidic peaks on LC and Fd probably was due to the lower level of deamidation. This new strategy could become a useful tool to reveal domain-specific charge heterogeneities profiles of a variety of therapeutic monoclonal antibodies in regulated environments. PMID:27038651

  18. Molecular activities, biosynthesis and evolution of triterpenoid saponins.

    Science.gov (United States)

    Augustin, Jörg M; Kuzina, Vera; Andersen, Sven B; Bak, Søren

    2011-04-01

    Saponins are bioactive compounds generally considered to be produced by plants to counteract pathogens and herbivores. Besides their role in plant defense, saponins are of growing interest for drug research as they are active constituents of several folk medicines and provide valuable pharmacological properties. Accordingly, much effort has been put into unraveling the modes of action of saponins, as well as in exploration of their potential for industrial processes and pharmacology. However, the exploitation of saponins for bioengineering crop plants with improved resistances against pests as well as circumvention of laborious and uneconomical extraction procedures for industrial production from plants is hampered by the lack of knowledge and availability of genes in saponin biosynthesis. Although the ability to produce saponins is rather widespread among plants, a complete synthetic pathway has not been elucidated in any single species. Current conceptions consider saponins to be derived from intermediates of the phytosterol pathway, and predominantly enzymes belonging to the multigene families of oxidosqualene cyclases (OSCs), cytochromes P450 (P450s) and family 1 UDP-glycosyltransferases (UGTs) are thought to be involved in their biosynthesis. Formation of unique structural features involves additional biosynthetical enzymes of diverse phylogenetic background. As an example of this, a serine carboxypeptidase-like acyltransferase (SCPL) was recently found to be involved in synthesis of triterpenoid saponins in oats. However, the total number of identified genes in saponin biosynthesis remains low as the complexity and diversity of these multigene families impede gene discovery based on sequence analysis and phylogeny. This review summarizes current knowledge of triterpenoid saponin biosynthesis in plants, molecular activities, evolutionary aspects and perspectives for further gene discovery. PMID:21333312

  19. Endogenously generated plasmin at the vascular wall injury site amplifies lysine binding site-dependent plasminogen accumulation in microthrombi.

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    Tomasz Brzoska

    Full Text Available The fibrinolytic system plays a pivotal role in the regulation of hemostasis; however, it remains unclear how and when the system is triggered to induce thrombolysis. Using intra-vital confocal fluorescence microscopy, we investigated the process of plasminogen binding to laser-induced platelet-rich microthrombi generated in the mesenteric vein of transgenic mice expressing green fluorescent protein (GFP. The accumulation of GFP-expressing platelets as well as exogenously infused Alexa Fluor 568-labeled Glu-plasminogen (Glu-plg on the injured vessel wall was assessed by measuring the increase in the corresponding fluorescence intensities. Glu-plg accumulated in a time-dependent manner in the center of the microthrombus, where phosphatidylserine is exposed on platelet surfaces and fibrin formation takes place. The rates of binding of Glu-plg in the presence of ε-aminocaproic acid and carboxypeptidase B, as well as the rates of binding of mini-plasminogen lacking kringle domains 1-4 and lysine binding sites, were significantly lower than that of Glu-plg alone, suggesting that the binding was dependent on lysine binding sites. Furthermore, aprotinin significantly suppressed the accumulation of Glu-plg, suggesting that endogenously generated plasmin activity is a prerequisite for the accumulation. In spite of the endogenous generation of plasmin and accumulation of Glu-plg in the center of microthrombi, the microthrombi did not change in size during the 2-hour observation period. When human tissue plasminogen activator was administered intravenously, Glu-plg further accumulated and the microthrombi were lysed. Glu-plg appeared to accumulate in the center of microthrombi in the early phase of microthrombus formation, and plasmin activity and lysine binding sites were required for this accumulation.

  20. Still NAAG'ing After All These Years: The Continuing Pursuit of GCPII Inhibitors.

    Science.gov (United States)

    Vornov, J J; Hollinger, K R; Jackson, P F; Wozniak, K M; Farah, M H; Majer, P; Rais, R; Slusher, B S

    2016-01-01

    Nearly two decades ago, Joe Coyle published a single-authored review with the provocative title, The Nagging Question of the Function of N-Acetylaspartylglutamate (Coyle, 1997). In this review, Coyle documented NAAG's localization to subpopulations of glutamatergic, cholinergic, GABAergic, and noradrenergic neurons, Ca(2+)-dependent release, mGlu3 receptor agonist and NMDA receptor antagonist activity, and cleavage by the glial enzyme glutamate carboxypeptidase II (GCPII). However, at the time of his review, NAAG's physiological function as a neurotransmitter remained elusive. Ironically his review was published months following the discovery of the first potent and selective GCPII inhibitor, 2-(phosphonomethyl)pentanedioc acid (2-PMPA) (Jackson et al., 1996). Over the ensuing decades, over a dozen independent laboratories used 2-PMPA and other GCPII inhibitors to elucidate two distinct neurotransmitter functions for NAAG. Under basal conditions, when GCPII activity is relatively low, intact NAAG dampens synaptic activity via presynaptic mGlu3 receptor activation and NMDA receptor blockade. However, under stimulated conditions, NAAG release and GCPII activity are enhanced resulting in excess glutamate generation, activating NMDA and other glutamate receptors, often pathologically. Diverse classes of GCPII inhibitors have been synthesized and shown to increase NAAG, decrease glutamate, and provide robust efficacy in many disease models wherein abnormal glutamatergic transmission is presumed pathogenic. In addition, over the past 20 years, basic questions regarding NAAG's synthesis, packaging into vesicles, and receptor selectivity profile have been eloquently elucidated. The purpose of this chapter is to summarize these advances and the promise of regulating NAAG metabolism through GCPII inhibition as a therapeutic strategy. PMID:27288079

  1. Mechanistic Inferences from the Binding of Ligands to LpxC, A Metal-Dependent Deacetylase

    International Nuclear Information System (INIS)

    The metal-dependent deacetylase LpxC catalyzes the first committed step of lipid A biosynthesis in Gram-negative bacteria. Accordingly, LpxC is an attractive target for the development of inhibitors that may serve as potential new antibiotics for the treatment of Gram-negative bacterial infections. Here, we report the 2.7 Angstroms resolution X-ray crystal structure of LpxC complexed with the substrate analogue inhibitor TU-514 and the 2.0 Angstroms resolution structure of LpxC complexed with imidazole. The X-ray crystal structure of LpxC complexed with TU-514 allows for a detailed examination of the coordination geometry of the catalytic zinc ion and other enzyme-inhibitor interactions in the active site. The hydroxamate group of TU-514 forms a bidentate chelate complex with the zinc ion and makes hydrogen bond interactions with conserved active site residues E78, H265, and T191. The inhibitor C-4 hydroxyl group makes direct hydrogen bond interactions with E197 and H58. Finally, the C-3 myristate moiety of the inhibitor binds in the hydrophobic tunnel of the active site. These intermolecular interactions provide a foundation for understanding structural aspects of enzyme-substrate and enzyme-inhibitor affinity. Comparison of the TU-514 complex with cacodylate and imidazole complexes suggests a possible substrate diphosphate binding site and highlights residues that may stabilize the tetrahedral intermediate and its flanking transition states in catalysis. Evidence of a catalytic zinc ion in the native zinc enzyme coordinated by H79, H238, D242, and two water molecules with square pyramidal geometry is also presented. These results suggest that the native state of this metallohydrolase may contain a pentacoordinate zinc ion, which contrasts with the native states of archetypical zinc hydrolases such as thermolysin and carboxypeptidase A

  2. Identification of latexin by a proteomic analysis in rat normal articular cartilage

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    Kouri Juan B

    2010-06-01

    Full Text Available Abstract Background Osteoarthritis (OA is characterized by degeneration of articular cartilage. Animal models of OA induced are a widely used tool in the study of the pathogenesis of disease. Several proteomic techniques for selective extraction of proteins have provided protein profiles of chondrocytes and secretory patterns in normal and osteoarthritic cartilage, including the discovery of new and promising biomarkers. In this proteomic analysis to study several proteins from rat normal articular cartilage, two-dimensional electrophoresis and mass spectrometry (MS were used. Interestingly, latexin (LXN was found. Using an immunohistochemical technique, it was possible to determine its localization within the chondrocytes from normal and osteoarthritic articular cartilage. Results In this study, 147 proteins were visualized, and 47 proteins were identified by MS. A significant proportion of proteins are involved in metabolic processes and energy (32%, as well as participating in different biological functions including structural organization (19%, signal transduction and molecular signaling (11%, redox homeostasis (9%, transcription and protein synthesis (6%, and transport (6%. The identified proteins were assigned to one or more subcellular compartments. Among the identified proteins, we found some proteins already recognized in other studies such as OA-associated proteins. Interestingly, we identified LXN, an inhibitor of mammalian carboxypeptidases, which had not been described in articular cartilage. Immunolabeling assays for LXN showed a granular distribution pattern in the cytoplasm of most chondrocytes of the middle, deep and calcified zones of normal articular cartilage as well as in subchondral bone. In osteoarthritic cartilage, LXN was observed in superficial and deep zones. Conclusions This study provides the first proteomic analysis of normal articular cartilage of rat. We identified LXN, whose location was demonstrated by

  3. Clinical utility of polymorphisms in one-carbon metabolism for breast cancer risk prediction

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    Shaik Mohammad Naushad

    2011-01-01

    Full Text Available This study addresses the issues in translating the laboratory derived data obtained during discovery phase of research to a clinical setting using a breast cancer model. Laboratory-based risk assessment indi-cated that a family history of breast cancer, reduced folate carrier 1 (RFC1 G80A, thymidylate synthase (TYMS 5’-UTR 28bp tandem repeat, methylene tetrahydrofolate reductase (MTHFR C677T and catecholamine-O-methyl transferase (COMT genetic polymorphisms in one-carbon metabolic pathway increase the risk for breast cancer. Glutamate carboxypeptidase II (GCPII C1561T and cytosolic serine hydroxymethyl transferase (cSHMT C1420T polymorphisms were found to decrease breast cancer risk. In order to test the clinical validity of this information in the risk prediction of breast cancer, data was stratified based on number of protective alleles into four categories and in each category sensitivity and 1-specificity values were obtained based on the distribution of number of risk alleles in cases and controls. Receiver operating characteristic (ROC curves were plotted and the area under ROC curve (C was used as a measure of discriminatory ability between cases and controls. In subjects without any protective allele, aberrations in one-carbon metabolism showed perfect prediction (C=0.93 while the predictability was lost in subjects with one protective allele (C=0.60. However, predictability increased steadily with increasing number of protective alleles (C=0.63 for 2 protective alleles and C=0.71 for 3 protective alleles. The cut-off point for discrimination was >4 alleles in all predictable combinations. Models of this kind can serve as valuable tools in translational re-search, especially in identifying high-risk individuals and reducing the disease risk either by life style modification or by medical intervention.

  4. Genome-wide survey of prokaryotic serine proteases: Analysis of distribution and domain architectures of five serine protease families in prokaryotes

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    Tripathi Lokesh P

    2008-11-01

    Full Text Available Abstract Background Serine proteases are one of the most abundant groups of proteolytic enzymes found in all the kingdoms of life. While studies have established significant roles for many prokaryotic serine proteases in several physiological processes, such as those associated with metabolism, cell signalling, defense response and development, functional associations for a large number of prokaryotic serine proteases are relatively unknown. Current analysis is aimed at understanding the distribution and probable biological functions of the select serine proteases encoded in representative prokaryotic organisms. Results A total of 966 putative serine proteases, belonging to five families, were identified in the 91 prokaryotic genomes using various sensitive sequence search techniques. Phylogenetic analysis reveals several species-specific clusters of serine proteases suggesting their possible involvement in organism-specific functions. Atypical phylogenetic associations suggest an important role for lateral gene transfer events in facilitating the widespread distribution of the serine proteases in the prokaryotes. Domain organisations of the gene products were analysed, employing sensitive sequence search methods, to infer their probable biological functions. Trypsin, subtilisin and Lon protease families account for a significant proportion of the multi-domain representatives, while the D-Ala-D-Ala carboxypeptidase and the Clp protease families are mostly single-domain polypeptides in prokaryotes. Regulatory domains for protein interaction, signalling, pathogenesis, cell adhesion etc. were found tethered to the serine protease domains. Some domain combinations (such as S1-PDZ; LON-AAA-S16 etc. were found to be widespread in the prokaryotic lineages suggesting a critical role in prokaryotes. Conclusion Domain architectures of many serine proteases and their homologues identified in prokaryotes are very different from those observed in eukaryotes

  5. Insertions and the emergence of novel protein structure: a structure-based phylogenetic study of insertions

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    Blouin Christian

    2007-11-01

    Full Text Available Abstract Background In protein evolution, the mechanism of the emergence of novel protein domain is still an open question. The incremental growth of protein variable regions, which was produced by stochastic insertions, has the potential to generate large and complex sub-structures. In this study, a deterministic methodology is proposed to reconstruct phylogenies from protein structures, and to infer insertion events in protein evolution. The analysis was performed on a broad range of SCOP domain families. Results Phylogenies were reconstructed from protein 3D structural data. The phylogenetic trees were used to infer ancestral structures with a consensus method. From these ancestral reconstructions, 42.7% of the observed insertions are nested insertions, which locate in previous insert regions. The average size of inserts tends to increase with the insert rank or total number of insertions in the variable regions. We found that the structures of some nested inserts show complex or even domain-like fold patterns with helices, strands and loops. Furthermore, a basal level of structural innovation was found in inserts which displayed a significant structural similarity exclusively to themselves. The β-Lactamase/D-ala carboxypeptidase domain family is provided as an example to illustrate the inference of insertion events, and how the incremental growth of a variable region is capable to generate novel structural patterns. Conclusion Using 3D data, we proposed a method to reconstruct phylogenies. We applied the method to reconstruct the sequences of insertion events leading to the emergence of potentially novel structural elements within existing protein domains. The results suggest that structural innovation is possible via the stochastic process of insertions and rapid evolution within variable regions where inserts tend to be nested. We also demonstrate that the structure-based phylogeny enables the study of new questions relating to the

  6. A Double-Blind, Randomised, Controlled Trial to Study the Effects of an Enteral Feed Supplemented with Glutamine, Arginine, and Omega-3 Fatty Acid in Predicted Acute Severe Pancreatitis

    Directory of Open Access Journals (Sweden)

    Callum B Pearce

    2006-07-01

    Full Text Available Context :Current best evidence is in favour of early institution of enteral feeding in acute severe pancreatitis with promising results from trials in immunonutrition on other patient groups. Objective: To identify which groups of patients and products are associated with benefit, we investigated immunonutrition in patients with predicted acute severe pancreatitis. Design :A randomised trial of a study feed containing glutamine, arginine, tributyrin and antioxidants versus an isocaloric isonitrogenous control feed was undertaken. Patients: Thirty-one patients with a diagnosis of acute pancreatitis predicted to develop severe disease: 15 study feeds and 16 control feeds. Interventions: Enteral feeding via nasojejunal tube for 3 days. If patients required further feeding the study was continued up to 15 days. Main outcome measures :Reduction in Creactive protein (CRP by 40 mg/L after 3 days of enteral feeding was the primary endpoint. Carboxypeptidase B activation peptide (CAPAP levels were taken at regular intervals. Results :After 3 days of feeding, in the study group 2/15 (13% of patients had reduced their CRP by 40 mg/L or more. In the control group 6/16 (38% of patients had reduced their CRP by this amount. This difference was found to be near the statistical significant limit (P=0.220. Conclusions :The cause of the unexpectedly higher CRP values in the study group is unclear. The rise in CRP was without a commensurate rise in CAPAP or outcome measures so there was no evidence that this represented pancreatic necrosis. The contrast between the CRP and CAPAP results is of interest and we believe that specific pancreatic indices such as CAPAP should be considered in larger future studies.

  7. Determination of digestive proteolytic profile in the larvae of Dyspessa palidata (Staudinger (Lepidoptera: Cossidae

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    M. Mardani-Talaee

    2015-12-01

    Full Text Available Digestive proteolytic profile was determined in the larvae of Dyspessa palidata (Staudinger, which is the most important pest of Alliaceae in Europe and Iran. Compartmentalisation of the proteolytic activities by considering soluble and membrane-bound fractions revealed that soluble fractions of the whole midgut preparations had higher general proteolytic activity than membrane-bound fractions. Also, four proteolytic bands were observed in the soluble fraction of the total midgut preparation in electrophoresis. Compartmentalisation of the specific proteases revealed presence of trypsin, elastase, aminoand carboxy peptidases in posterior midgut but the highest activities of other proteases were found in anterior midgut. The highest activity of general protease was found at pHs of 6 and 8. Also, pH dependency of trypsin, chymotrypsin and elastase were found at values of 8, 7-8 and 9 but cathepsins had the optimal pH at 6. Exopeptidases showed the optimal value at pH of 7 although carboxypeptidase showed same activity at values of 6 and 7. The inhibitory concentrations 50% (IC50 of AEBSF.HCL on trypsin, chymotrypsin and elastase proteases were found to be 3.69, 3.31 and 4.09 mM, respectively. IC50 concentrations of TLCK, SBTI and TPCK significantly inhibited trypsin and chymotrypsin activities. IC50 of E-64 were 3.67 and 4.16 mM on cathepsin B and L but cystatin revealed 5.22 and 4.48 mM concentrations on cathepsin B and L, respectively. EDTA and phenathroline as metalloproteinase inhibitors had IC50 of 3.25 and 3.91 mM on general proteolytic activity. Results of the current study revealed larvae of D. palidata utilised different proteases to increase digestive efficiency when they fed on the host plants containing several toxic molecules.

  8. Structure of the Bacteriophage [phi]KZ Lytic Transglycosylase gp144

    Energy Technology Data Exchange (ETDEWEB)

    Fokine, Andrei; Miroshnikov, Konstantin A.; Shneider, Mikhail M.; Mesyanzhinov, Vadim V.; Rossmann, Michael G. (SOIBC); (Purdue)

    2008-04-02

    Lytic transglycosylases are enzymes that act on the peptidoglycan of bacterial cell walls. They cleave the glycosidic linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anhydromuramoyl product. The x-ray structure of the lytic transglycosylase gp144 from the Pseudomonas bacteriophage {phi}KZ has been determined to 2.5-{angstrom} resolution. This protein is probably employed by the bacteriophage in the late stage of the virus reproduction cycle to destroy the bacterial cell wall to release the phage progeny. {phi}KZ gp144 is a 260-residue {alpha}-helical protein composed of a 70-residue N-terminal cell wall-binding domain and a C-terminal catalytic domain. The fold of the N-terminal domain is similar to the peptidoglycan-binding domain from Streptomyces albus G d-Ala-d-Ala carboxypeptidase and to the N-terminal prodomain of human metalloproteinases that act on extracellular matrices. The C-terminal catalytic domain of gp144 has a structural similarity to the catalytic domain of the transglycosylase Slt70 from Escherichia coli and to lysozymes. The gp144 catalytic domain has an elongated groove that can bind at least five sugar residues at sites A-E. As in other lysozymes, the peptidoglycan cleavage (catalyzed by Glu{sup 115} in gp144) occurs between sugar-binding subsites D and E. The x-ray structure of the {phi}KZ transglycosylase complexed with the chitotetraose (N-acetylglucosamine){sub 4} has been determined to 2.6-{angstrom} resolution. The N-acetylglucosamine residues of the chitotetraose bind in sites A-D.

  9. Ganglioside inhibition of 125I-plasmin binding to colorectal carcinoma cells

    International Nuclear Information System (INIS)

    The pre-incubation of human colorectal carcinoma cells SW 1116 with 25 to 100 uM purified gangliosides resulted in 35-60% inhibition of specific 125I-plasmin binding to the cell surface. After 5 to 6 days in culture, tumor cells were pre-incubated at 4 degrees for 1 to 4 h followed by post-incubation with 125I-plasmin by techniques previously described. At 25 uM the capacity for inhibition of plasmin binding was GT1b greater than GQ1b greater than or equal to GD1a greater than GM1 less than or equal to GgOse 4Cer. Thus a terminal sialyl moiety appears to be necessary (p less than 0.05) although exogenous N-acetyl neuraminic acid was ineffective (p greater than 0.05), indicating a role for the lipid portion of the ganglioside. Other (glyco)lipids such as sphingosine, fucolipid H-1 and sulfatide were without significant effect. The inhibition could not be reversed by the presence of 10 mM Ca+2, EDTA, pre-treatment of the cell with carboxypeptidase or pretreatment of plasmin with neuraminidases. The inhibition was however reversed by post-incubation in control medium without exogenous ganglioside. Cell counts determined prior to, and after ganglioside incubation showed that the effect was not due to cell death or detachment from the culture surface. The dissociation constant for 125I-plasmin binding was 5.6 x 10(-8) M (700,000 sites/cell), but in the presence of trisialoganglioside (GT1b), Scatchard plots suggested diversification of binding sites with 280,000 sites/cell at Kd 2.6 x 10(-8) M and 820,000 sites/cell at Kd 2.1 x 10(-7) M. Another interpretation of the Scatchard plot in the presence of ganglioside was that the glycolipid imposed negative cooperativity on plasmin binding to the cell surface. These results suggest that certain gangliosides can affect tumor cell invasiveness by altering protease binding to the cell surface

  10. Gender differences in genetic risk profiles for cardiovascular disease.

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    Kaisa Silander

    Full Text Available BACKGROUND: Cardiovascular disease (CVD incidence, complications and burden differ markedly between women and men. Although there is variation in the distribution of lifestyle factors between the genders, they do not fully explain the differences in CVD incidence and suggest the existence of gender-specific genetic risk factors. We aimed to estimate whether the genetic risk profiles of coronary heart disease (CHD, ischemic stroke and the composite end-point of CVD differ between the genders. METHODOLOGY/PRINCIPAL FINDINGS: We studied in two Finnish population cohorts, using the case-cohort design the association between common variation in 46 candidate genes and CHD, ischemic stroke, CVD, and CVD-related quantitative risk factors. We analyzed men and women jointly and also conducted genotype-gender interaction analysis. Several allelic variants conferred disease risk for men and women jointly, including rs1801020 in coagulation factor XII (HR = 1.31 (1.08-1.60 for CVD, uncorrected p = 0.006 multiplicative model. Variant rs11673407 in the fucosyltransferase 3 gene was strongly associated with waist/hip ratio (uncorrected p = 0.00005 in joint analysis. In interaction analysis we found statistical evidence of variant-gender interaction conferring risk of CHD and CVD: rs3742264 in the carboxypeptidase B2 gene, p(interaction = 0.009 for CHD, and rs2774279 in the upstream stimulatory factor 1 gene, p(interaction = 0.007 for CHD and CVD, showed strong association in women but not in men, while rs2069840 in interleukin 6 gene, p(interaction = 0.004 for CVD, showed strong association in men but not in women (uncorrected p-values. Also, two variants in the selenoprotein S gene conferred risk for ischemic stroke in women, p(interaction = 0.003 and 0.007. Importantly, we identified a larger number of gender-specific effects for women than for men. CONCLUSIONS/SIGNIFICANCE: A false discovery rate analysis suggests that we may expect half of the reported

  11. Structural and functional characterization of human and murine C5a anaphylatoxins

    Energy Technology Data Exchange (ETDEWEB)

    Schatz-Jakobsen, Janus Asbjørn; Yatime, Laure, E-mail: lay@mb.au.dk; Larsen, Casper [Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus (Denmark); Petersen, Steen Vang [Aarhus University, Bartholin Building, Wilhelm Meyers Allé 4, DK-8000 Aarhus (Denmark); Klos, Andreas [Medical School Hannover, Hannover (Germany); Andersen, Gregers Rom, E-mail: lay@mb.au.dk [Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus (Denmark)

    2014-06-01

    The structure of the human C5aR antagonist, C5a-A8, reveals a three-helix bundle conformation similar to that observed for human C5a-desArg, whereas murine C5a and C5a-desArg both form the canonical four-helix bundle. These conformational differences are discussed in light of the differential C5aR activation properties observed for the human and murine complement anaphylatoxins across species. Complement is an ancient part of the innate immune system that plays a pivotal role in protection against invading pathogens and helps to clear apoptotic and necrotic cells. Upon complement activation, a cascade of proteolytic events generates the complement effectors, including the anaphylatoxins C3a and C5a. Signalling through their cognate G-protein coupled receptors, C3aR and C5aR, leads to a wide range of biological events promoting inflammation at the site of complement activation. The function of anaphylatoxins is regulated by circulating carboxypeptidases that remove their C-terminal arginine residue, yielding C3a-desArg and C5a-desArg. Whereas human C3a and C3a-desArg adopt a canonical four-helix bundle fold, the conformation of human C5a-desArg has recently been described as a three-helix bundle. Here, the crystal structures of an antagonist version of human C5a, A8{sup Δ71–73}, and of murine C5a and C5a-desArg are reported. Whereas A8{sup Δ71–73} adopts a three-helix bundle conformation similar to human C5a-desArg, the two murine proteins form a four-helix bundle. A cell-based functional assay reveals that murine C5a-desArg, in contrast to its human counterpart, exerts the same level of activition as murine C5a on its cognate receptor. The role of the different C5a conformations is discussed in relation to the differential activation of C5a receptors across species.

  12. Denaturation of proteins by SDS and tetraalkylammonium dodecyl sulfates.

    Science.gov (United States)

    Lee, Andrew; Tang, Sindy K Y; Mace, Charles R; Whitesides, George M

    2011-09-20

    This article describes the use of capillary electrophoresis (CE) to examine the influence of different cations (C(+); C(+) = Na(+) and tetra-n-alkylammonium, NR(4)(+), where R = Me, Et, Pr, and Bu) on the rates of denaturation of bovine carbonic anhydrase II (BCA) in the presence of anionic surfactant dodecylsulfate (DS(-)). An analysis of the denaturation of BCA in solutions of Na(+)DS(-) and NR(4)(+)DS(-) (in Tris-Gly buffer) indicated that the rates of formation of complexes of denatured BCA with DS(-) (BCA(D)-DS(-)(n,sat)) are indistinguishable and independent of the cation below the critical micellar concentration (cmc) and independent of the total concentration of DS(-) above the cmc. At concentrations of C(+)DS(-) above the cmc, BCA denatured at rates that depended on the cation; the rates decreased by a factor >10(4) in the order of Na(+) ≈ NMe(4)(+) > NEt(4)(+) > NPr(4)(+) > NBu(4)(+), which is the same order as the values of the cmc (which decrease from 4.0 mM for Na(+)DS(-) to 0.9 mM for NBu(4)(+)DS(-) in Tris-Gly buffer). The relationship between the cmc values and the rates of formation of BCA(D)-DS(-)(n,sat()) suggested that the kinetics of denaturation of BCA involve the association of this protein with monomeric DS(-) rather than with micelles of (C(+)DS(-))(n). A less-detailed survey of seven other proteins (α-lactalbumin, β-lactoglobulin A, β-lactoglobulin B, carboxypeptidase B, creatine phosphokinase, myoglobin, and ubiquitin) showed that the difference between Na(+)DS(-) and NR(4)(+)DS(-) observed with BCA was not general. Instead, the influence of NR(4)(+) on the association of DS(-) with these proteins depended on the protein. The selection of the cation contributed to the properties (including the composition, electrophoretic mobility, and partitioning behavior in aqueous two-phase systems) of aggregates of denatured protein and DS(-). These results suggest that the variation in the behavior of NR(4)(+)DS(-) with changes in R may be

  13. Transcriptomic profiling of Aspergillus flavus in response to 5-azacytidine.

    Science.gov (United States)

    Lin, Jian-Qing; Zhao, Xi-Xi; Zhi, Qing-Qing; Zhao, Ming; He, Zhu-Mei

    2013-07-01

    Aspergillus flavus is a common saprophyte and opportunistic pathogen producing aflatoxin (AF) and many other secondary metabolites. 5-Azacytidine (5-AC), a derivative of the nucleoside cytidine, is widely used for studies in epigenetics and cancer biology as an inactivator of DNA methyltransferase and is also used for studying secondary metabolism in fungi. Our previous studies showed that 5-AC affects development and inhibits AF production in A. flavus, and that A. flavus lacks DNA methylation. In this study, an RNA-Seq approach was applied to explore the mechanism of 5-AC's effect on A. flavus. We identified 240 significantly differentially expressed (Q-value<0.05) genes after 5-AC treatment, including two backbone genes respectively in secondary metabolite clusters #27 and #35. These two clusters are involved in development or survival of sclerotia. GO functional enrichment analysis showed that these significantly differentially expressed genes were mainly involved in catalytic activity and proteolytic functions. The expressed transcripts of most genes in the AF biosynthetic gene cluster in A. flavus showed no significant changes after treatment with 5-AC and were expressed at low levels, and the transcription regulator genes aflR and aflS in this cluster did not show differential expression relative to the sample without 5-AC treatment. We found that the veA gene, which encodes protein bridges VelB and LaeA, decreased profoundly the expressed transcripts, and brlA, which encodes an early regulator of development, increased its transcripts in A. flavus after 5-AC treatment. Our data support a model whereby 5-AC affects development through increasing the expression of brlA by depressing the expression of veA and AF production through suppressing veA expression and dysregulating carboxypeptidase activity, which then prevents the aflatoxisomes (vesicles) from performing their normal function in AF formation. Furthermore, the suppressed veA expression weakens or

  14. Some physical and chemical properties of the smooth muscle inhibitory factor in extracts of the bovine retractor penis muscle.

    Science.gov (United States)

    Gillespie, J S; Hunter, J C; Martin, W

    1981-06-01

    1. A method of extracting and partially purifying a smooth muscle inhibitory factor from the bovine retractor penis is described. This consists of extraction in methanol followed by adsorption on an anion exchange resin, elution from the resin with 500 mM-sodium chloride solution and, if necessary, removal of adenine nucleotides by adsorption on alumina. 2. The inhibitory factor exists in a stable pharmacologically inactive form and an unstable pharmacologically active form. Conversion to the active form is by a brief exposure to acid at pH 2.0. 3. The inhibitory factor is insoluble in ether or acetone but soluble in methanol. Anhydrous methanol, however, irreversibly destroys pharmacological activity especially if the inhibitory factor is in the active form. This effect of methanol is prevented by the presence of 20-30-% water. 4. The inhibitory factor binds to an anion exchange resin but not to a cation exchange resin. It can be eluted from the resin by 500 mM-sodium chloride solution. 5. The molecular weight of the inhibitory factor, as judged by the ability to pass ultrafiltration membranes, is about 500. 6. Inhibitory activity is unaffected by the proteases trypsin, subtilisin or pepsin or by leucine aminopeptidase, pyroglutamate aminopeptidase or carboxypeptidase. The inhibitory effect of the extract and the inhibitory response to stimulation of the non-adrenergic, non-cholinergic nerves are also unaffected by the protease inhibitor, aprotinin. The active material, therefore, is unlikely to be a peptide. 7. Inhibitory activity is abolished by exposure of the extracts to periodic acid or sodium periodate. Acetic anhydride in pyridine also abolishes activity but the vehicle pyridine is also effective. 8. Sodium borohydride but not borate abolishes inhibitory activity when added to the acid-activated material at pH 2.0 but has no effect or may even potentiate activity if added to the stable inactive form at pH 9.0. When added to the acid-activated but

  15. Crystal structure of cathepsin A, a novel target for the treatment of cardiovascular diseases

    Energy Technology Data Exchange (ETDEWEB)

    Schreuder, Herman A., E-mail: herman.schreuder@sanofi.com; Liesum, Alexander, E-mail: alexander.liesum@sanofi.com; Kroll, Katja, E-mail: katja.kroll@sanofi.com; Böhnisch, Britta, E-mail: britta.boehnisch@sanofi.com; Buning, Christian, E-mail: christian.buning@sanofi.com; Ruf, Sven, E-mail: sven.ruf@sanofi.com; Sadowski, Thorsten, E-mail: thorsten.sadowski@sanofi.com

    2014-03-07

    Graphical abstract: - Highlights: • The structures of active cathepsin A and the inactive precursor are very similar. • The only major difference is the absence of a 40 residue activation domain. • The termini of the active catalytic core are held together by a disulfide bond. • Compound 1 reacts with the catalytic Ser150, building a tetrahedral intermediate. • Compound 2 is cleaved by the enzyme and a fragment remained bound. - Abstract: The lysosomal serine carboxypeptidase cathepsin A is involved in the breakdown of peptide hormones like endothelin and bradykinin. Recent pharmacological studies with cathepsin A inhibitors in rodents showed a remarkable reduction in cardiac hypertrophy and atrial fibrillation, making cathepsin A a promising target for the treatment of heart failure. Here we describe the crystal structures of activated cathepsin A without inhibitor and with two compounds that mimic the tetrahedral intermediate and the reaction product, respectively. The structure of activated cathepsin A turned out to be very similar to the structure of the inactive precursor. The only difference was the removal of a 40 residue activation domain, partially due to proteolytic removal of the activation peptide, and partially by an order–disorder transition of the peptides flanking the removed activation peptide. The termini of the catalytic core are held together by the Cys253–Cys303 disulfide bond, just before and after the activation domain. One of the compounds we soaked in our crystals reacted covalently with the catalytic Ser150 and formed a tetrahedral intermediate. The other compound got cleaved by the enzyme and a fragment, resembling one of the natural reaction products, was found in the active site. These studies establish cathepsin A as a classical serine proteinase with a well-defined oxyanion hole. The carboxylate group of the cleavage product is bound by a hydrogen-bonding network involving one aspartate and two glutamate side chains

  16. Comparative Transcriptome and iTRAQ Proteome Analyses of Citrus Root Responses to Candidatus Liberibacter asiaticus Infection.

    Directory of Open Access Journals (Sweden)

    Yun Zhong

    Full Text Available Root samples of 'Sanhu' red tangerine trees infected with and without Candidatus Liberibacter asiaticus (CLas were collected at 50 days post inoculation and subjected to RNA-sequencing and isobaric tags for relative and absolute quantification (iTRAQ to profile the differentially expressed genes (DEGs and proteins (DEPs, respectively. Quantitative real-time PCR was subsequently used to confirm the expression of 16 selected DEGs. Results showed that a total of 3956 genes and 78 proteins were differentially regulated by HLB-infection. Among the most highly up-regulated DEPs were sperm specific protein 411, copper ion binding protein, germin-like proteins, subtilisin-like proteins and serine carboxypeptidase-like 40 proteins whose transcript levels were concomitantly up-regulated as shown by RNA-seq data. Comparison between our results and those of the previously reported showed that known HLB-modulated biological pathways including cell-wall modification, protease-involved protein degradation, carbohydrate metabolism, hormone synthesis and signaling, transcription activities, and stress responses were similarly regulated by HLB infection but different or root-specific changes did exist. The root unique changes included the down-regulation in genes of ubiquitin-dependent protein degradation pathway, secondary metabolism, cytochrome P450s, UDP-glucosyl transferases and pentatricopeptide repeat containing proteins. Notably, nutrient absorption was impaired by HLB-infection as the expression of the genes involved in Fe, Zn, N and P adsorption and transportation were significantly changed. HLB-infection induced some cellular defense responses but simultaneously reduced the biosynthesis of the three major classes of secondary metabolites, many of which are known to have anti-pathogen activities. Genes involved in callose deposition were up-regulated whereas those involved in callose degradation were also up-regulated, indicating that the sieve tube

  17. Crystal structure of cathepsin A, a novel target for the treatment of cardiovascular diseases

    International Nuclear Information System (INIS)

    Graphical abstract: - Highlights: • The structures of active cathepsin A and the inactive precursor are very similar. • The only major difference is the absence of a 40 residue activation domain. • The termini of the active catalytic core are held together by a disulfide bond. • Compound 1 reacts with the catalytic Ser150, building a tetrahedral intermediate. • Compound 2 is cleaved by the enzyme and a fragment remained bound. - Abstract: The lysosomal serine carboxypeptidase cathepsin A is involved in the breakdown of peptide hormones like endothelin and bradykinin. Recent pharmacological studies with cathepsin A inhibitors in rodents showed a remarkable reduction in cardiac hypertrophy and atrial fibrillation, making cathepsin A a promising target for the treatment of heart failure. Here we describe the crystal structures of activated cathepsin A without inhibitor and with two compounds that mimic the tetrahedral intermediate and the reaction product, respectively. The structure of activated cathepsin A turned out to be very similar to the structure of the inactive precursor. The only difference was the removal of a 40 residue activation domain, partially due to proteolytic removal of the activation peptide, and partially by an order–disorder transition of the peptides flanking the removed activation peptide. The termini of the catalytic core are held together by the Cys253–Cys303 disulfide bond, just before and after the activation domain. One of the compounds we soaked in our crystals reacted covalently with the catalytic Ser150 and formed a tetrahedral intermediate. The other compound got cleaved by the enzyme and a fragment, resembling one of the natural reaction products, was found in the active site. These studies establish cathepsin A as a classical serine proteinase with a well-defined oxyanion hole. The carboxylate group of the cleavage product is bound by a hydrogen-bonding network involving one aspartate and two glutamate side chains

  18. Key role of mast cells and their major secretory products in inflammatory bowel disease

    Institute of Scientific and Technical Information of China (English)

    Shao-Heng He

    2004-01-01

    Hirtoncally, mast cells were known as a key cell type involved in type I hypersensitivity Until last two decades, this cell type was recognized to be widely involved in a number of non-allergic diseases including inflammatory bowel disease (IBD). Markedly increased numbers of mast cells were observed in the mucosa of the iieum and colon of patients with IBD, which was accompanied by great changes of the content in mart cells such as dramatically increaed expression of TNFα, IL-16 and substance P.The evidence of mast cell degranulation was found in the wall of intestine from patients with IBD with immunohistochemistry technique. The highly elevated histamine and tryptase levels were detected in mucosa of petienta with IBD, strongly suggesting that mast cell degranulation is involved in the pathogenesis of IBD.However, Iittle is known of the actions of histamine, tryptase,chymase and carboxypeptidase in IBD. Over the lart decade,heparin has been used to treat IBD in clinical practice. The low molecular weight heparin (LMWH) was effective as adjuvant therapy, and the petienis showed good clinical and laberatory respense with no senous advere effectd. The roles of PGD2, LTC4, PAF and mast cell cytokines in IBD were also discussed. Recently, a series of experiments with dispersed colon mast cells suggested there should be at least two pathways in man for mast colls to amplify their own activationdegranulation signals in an autocrine or paracrine mannec.The hypethesis is that mast cell secretogogues induce mart cell degranulation, release histamine, then stimulate the adjacent mast cells or positively feedback to further stimulate its host mast cells through H1 recepton.Whereas released tryptase acts similarly to hirtamine, but activates mart cells through its receptor PAR-2. The connections between current anti-IBD therapies or potential therapies for IBD with mast cells were discussed, implicating further that mast cell is a key cell type that is involved in the

  19. Epigenetic modulation of RFC1, MHC2TA and HLA-DR in systemic lupus erythematosus: association with serological markers and six functional polymorphisms of one-carbon metabolic pathway.

    Science.gov (United States)

    Rupasree, Yedluri; Naushad, Shaik Mohammad; Rajasekhar, Liza; Kutala, Vijay Kumar

    2014-02-15

    The current study was conducted to elucidate the effect of genetic variations in one-carbon metabolism on the epigenetic regulation of major histocompatibility complex II transactivator (MHC2TA), reduced folate carrier 1 (RFC1/SLC19A1) and human leukocyte antigen (HLA)-DR in systemic lupus erythematosus (SLE). PCR-RFLP/AFLP, bisulfite-sequencing and real-time PCR approaches were used for genetic, epigenetic and expression analysis respectively. SLE cases exhibited elevated plasma homocysteine levels compared to healthy controls (24.93 ± 1.3 vs. 11.67 ± 0.48 μmol/l), while plasma folate levels showed no association (7.10 ± 2.49 vs. 7.64 ± 2.09 ng/ml). The RFC1 80G>A polymorphism showed 1.32-fold risk (95% CI: 1.02-1.72) for SLE, while glutamate carboxypeptidase II (GCPII) 1561C>T showed reduced risk (OR: 0.47, 95% CI: 0.24-0.90). The expression of RFC1 (0.37 ± 0.09 vs. 0.60 ± 0.17) and HLA-DR (0.68 ± 0.17 vs. 0.98 ± 0.02) was down regulated in the SLE cases. The hypermethylation of RFC1 as observed in the current study may contribute for its down regulation. Plasma folate and thymidylate synthase (TYMS) 5'-UTR 28 bp tandem repeat showed an inverse association with methylation of RFC1 and MHC2TA. SLE cases with hypocomplementemia showed hypermethylation of RFC1, hypomethylation/up regulation of MHC2TA and down regulation of HLA-DR. The hypermethylation of MHC2TA and down regulation of RFC1, MHC2TA and HLA-DR were observed in anti-cardiolipin antibody positive SLE cases. The up regulation of RFC1 and HLA-DR was observed in anti-dsDNA antibody positive SLE cases. The hypomethylation/upregulation of RFC1 and MHC2TA was observed in anti-RNP antibody positive cases. To conclude, one-carbon genetic variants influence epigenetic of MHC2TA and RFC1, thus contributing to phenotypic heterogeneity of SLE. PMID:24333266

  20. Genetic associations of type 2 diabetes with islet amyloid polypeptide processing and degrading pathways in asian populations.

    Directory of Open Access Journals (Sweden)

    Vincent Kwok Lim Lam

    Full Text Available Type 2 diabetes (T2D is a complex disease characterized by beta cell dysfunctions. Islet amyloid polypeptide (IAPP is highly conserved and co-secreted with insulin with over 40% of autopsy cases of T2D showing islet amyloid formation due to IAPP aggregation. Dysregulation in IAPP processing, stabilization and degradation can cause excessive oligomerization with beta cell toxicity. Previous studies examining genetic associations of pathways implicated in IAPP metabolism have yielded conflicting results due to small sample size, insufficient interrogation of gene structure and gene-gene interactions. In this multi-staged study, we screened 89 tag single nucleotide polymorphisms (SNPs in 6 candidate genes implicated in IAPP metabolism and tested for independent and joint associations with T2D and beta cell dysfunctions. Positive signals in the stage-1 were confirmed by de novo and in silico analysis in a multi-centre unrelated case-control cohort. We examined the association of significant SNPs with quantitative traits in a subset of controls and performed bioinformatics and relevant functional analyses. Amongst the tag SNPs, rs1583645 in carboxypeptidase E (CPE and rs6583813 in insulin degrading enzyme (IDE were associated with 1.09 to 1.28 fold increased risk of T2D (P Meta = 9.4×10(-3 and 0.02 respectively in a meta-analysis of East Asians. Using genetic risk scores (GRS with each risk variant scoring 1, subjects with GRS≥3 (8.2% of the cohort had 56% higher risk of T2D than those with GRS = 0 (P = 0.01. In a subcohort of control subjects, plasma IAPP increased and beta cell function index declined with GRS (P = 0.008 and 0.03 respectively. Bioinformatics and functional analyses of CPE rs1583645 predicted regulatory elements for chromatin modification and transcription factors, suggesting differential DNA-protein interactions and gene expression. Taken together, these results support the importance of dysregulation of IAPP

  1. 中国红树林最北缘引种区不同季节和树龄秋茄生理特征比较%Comparison of Physiological Characteristics of Different Aged Transplanted Kandelia obovata Trees in Summer and Winter in the Northest Transplanted Area of China

    Institute of Scientific and Technical Information of China (English)

    马小伟; 郑春芳; 刘伟成; 施孟如; 仇建标; 彭欣; 黄丽; 陈少波

    2013-01-01

    Ximen island of Yueqing bay, the northernmost boundary of artificially planted mangrove in China, was chosen as a study site. Changes of photosynthetic pigment content, the antioxidation system, proteinases of different aged Kandelia obovata (aged 5, 9 and 54 years old) in summer and winter were studied. The results obtained were as follows. (1) Low temperature in winter reduced the leaf chlorophyll content, but increased leaf chlorophyll a to chlorophyll b (chl a/b) ratio, contents of malondialdehyde (MDA), soluble sugar, soluble protein and free amino acid, and enhanced activities of endopeptidase and carboxypeptidase in leaf. (2) The contents of the soluble sugar, soluble protein and free amino acid, and the activities of endopeptidase, carboxypeptidase in leaf were decreased with increasing age in summer and winter. (3) In summer, the content of the chlorophyll of the Kandelia obovata aged 5, 9 years old was more higher than the Kandelia obovata aged 54 years old. The super oxygen dehydrogenises (SOD) activity and mai ildehyde (MDA) content were increased with increasing age. However, the peroxidase (POD) activity was decreased with ,, .asing age. (4) In winter, the contents of leaf chlorophyll and MDA were decreased with increased age, while of the POD activity and Chla/Chlb ratio in leaf were increased with increasing age. Also, the SOD activity in leaf decreased and then increased with increasing age. In short, Kandelia obovata with 5 years old and 9 years old could vigorously grow, while 54 years old mangrove keep metabolism stability in summer. In winter, the physiological response of 5-year and 9-year old Kandelia obovata to low temperature was more sensitive than that of 54 years old, but 5-year and 9-year old trees could survive safely the winter through adjusting their carbon and nitrogen metabolism.%以中国红树林引种最北缘的乐清湾西门岛海域为研究地点,研究了夏季、冬季5年生、9年生、54年生秋茄植株的光合色

  2. Lasso peptides: an intriguing class of bacterial natural products.

    Science.gov (United States)

    Hegemann, Julian D; Zimmermann, Marcel; Xie, Xiulan; Marahiel, Mohamed A

    2015-07-21

    mechanism through which the precursor peptide is enzymatically processed into a mature lasso peptide and crucial residues for enzymatic recognition. Furthermore, we highlight new insights considering the protease and thermal stability of lasso peptides and discuss why seven amino acid residue rings are likely to be the lower limit feasible for this compound class. To elucidate their fascinating three-dimensional structures, NMR spectroscopy is commonly employed. Therefore, the general methodology to elucidate these structures by NMR will be discussed and pitfalls for these approaches are highlighted. In addition, new tools provided by recent investigations to assess and prove the lasso topology without a complete structure elucidation will be summarized. These include techniques like ion mobility-mass spectrometry and a combined approach of thermal and carboxypeptidase treatment with subsequent LC-MS analysis. Nevertheless, even though much was learned about these compounds in recent years, their true native function and the exact enzymatic mechanism of their maturation remain elusive. PMID:26079760

  3. Simulations of chemical catalysis

    Science.gov (United States)

    Smith, Gregory K.

    dissociative, metaphosphate-like structure, stabilized by the Mg(II) ion and several hydrogen bonds. The calculated free-energy barrier is consistent with experimental values. Project 3 - Bacterial Enzyme Anthrax Lethal Factor In this dissertation, we report a hybrid quantum mechanical and molecular mechanical study of the catalysis of anthrax lethal factor, an important first step in designing inhibitors to help treat this powerful bacterial toxin. The calculations suggest that the zinc peptidase uses the same general base-general acid mechanism as in thermolysin and carboxypeptidase A, in which a zinc-bound water is activated by Glu687 to nucleophilically attack the scissile carbonyl carbon in the substrate. The catalysis is aided by an oxyanion hole formed by the zinc ion and the side chain of Tyr728, which provide stabilization for the fractionally charged carbonyl oxygen. Project 4 - Methanol Steam Reforming on PdZn alloy Recent experiments suggested that PdZn alloy on ZnO support is a very active and selective catalyst for methanol steam reforming (MSR). Plane-wave density functional theory calculations were carried out on the initial steps of MSR on both PdZn and ZnO surfaces. Our calculations indicate that the dissociation of both methanol and water is highly activated on flat surfaces of PdZn such as (111) and (100), while the dissociation barriers can be lowered significantly by surface defects, represented here by the (221), (110), and (321) faces of PdZn. The corresponding processes on the polar Zn-terminated ZnO(0001) surfaces are found to have low or null barriers. Implications of these results for both MSR and low temperature mechanisms are discussed.

  4. Quantitative proteomic analysis on the serum of patients with medicamentose-like dermatitis induced by occupational trichloroethylene exposure%职业性三氯乙烯药疹样皮炎患者血清蛋白质差异表达分析

    Institute of Scientific and Technical Information of China (English)

    黄振烈; 越飞; 黄汉林; 杨杏芬; 夏丽华; 陈慈珊; 邱新香; 黄建勋; 李来玉

    2011-01-01

    Objective To compare the proteome of the serum of patients with medicamentose-like dermatitis due to occupational trichloroethylene exposure(OMDTE) in acute and recovery stages. Methods After the samples were collected and pretreated, the expression of protein in serum was analyzed by 2-dimensional electrophoresis (2-DE) and differentially expressed protein spots were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry ( MALDI-TOF-TOF/ MS). Results 31 proteins with altered modifications were separated and identified by 2-DE combined with MALDI-TOF-TOF/ MS. Compared with the serum proteome in the recovery stage, proteins showed up-regulated expression in acute stage included S100 cal-cium-binding protein A8, calprotectin, amyloid related serum protein SAA, leucine aminopeptidase, plasma glutathione peroxidase, etc. However, retinol binding protein, acetyl-CoA carboxylase and carboxypeptidase N were down-regulated. The function of these proteins involved in inflammatory responses, oxidative stress, retinol metabolism, fatty acid metabolism and regulation of kinins and anaphylatoxins. Conclusion The identified proteins provided target molecules for the further study on mechanisms of OMDTE and can be used as potential biomarkers for the disease.%目的 比较职业性三氯乙烯药疹样皮炎(OMDTE)患者发病急性期与治愈后的血清蛋白质表达谱.方法 患者血清经前处理后,双向凝胶电泳分离蛋白质,软件分析凝胶图像,基质辅助激光解吸电离飞行时间串联质谱鉴定差异表达蛋白斑点.结果 与治愈后的血清蛋白质表达谱比较,在发病急性期发现41个明显差异表达的蛋白斑点,鉴定出31个蛋白.上调的蛋白有S100钙结合蛋白A8、钙网蛋白、血清淀粉样蛋白A、亮氨酸氨基肽酶、谷胱甘肤过氧化物酶等;下调的蛋白有视黄醇结合蛋白、乙酰辅酶A羧化酶、羧肽酶N等;涉及的功能通路包括炎症反应、氧